U.S. patent application number 14/180181 was filed with the patent office on 2014-09-11 for human g protein-coupled receptor and modulators thereof for the treatment of atherosclerosis and atherosclerotic disease and for the treatment of conditions related to mcp-1 expression.
This patent application is currently assigned to ARENA PHARMACEUTICALS, INC.. The applicant listed for this patent is ARENA PHARMACEUTICALS, INC.. Invention is credited to Joel Gatlin, Yaron Hakak, Chen W. Liaw, David J. Unett.
Application Number | 20140255310 14/180181 |
Document ID | / |
Family ID | 37547612 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255310 |
Kind Code |
A1 |
Hakak; Yaron ; et
al. |
September 11, 2014 |
Human G Protein-Coupled Receptor and Modulators Thereof for the
Treatment of Atherosclerosis and Atherosclerotic Disease and for
the Treatment of Conditions Related to MCP-1 Expression
Abstract
The present invention relates to methods of using a G
protein-coupled receptor (GPCR) to identify whether a candidate
compound is a modulator of atherogenesis. In certain embodiments,
the GPCR couples to Gi. In certain embodiments, the GPCR is human.
Agonists of the invention are useful as therapeutic agents for the
prevention or treatment of atherosclerosis and atherosclerotic
disease, including coronary artery disease, myocardial infarction,
peripheral arterial disease, and ischemic stroke. Agonists of the
invention are additionally useful as therapeutic agents for the
prevention or treatment of conditions related to MCP-1 expression,
including but not limited to rheumatoid arthritis, Crohn's disease,
and multiple sclerosis.
Inventors: |
Hakak; Yaron; (San Diego,
CA) ; Unett; David J.; (San Diego, CA) ;
Gatlin; Joel; (San Diego, CA) ; Liaw; Chen W.;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARENA PHARMACEUTICALS, INC. |
San Diego |
CA |
US |
|
|
Assignee: |
ARENA PHARMACEUTICALS, INC.
San Diego
CA
|
Family ID: |
37547612 |
Appl. No.: |
14/180181 |
Filed: |
February 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11991232 |
Oct 7, 2009 |
8691498 |
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PCT/US2006/033651 |
Aug 29, 2006 |
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14180181 |
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60714137 |
Sep 2, 2005 |
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Current U.S.
Class: |
424/9.2 ;
435/6.12; 435/6.13; 435/7.21; 514/21.2; 530/350 |
Current CPC
Class: |
G01N 33/74 20130101;
C07K 14/47 20130101; C07K 14/723 20130101; G01N 2800/32 20130101;
A61P 9/10 20180101; A61K 49/0008 20130101; G01N 2500/00 20130101;
G01N 33/5041 20130101; A61K 38/1709 20130101; G01N 33/6893
20130101; G01N 2800/323 20130101; G01N 2333/726 20130101; A61P
29/00 20180101 |
Class at
Publication: |
424/9.2 ;
514/21.2; 530/350; 435/7.21; 435/6.12; 435/6.13 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/68 20060101 G01N033/68; A61K 49/00 20060101
A61K049/00; C07K 14/47 20060101 C07K014/47; A61K 38/17 20060101
A61K038/17 |
Claims
1-57. (canceled)
58. A method comprising: (a) contacting a candidate compound with a
G protein-coupled receptor (GPCR) comprising an amino acid sequence
having at least 80% identity to SEQ ID NO:2, wherein said GPCR is
present on a cell or isolated membrane thereof; (b) determining the
ability of the compound to inhibit or stimulate said GPCR; and (c)
determining if said compound modulates atherogenesis.
59. The method of claim 58, wherein said determining element (c)
comprises administering said compound to a mammal.
60. The method of claim 59, wherein said determining element (b)
comprises detecting stimulation of said GPCR and element (c)
comprises detecting inhibition of atherogenesis.
61. The method of claim 59, wherein said determining element (c)
comprises determining if said compound treats or prevents
atherosclerosis, coronary artery disease, myocardial infarction,
peripheral artery disease or ischemic stroke.
62. A compound that stimulates a G protein-coupled receptor
comprising an amino acid having at least 80% identity to SEQ ID
NO:2, wherein said compound is identified using the method of claim
58.
63. A pharmaceutical composition comprising the compound of claim
62 and a pharmaceutically acceptable carrier.
64. The pharmaceutical composition of claim 63, further comprising
a second pharmaceutically active agent.
65. A method of treating or preventing an atherosclerotic disease,
comprising administering the pharmaceutical composition of claim 63
to a mammal having said atherosclerotic disease.
66. A method comprising: (a) contacting a compound with a GPCR
comprising an amino acid sequence having at least 80% identity to
SEQ ID NO:2; (b) determining the ability of the compound to bind to
said GPCR; and (c) determining if said compound modulates
atherogenesis.
67. The method of claim 66, wherein said determining element (c)
comprises contacting said compound with a macrophage and
determining if said compound modulates ATP-binding cassette
transporter 1 (ABCA1) expression.
68. The method of claim 66, wherein said determining element (c)
comprises contacting said compound with a macrophage and
determining if said compound modulates monocyte chemoattractant
protein-1 (MCP-1) expression.
69. The method of claim 66, wherein said determining element (c)
comprises determining if said compound treats or prevents
atherosclerosis, coronary artery disease, myocardial infarction,
peripheral artery disease or ischemic stroke.
70. The method of claim 66, wherein said method further comprises
admixing said compound with a pharmaceutically acceptable carrier
to produce a pharmaceutical composition.
71. A method of treating or preventing an atherosclerotic disease,
comprising administering the pharmaceutical composition of claim 70
to a mammal having said atherosclerotic disease.
72. A method of preparing a pharmaceutical composition, comprising
admixing a compound that has been determined to modulate
atherogenesis using the method of claim 66 with a pharmaceutically
acceptable carrier.
73. A method comprising: (a) contacting a candidate compound with a
GPCR comprising an amino acid sequence having at least 80% identity
to SEQ ID NO:2, wherein said GPCR is present on a cell or isolated
membrane thereof; (b) determining the ability of the compound to
inhibit or stimulate said GPCR; and (c) determining if said
compound modulates inflammation.
74. A compound that stimulates a G protein-coupled receptor
comprising an amino acid sequence having at least 80% identity to
SEQ ID NO:2, wherein said compound is identified using the method
of claim 73.
75. A pharmaceutical composition comprising the compound of claim
74 and a pharmaceutically acceptable carrier.
76. A method of treating inflammation, comprising administering the
pharmaceutical composition of claim 75 to a mammal having
inflammation.
77. The method of claim 73, wherein said method further comprises
admixing said compound with a pharmaceutically acceptable carrier
to produce a pharmaceutical composition.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of using a G
protein-coupled receptor (GPCR) to identify whether a candidate
compound is a modulator of atherogenesis. In certain embodiments,
the GPCR couples to Gi. In certain embodiments, the GPCR is human.
Agonists of the invention are useful as therapeutic agents for
inhibiting atherogenesis and for the prevention or treatment of
atherosclerosis and atherosclerotic disease, including coronary
artery disease, myocardial infarction, peripheral arterial disease,
and ischemic stroke. Agonists of the invention are additionally
useful as therapeutic agents for the prevention or treatment of
conditions related to MCP-1 expression, including but not limited
to rheumatoid arthritis, Crohn's disease, and multiple
sclerosis.
BACKGROUND OF THE INVENTION
[0002] The following discussion is intended to facilitate the
understanding of the invention, but is not intended nor admitted to
be prior art to the invention.
[0003] A. Atherosclerosis
[0004] Atherosclerosis is a complex disease that is characterized
by cholesterol deposition and monocyte infiltration into the
subendothelial space, resulting in foam cell formation.
Cardiovascular disease (mainly atherosclerosis) accounts for 35% of
all deaths in the U.S.A. and similar Western countries. The
monocyte/macrophage plays key roles both in the initiation and
progression of atherosclerosis; for example, hypercholesterolemic
mice become extremely resistant to atherosclerosis if they are bred
to macrophage-deficient mice [Smith et al, PNAS (1995)
92:8264-8268].
[0005] ATP-binding cassette transporter 1 (ABCA1) controls
apoAI-mediated cholesterol efflux from macrophages. Expression of
ABCA1 is induced during monocyte differentiation into macrophages.
ABCA1 protein is dramatically decreased in human atheroma in
comparison to nonlesional tissue [Forcheron et al, Arterioscler
Thromb Vasc Biol (2005) 25:1711-1717]. Inactivation of ABCA1 in
macrophages markedly increases atherosclerosis and foam cell
accumulation in ApoE.sup.-/- mice [Aiello et al, Arterioscler
Thromb Vasc Biol (2002) 22:630-637]. ABCA1 upregulation in
macrophages inhibits the progression of atherosclerotic lesions
[Van Eck et al, Arterioscler Thromb Vasc Biol (2006)
26:929-934].
[0006] Monocyte chemoattractant protein (MCP-1) is a key mediator
of monocyte trafficking. In situ hybridization carried out on
atherosclerotic human arteries detected MCP-1 mRNA macrophage-rich
regions of atherosclerotic lesions but not in sublesional medial
smooth muscle cells or in normal arteries [Yla-Herttuala et al,
PNAS (1991) 88:5252-5256; Lutgens et al, Circulation (2005)
111:3443-3452]. MCP-1 expression by macrophages increases the
progression of atherosclerosis by increasing both macrophage
numbers and oxidized lipid accumulation [Aiello et al, Arterioscler
Thromb Vasc Biol (1999) 19:1518-1525]. Use of knockout mice has
implicated MCP-1 in attracting macrophage recruitment in
atherosclerosis. Atherosclerosis is essentially abolished in
MCP-1.sup.-/- mice indicating that MCP-1 is absolutely required for
atherosclerois from its earliest stages [Gu et al, Mol Cell (1998)
2:275-281]. Using a dominant-negative mutant of MCP-1, it has been
shown that vascular inflammation mediated by MCP-1 has a central
role in the development of atherosclerosis, and plaque
destabilization, leading to acute myocardial ischemia [Egashira,
Hypertension (2003) 41:834-841].
[0007] Conditions related to expression of MCP-1 in
monocytes/macrophages additional to atherosclerosis and
atherosclerotic disease include, but are not limited to, rheumatoid
arthritis [see, e.g., Koch et al, J Clin Invest (1992) 90:772-779;
Dawson et al, Expert Opin Ther Targets (2003) 7:35-48].
[0008] An agent that decreases expression of MCP-1 or increases
expression of ABCA1 is useful in the treatment of atherosclerosis
and atherosclerotic disease, and an agent which is a small molecule
is further advantageous.
[0009] B. GPR84
[0010] GPR84 is a GPCR asserted to be selectively expressed in
granulocytes [Yousefi et al., J Leukoc Biol (2001) 69:1045-1052].
GPR84 has been conjectured to have a role in regulating early IL-4
gene expression in activated T cells [Venkataraman et al, Immunol
Lett (2005) 101:144-153].
[0011] C. G Protein-Coupled Receptors
[0012] Although a number of receptor classes exist in humans, by
far the most abundant and therapeutically relevant is represented
by the G protein-coupled receptor (GPCR) class. It is estimated
that there are some 30,000-40,000 genes within the human genome,
and of these, approximately 2% are estimated to code for GPCRs.
[0013] GPCRs represent an important area for the development of
pharmaceutical products: from approximately 20 of the 100 known
GPCRs, approximately 60% of all prescription pharmaceuticals have
been developed. For example, in 1999, of the top 100 brand name;
prescription drugs, the following drugs interact with GPCRs (the
primary diseases and/or disorders treated related to the drug is
indicated in parentheses):
TABLE-US-00001 Claritin .RTM. (allergies) Prozac .RTM. (depression)
Vasotec .RTM. (hypertension) Paxil .RTM. (depression) Zoloft .RTM.
(depression) Zyprexa .RTM.(psychotic disorder) Cozaar .RTM.
(hypertension) Imitrex .RTM. (migraine) Zantac .RTM. (reflux)
Propulsid .RTM. (reflux disease) Risperdal .RTM. (schizophrenia)
Serevent .RTM. (asthma) Pepcid .RTM. (reflux) Gaster .RTM. (ulcers)
Atrovent .RTM. (bronchospasm) Effexor .RTM. (depression) Depakote
.RTM. (epilepsy) Cardura .RTM.(prostatic hypertrophy) Allegra .RTM.
(allergies) Lupron .RTM. (prostate cancer) Zoladex .RTM. (prostate
cancer) Diprivan .RTM. (anesthesia) BuSpar .RTM. (anxiety) Ventolin
.RTM. (bronchospasm) Hytrin .RTM. (hypertension) Wellbutrin .RTM.
(depression) Zyrtec .RTM. (rhinitis) Plavix .RTM. (MI/stroke)
Toprol-XL .RTM. (hypertension) Tenormin .RTM. (angina) Xalatan
.RTM. (glaucoma) Singulair .RTM. (asthma) Diovan .RTM.
(hypertension) Hamal .RTM. (prostatic hyperplasia) (Med Ad News
1999 Data).
[0014] GPCRs share a common structural motif, having seven
sequences of between 22 to 24 hydrophobic amino acids that form
seven alpha helices, each of which spans the membrane (each span is
identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2
(TM-2), etc.). The transmembrane helices are joined by strands of
amino acids between transmembrane-2 and transmembrane-3,
transmembrane-4 and transmembrane-5, and transmembrane-6 and
transmembrane-7 on the exterior, or "extracellular" side, of the
cell membrane (these are referred to as "extracellular" regions 1,
2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane
helices are also joined by strands of amino acids between
transmembrane-1 and transmembrane-2, transmembrane-3 and
transmembrane-4, and transmembrane-5 and transmembrane-6 on the
interior, or "intracellular" side, of the cell membrane (these are
referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and
IC-3), respectively). The "carboxy" ("C") terminus of the receptor
lies in the intracellular space within the cell, and the "amino"
("N") terminus of the receptor lies in the extracellular space
outside of the cell.
[0015] Generally, when a ligand binds with the receptor (often
referred to as "activation" of the receptor), there is a change in
the conformation of the receptor that facilitates coupling between
the intracellular region and an intracellular "G-protein." It has
been reported that GPCRs are "promiscuous" with respect to G
proteins, i.e., that a GPCR can interact with more than one G
protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although
other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G
proteins that have been identified. Ligand-activated GPCR coupling
with the G-protein initiates a signaling cascade process (referred
to as "signal transduction"). Under normal conditions, signal
transduction ultimately results in cellular activation or cellular
inhibition. Although not wishing to be bound to theory, it is
thought that the IC-3 loop as well as the carboxy terminus of the
receptor interact with the G protein.
[0016] Gs-coupled GPCRs elevate-intracellular cAMP levels. GPCRs
coupled to Gi, Go, or Gz lower intracellular cAMP levels.
Gq-coupled GPCRs elevate intracellular IP.sub.3 and Ca.sup.2+
levels.
[0017] There are also promiscuous G proteins, which appear to
couple several classes of GPCRs to the phospholipase C pathway,
such as G15 or G16 [Offermanns & Simon, J Biol Chem (1995)
270:15175-80], or chimeric G proteins designed to couple a large
number of different GPCRs to the same pathway, e.g. phospholipase C
[Milligan & Rees, Trends in Pharmaceutical Sciences (1999)
20:118-24]. A GPCR coupled to the phospholipase C pathway elevates
intracellular IP.sub.3 and Ca.sup.2+ levels.
[0018] Under physiological conditions, GPCRs exist in the cell
membrane in equilibrium between two different conformations: an
"inactive" state and an "active" state. A receptor in an inactive
state is unable to link to the intracellular signaling transduction
pathway to initiate signal transduction leading to a biological
response. Changing the receptor conformation to the active state
allows linkage to the transduction pathway (via the G-protein) and
produces a biological response.
[0019] A receptor may be stabilized in an active state by a ligand
or a compound such as a drug. Recent discoveries, including but not
exclusively limited to modifications to the amino acid sequence of
the receptor, provide means other than ligands or drugs to promote
and stabilize the receptor in the active state conformation. These
means effectively stabilize the receptor in an active state by
simulating the effect of a ligand binding to the receptor.
Stabilization by such ligand-independent means is termed
"constitutive receptor activation."
SUMMARY OF THE INVENTION
[0020] Nucleotide sequence encoding human GPR84 polypeptide is
given in SEQ ID NO:1. The amino acid sequence of said encoded human
GPR84 polypeptide is given in SEQ ID NO:2. Nucleotide sequence
encoding mouse GPR84 polypeptide is given in SEQ ID NO:3. The amino
acid sequence of said encoded mouse GPR84 polypeptide is given in
SEQ ID NO:4. Nucleotide sequence encoding rat GPR84 polypeptide is
given in SEQ ID NO:5. The amino acid sequence of said encoded rat
GPR84 polypeptide is given in SEQ ID NO:6. Nucleotide sequence
encoding amino acids 2-396 of SEQ ID NO:2 fused N-terminally with a
hemagglutinin (HA) epitope tag is given in SEQ ID NO: 19. The amino
acid sequence of said encoded amino acids 2-396 of SEQ ID NO:2
fused N-terminally with a hemagglutinin (HA) epitope tag is given
in SEQ ID NO:20.
[0021] Applicants have shown that GPR84 is expressed endogenously
in monocytes/macrophages. Applicants have shown that activation of
GPR84 leads to decreased levels of intracellular cAMP, consistent
with GPR84 being Gi-coupled. Applicants have shown that an agonist
of GPR84 selectively modulates cytokine expression in
monocytes/macrophages, including decreasing MCP-1 expression.
Applicants have shown that an agonist of GPR84 increases expression
of ABCA1 in monocytes/macrophages. The present invention features
methods relating to GPR84 for identifying a candidate compound as a
modulator of atherogenesis, as an agonist for use as a
pharmaceutical agent for atherosclerosis or an atherosclerotic
disease, as a compound for the prevention or treatment of
atherosclerosis or an atherosclerotic disease, or as a compound for
the prevention or treatment of a condition related to MCP-1
expression. The present invention additionally features methods of
using an agonist of GPR84 for preventing or treating
atherosclerosis or an atherosclerotic disease in a mammal. The
present invention additionally features methods of using an agonist
of GPR84 for preventing or treating a condition related to MCP-1
expression in a mammal. Agonists of the invention are useful as
therapeutic agents for inhibiting atherogenesis and for the
prevention or treatment of atherosclerosis and atherosclerotic
disease, including coronary artery disease, myocardial infarction,
peripheral arterial disease, and ischemic stroke. Agonists of the
invention are additionally useful as therapeutic agents for the
prevention or treatment of conditions related to MCP-1 expression,
including but not limited to rheumatoid arthritis, Crohn's disease,
and multiple sclerosis.
[0022] In a first aspect, the invention features a method of
identifying a candidate compound as a modulator of atherogenesis,
comprising the steps of: [0023] (a) contacting the candidate
compound with a GPCR comprising an amino acid sequence selected
from the group consisting of: [0024] (i) the amino acid sequence of
SEQ ID NO:2; [0025] (ii) amino acids 2-396 of SEQ ID NO:2; [0026]
(iii) amino acids 2-396 of SEQ ID NO:2, wherein the GPCR does not
comprise amino acids 1-396 of SEQ ID NO:2; [0027] (iv) the amino
acid sequence of SEQ ID NO:20; [0028] (v) the amino acid sequence
of a G protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0029]
(vi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0030] (vii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:2; [0031] (viii) the amino acid sequence
of SEQ ID NO:4; [0032] (ix) amino acids-2-396 of SEQ ID NO:4;
[0033] (x) amino acids 2-396 of SEQ ID NO:4 wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:4; [0034] (xi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0035] (xii) the amino acid sequence of
an endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0036] (xiii) the amino acid sequence of SEQ ID NO:6;
[0037] (xiv) amino acids 2-396 of SEQ ID NO:6; [0038] (xv) amino
acids 2-396 of SEQ ID NO:6, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:6; [0039] (xvi) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0040] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0041] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0042] (b) determining the ability of the compound to
inhibit or stimulate functionality of the GPCR, [0043] wherein the
ability of the compound to inhibit or stimulate functionality of
the GPCR is indicative of the compound being a modulator of
atherogenesis.
[0044] The invention also features a method of identifying a
candidate compound as a modulator of MCP-1 expression, comprising
the steps of: [0045] (a) contacting the candidate compound with a
GPCR comprising an amino acid sequence selected from the group
consisting of: [0046] (i) the amino acid sequence of SEQ ID NO:2;
[0047] (ii) amino acids 2-396 of SEQ ID NO:2; [0048] (iii) amino
acids 2-396 of SEQ ID NO:2, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:2; [0049] (iv) the amino acid
sequence of SEQ ID NO:20; [0050] (v) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0051]
(vi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0052] (vii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:2; [0053] (viii) the amino acid sequence
of SEQ ID NO:4; [0054] (ix) amino acids 2-396 of SEQ ID NO:4;
[0055] (x) amino acids 2-396 of SEQ ID NO:4 wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:4; [0056] (xi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0057] (xii) the amino acid sequence of
an endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0058] (xiii) the amino acid sequence of SEQ ID NO:6;
[0059] (xiv) amino acids 2-396 of SEQ ID NO:6; [0060] (xv) amino
acids 2-396 of SEQ ID NO:6, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:6; [0061] (xvi) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0062] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0063] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0064] (b) determining the ability of the compound to
inhibit or stimulate functionality of the GPCR, [0065] wherein the
ability of the compound to inhibit or stimulate functionality of
the GPCR is indicative of the compound being a modulator of MCP-1
expression.
[0066] The invention also features a method of identifying a
candidate compound as a modulator of ABCA1 expression, comprising
the steps of: [0067] (a) contacting the candidate compound with a
GPCR comprising an amino acid sequence selected from the group
consisting of: [0068] (i) the amino acid sequence of SEQ ID NO:2;
[0069] (ii) amino acids 2-396 of SEQ ID NO:2; [0070] (iii) amino
acids 2-396 of SEQ ID NO:2, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:2; [0071] (iv) the amino acid
sequence of SEQ ID NO:20; [0072] (v) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0073]
(vi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0074] (vii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:2; [0075] (viii) the amino acid sequence
of SEQ ID NO:4; [0076] (ix) amino acids 2-396 of SEQ ID NO:4;
[0077] (x) amino acids 2-396 of SEQ ID NO:4 wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:4; [0078] (xi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0079] (xii) the amino acid sequence of
an endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0080] (xiii) the amino acid sequence of SEQ ID NO:6;
[0081] (xiv) amino acids 2-396 of SEQ ID NO:6; [0082] (xv) amino
acids 2-396 of SEQ ID NO:6, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:6; [0083] (xvi) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0084] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0085] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0086] (b) determining the ability of the compound to
inhibit or stimulate functionality of the GPCR, [0087] wherein the
ability of the compound to inhibit or stimulate functionality of
the GPCR is indicative of the compound being a modulator of ABCA1
expression.
[0088] The invention also features a method of identifying a
candidate compound as an agonist for use as a pharmaceutical agent
for atherosclerosis or an atherosclerotic disease, comprising the
steps of: [0089] (a) contacting the candidate compound with a GPCR
comprising an amino acid sequence selected from the group
consisting of: [0090] (i) the amino acid sequence of SEQ ID NO:2;
[0091] (ii) amino acids 2-396 of SEQ ID NO:2; [0092] (iii) amino
acids 2-396 of SEQ ID NO:2, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:2; [0093] (iv) the amino acid
sequence of SEQ ID NO:20; [0094] (v) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0095]
(vi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0096] (vii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:2; [0097] (viii) the amino acid sequence
of SEQ ID NO:4; [0098] (ix) amino acids 2-396 of SEQ ID NO:4;
[0099] (x) amino acids 2-396 of SEQ ID NO:4 wherein the GPCR does
not comprise amino acids-1-396 of SEQ ID NO:4; [0100] (xi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0101] (xii) the amino acid sequence of
an endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0102] (xiii) the amino acid sequence of SEQ ID NO:6;
[0103] (xiv) amino acids 2-396 of SEQ ID NO:6; [0104] (xv) amino
acids 2-396 of SEQ ID NO:6, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:6; [0105] (xvi) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0106] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0107] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0108] (b) determining the ability of the compound to
stimulate functionality of the GPCR, wherein the ability of the
compound to stimulate functionality of the GPCR is indicative of
the compound being an agonist for use as a pharmaceutical agent for
atherosclerosis or an atherosclerotic disease.
[0109] In some embodiments, the agonist for use as a pharmaceutical
agent for atherosclerosis or an atherosclerotic disease is an
agonist for use as a pharmaceutical agent for atherosclerosis. In
some embodiments, the agonist for use as a pharmaceutical agent for
atherosclerosis or an atherosclerotic disease is an agonist for use
as a pharmaceutical agent for an atherosclerotic disease. In some
embodiments, the atherosclerotic disease is selected from the group
consisting of coronary artery disease, myocardial infarction,
peripheral artery disease, and ischemic stroke.
[0110] The invention also features a method of identifying a
compound for inhibiting atherogenesis, comprising the steps of:
[0111] (a) contacting a candidate candidate compound with a GPCR
comprising an amino acid sequence selected from the group
consisting of: [0112] (i) the amino acid sequence of SEQ ID NO:2;
[0113] (ii) amino acids 2-396 of SEQ ID NO:2; [0114] (iii) amino
acids 2-396 of SEQ ID NO:2, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:2; [0115] (iv) the amino acid
sequence of SEQ ID NO:20; [0116] (v) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0117]
(vi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0118] (vii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:2; [0119] (viii) the amino acid sequence
of SEQ ID NO:4; [0120] (ix) amino acids 2-396 of SEQ ID NO:4;
[0121] (x) amino acids 2-396 of SEQ ID NO:4 wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:4; [0122] (xi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0123] (xii) the amino acid sequence of
an endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0124] (xiii) the amino acid sequence of SEQ ID NO:6;
[0125] (xiv) amino acids 2-396 of SEQ ID NO:6; [0126] (xv) amino
acids 2-396 of SEQ ID NO:6, wherein the GPCR does not comprise
amino acids 1-396 of SEQ ID NO:6; [0127] (xvi) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0128] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0129] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0130] (b) determining the ability of the candidate
compound to stimulate functionality of the GPCR, [0131] wherein the
ability of the candidate compound to stimulate functionality of the
GPCR is indicative of the candidate compound being a compound for
inhibiting atherogenesis.
[0132] The invention also features a method of identifying a
compound for the prevention or treatment of atherosclerosis or an
atherosclerotic disease, comprising the steps of: [0133] (a)
contacting a candidate candidate compound with a GPCR comprising an
amino acid sequence selected from the group consisting of: [0134]
(i) the amino, acid sequence of SEQ ID NO:2; [0135] (ii) amino
acids 2-396 of SEQ ID NO:2; [0136] (iii) amino acids 2-396 of SEQ
ID NO:2, wherein the GPCR does not comprise amino acids 1-396 of
SEQ ID NO:2; [0137] (iv) the amino acid sequence of SEQ ID NO:20;
[0138] (v) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using specific primers SEQ ID
NO:7 and SEQ ID NO:8; [0139] (vi) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:1; [0140]
(vii) the amino acid sequence of an endogenous G protein-coupled
receptor having at least about 75% identity, at least about 80%
identity, at least about 85% identity, at least about 90% identity,
or at least about 95% identity to SEQ ID NO:2; [0141] (viii) the
amino acid sequence of SEQ ID NO:4; [0142] (ix) amino acids 2-396
of SEQ ID NO:4; [0143] (x) amino acids 2-396 of SEQ ID NO:4 wherein
the GPCR does not comprise amino acids 1-396 of SEQ ID NO:4; [0144]
(xi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:3; [0145] (xii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:4; [0146] (xiii) the amino acid sequence
of SEQ ID NO:6; [0147] (xiv) amino acids 2-396 of SEQ ID NO:6;
[0148] (xv) amino acids 2-396 of SEQ ID NO:6, wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:6; [0149] (xvi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0150] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0151] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0152] (b) determining the ability of the candidate
compound to stimulate functionality of the GPCR, [0153] wherein the
ability of the candidate compound to stimulate functionality of the
GPCR is indicative of the candidate compound being a compound for
the prevention or treatment of atherosclerosis or an
atherosclerotic disease.
[0154] In some embodiments, the compound for the prevention or
treatment of atherosclerosis or an atherosclerotic disease is a
compound for the prevention or treatment of atherosclerosis. In
some embodiments, the compound for the prevention or treatment of
atherosclerosis or an atherosclerotic disease is a compound for the
prevention or treatment of an atherosclerotic disease.
[0155] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral artery disease, and ischemic stroke.
[0156] The invention also features a method of identifying a
compound for the prevention or treatment of a condition related to
MCP-1 expression, comprising the steps of: [0157] (a) contacting a
candidate candidate compound with a GPCR comprising an amino acid
sequence selected from the group consisting of: [0158] (i) the
amino acid sequence of SEQ ID NO:2; [0159] (ii) amino acids 2-396
of SEQ ID NO: 2; [0160] (iii) amino acids 2-396 of SEQ ID NO:2,
wherein the GPCR does not comprise amino acids 1-396 of SEQ ID
NO:2; [0161] (iv) the amino acid sequence of SEQ ID NO:20; [0162]
(v) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using specific primers SEQ ID
NO:7 and SEQ ID NO:8; [0163] (vi) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:1; [0164]
(vii) the amino acid sequence of an endogenous G protein-coupled
receptor having at least about 75% identity, at least about 80%
identity, at least about 85% identity, at least about 90% identity,
or at least about 95% identity to SEQ ID NO:2; [0165] (viii) the
amino acid sequence of SEQ ID NO:4; [0166] (ix) amino acids 2-396
of SEQ ID NO:4; [0167] (x) amino acids 2-396 of SEQ ID NO:4 wherein
the GPCR does not comprise amino acids 1-396 of SEQ ID NO:4; [0168]
(xi) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:3; [0169] (xii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:4; [0170] (xiii) the amino acid sequence
of SEQ ID NO:6; [0171] (xiv) amino acids 2-396 of SEQ ID NO:6;
[0172] (xv) amino acids 2-396 of SEQ ID NO:6, wherein the GPCR does
not comprise amino acids 1-396 of SEQ ID NO:6; [0173] (xvi) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0174] (xvii) the amino acid
sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:6; [0175] or a variant or biologically
active fragment thereof; wherein the receptor couples to a G
protein; and [0176] (b) determining the ability of the candidate
compound to stimulate functionality of the GPCR, [0177] wherein the
ability of the candidate compound to stimulate functionality of the
GPCR is indicative of the candidate compound being a compound for
the prevention or treatment of a condition related to MCP-1
expression.
[0178] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis.
[0179] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, insulin
resistance, Type 1 diabetes, Type 2 diabetes, metabolic syndrome,
obesity, lower than normal HDL-cholesterol, hypertension,
hyperlipidemia, ischemic heart disease, congestive heart failure,
osteoporosis, restenosis, septic shock, ischemia/reperfusion
injury, disseminated intravascular coagulation, psoriasis, allergic
inflammation, asthma, systemic lupus erythematosus, acute
transplant rejection, chronic hepatitis, interstitial lung disease,
idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome,
interstitial nephritis, hepatic steatosis, chronic obstructive
pulmonary disease, higher than normal osteoclastogenesis, multiple
sclerosis, ischemic stroke, Parkinson's disease, prion-associated
disease, excitotoxic injury, mild cognitive impairment (MCI) and
Alzheimer's disease.
[0180] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder.
[0181] In some embodiments, the G protein is Gi.
[0182] In some embodiments, the G protein is Gq(del)/Gi chimeric G
protein.
[0183] In some embodiments, said contacting is carried out in the
absence of a known ligand of the receptor. In some embodiments,
said contacting is carried out in the absence of a known agonist of
the receptor. In some embodiments, said identifying is directly
identifying.
[0184] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
contacting is carried out in the presence of a known agonist of the
GPCR. In some embodiments, the known agonist of the GPCR is a
compound selected from Table 1. In some embodiments, the known
agonist is Compound 1.
[0185] In some embodiments, the human DNA human genomic DNA.
[0186] In some embodiments, the human DNA is human cDNA derived
from a tissue or cell type that expresses GPR84. In some
embodiments, the human cDNA is derived from a leukocyte. In some
embodiments, the human cDNA is derived from a monocyte/macrophage.
In certain embodiments, the human cDNA is derived from a
granulocyte.
[0187] In some embodiments, the G protein-coupled receptor encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 is an
endogenous GPCR. In some embodiments, the G protein-coupled
receptor encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:5 can decrease a level of intracellular cAMP in response to a
compound selected from Table 1. In some embodiments, the G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1, SEQ
ID NO:3 or SEQ ID NO:5 can increase a level of intracellular
IP.sub.3 accumulation in a cell comprising Gq(del)/Gi chimeric G
protein in response to a compound selected from Table 1. In some
embodiments, the G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 can cause
melanophore cells to undergo pigment aggregation in response to a
compound selected from Table 1. In some embodiments, the G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1, SEQ
ID NO:3 or SEQ ID NO:5 which can decrease a level of intracellular
cAMP, which can increase a level of intracellular IP.sub.3
accumulation in a cell comprising Gq(del)/Gi chimeric G protein, or
which can cause melanophore cells to undergo pigment aggregation in
response to a compound selected from Table 1 exhibits a detectable
level of constitutive activity. In some embodiments, the compound
selected from Table 1 is Compound 1.
[0188] In some embodiments, the ability of the compound to
stimulate functionality of the GPCR is indicative of the compound
being an inhibitor of atherogenesis. In some embodiments, the
ability of the compound to stimulate functionality of the GPCR is
indicative of the compound being a compound that decreases
atherogenesis.
[0189] In some embodiments, the ability of the compound to
stimulate functionality of the GPCR is indicative of the compound
being a compound that decreases MCP-1 expression in
monocytes/macrophages.
[0190] In some embodiments, the ability of the compound to
stimulate functionality of the GPCR is indicative of the compound
being a compound that increases ABCA1 expression in
monocytes/macrophages.
[0191] In some embodiments, the MCP-1 expression comprises MCP-1
expression in monocytes/macrophages.
[0192] In some embodiments, the MCP-1 expression is in a
monocyte/macrophage.
[0193] In some embodiments, the ABCA1 expression is in a
monocyte/macrophage.
[0194] In some embodiments, the GPCR is recombinant.
[0195] In some embodiments, the GPCR is endogenous.
[0196] In some embodiments, the GPCR is a mammalian GPR84.
[0197] In some embodiments, the GPCR is constitutively active. In
some embodiments, the GPCR that is endogenous is constitutively
active. In some embodiments, the GPCR that is a mammalian GPR84 is
constitutively active. In some embodiments, the mammalian GPR84 is
a human GPR84. In some embodiments, the human GPR84 is SEQ ID NO:2
or an allele thereof.
[0198] In some embodiments, the GPCR exhibits a detectable level of
constitutive activity. In some embodiments, the GPCR that is
endogenous exhibits a detectable level of constitutive activity. In
some embodiments, the GPCR that is a mammalian GPR84 exhibits a
detectable level of constitutive activity. In some embodiments, the
mammalian GPR84 is a human GPR84. In some embodiments, the human
GPR84 is SEQ ID NO:2 or an allele thereof.
[0199] In some embodiments, said contacting comprises contacting
with a host cell comprising the GPCR or with a host cell membrane
that comprises the GPCR, wherein said host cell comprises an
expression vector comprising a polynucleotide encoding the GPCR. In
certain embodiments, the host cell is a eukaryotic host cell. In
some embodiments, the eukaryotic host cell is a mammalian host
cell. In some embodiments, the eukaryotic host cell is a
melanophore host cell. In some embodiments, the eukaryotic host
cell is a yeast host cell. In some embodiments, the mammalian host
cell is a non-myeloid host cell. In some embodiments, the mammalian
host cell is not identical to a monocyte or to a cell derived from
a monocyte. In some embodiments, the mammalian host cell is a
myeloid host cell. In some embodiments, the mammalian host cell is
identical a monocyte or to a cell derived from a monocyte.
[0200] In some embodiments, said determining is by a process
comprising the measurement of a level of a second messenger.
[0201] In some embodiments, said determining is by a process
comprising the measurement of a level of a second messenger
selected from the group consisting of cyclic AMP (cAMP), cyclic GMP
(cGMP), inositol 1,4,5-triphosphate (IP.sub.3), diacylglycerol
(DAG), MAP kinase activity, MAPK/ERK kinase kinase-1 (MEKK1)
activity, and Ca.sup.2+. In some embodiments, said second messenger
is cAMP. In some embodiments, the level of intracellular cAMP is
decreased. In some embodiments relating to Gq(del)/Gi chimeric G
protein, said second messenger is IP.sub.3. In some embodiments,
the level of intracellular IP.sub.3 is increased. In some
embodiments relating to Gq(del)/Gi chimeric G protein, said second
messenger is Ca.sup.2+ In some embodiments, the level of
intracellular Ca.sup.2+ is increased. In some embodiments, the
Ca.sup.2+ measurement is carried out by fluorometric imaging plate
reader (FLIPR) assay.
[0202] In some embodiments, said determining is through the use of
a Melanophore assay. In some embodiments, the melanophore cells
undergo pigment aggregation.
[0203] In some embodiments, said determining is through the
measurement of GTP.gamma.S binding to membrane comprising the GPCR.
In some embodiments, GTP.gamma.S binding to membrane comprising the
GPCR is increased.
[0204] In some embodiments, said determining is through CRE
reporter assay. In some embodiments relating to Gq(del)/Gi chimeric
G protein, said determining is through AP1-reporter assay. In some
embodiments relating to Gq(del)/Gi chimeric G protein, said
determining is through SRF-reporter assay.
[0205] In some embodiments, said determining is carried out with
membrane comprising the GPCR.
[0206] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the GPCR. In some
embodiments, the agonist, partial agonist, inverse agonist, or
antagonist of the GPCR is an agonist, partial agonist, inverse
agonist, or antagonist of a mammalian GPR84. In some embodiments,
the mammalian GPR84 is a human GPR84. In some embodiments, the
modulator is an agonist of the GPCR. In some embodiments, the
modulator is a partial agonist of the GPCR.
[0207] In some embodiments, the candidate compound is a small
molecule. In some embodiments, the candidate compound is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the candidate compound is not a polypeptide. In some
embodiments, the candidate compound is not a lipid. In certain
embodiments, the candidate compound is not identical to a compound
in Table 1. In certain embodiments, the candidate compound is not
identical to Compound 1. In certain embodiments, the candidate
compound is non-endogenous. In certain embodiments, the candidate
compound is not endogenous. In some embodiments, the candidate
compound is not material that a prokaryote or eukaryote naturally
produces. In some embodiments, the candidate compound is not
material that a prokaryote naturally produces. In some embodiments,
the candidate compound is not material that a eukaryote naturally
produces.
[0208] In some embodiments, the method further comprises the step
of comparing the modulation of the receptor caused by the candidate
compound to a second modulation of the receptor caused by
contacting the receptor with a known modulator of the receptor.
[0209] In some embodiments, said method further comprises synthesis
of the modulator or agonist or identified compound.
[0210] In some embodiments, said method further comprises:
optionally, determining the structure of the modulator or agonist
or identified compound; and providing the modulator or agonist or
identified compound or the name or structure of the modulator or
agonist or identified compound.
[0211] In some embodiments, said method further comprises:
optionally, determining the structure of the modulator or agonist
or identified compound; optionally, providing the modulator or
agonist or identified compound or the name or structure of the
modulator or agonist or identified compound; and producing or
synthesizing the modulator or agonist or identified compound.
[0212] In some embodiments, said method further comprises
formulating the modulator of atherogenesis, the modulator of MCP-1
expression, the modulator of ABCA1 expression, the agonist for use
as a pharmaceutical agent for atherosclerosis or an atherosclerotic
disease, the compound for the prevention or treatment of
atherosclerosis or an atherosclerotic disease, or the compound for
the prevention or treatment of a condition related to MCP-1
expression into a pharmaceutical. In some embodiments, said method
further comprises formulating the modulator of atherogenesis, the
modulator of MCP-1 expression, the modulator of ABCA1 expression,
the agonist for use as a pharmaceutical agent for atherosclerosis
or an atherosclerotic disease, the compound for the prevention or
treatment of atherosclerosis or an atherosclerotic disease, or the
compound for the prevention or treatment of a condition related to
MCP-1 expression into a pharmaceutical composition.
[0213] In a second aspect, the invention features a modulator
identifiable according to a method of the first aspect.
[0214] In certain embodiments, the modulator is a modulator of
atherogenesis or a modulator of MCP-1 expression or a modulator of
ABCA1 expression.
[0215] In some embodiments, the modulator is identified according
to a method of the first aspect.
[0216] In some embodiments, the modulator is an agonist, a partial
agonist, an inverse agonist, or an antagonist of the GPCR. In some
embodiments, the agonist, partial agonist, inverse agonist, or
antagonist of the GPCR is an agonist, partial agonist, inverse
agonist, or antagonist of a mammalian GPR84. In some embodiments,
the mammalian GPR84 is a human GPR84. In some embodiments, the
modulator of the human GPR84 is a modulator of SEQ ID NO:2. In some
embodiments, the modulator of the human GPR84 is a modulator of SEQ
ID NO:20.
[0217] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0218] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0219] In certain embodiments, the modulator is an agonist of the
GPCR. In some embodiments, the agonist of the GPCR is an agonist of
a mammalian GPR84. In some embodiments, the mammalian GPR84 is a
human GPR84. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:2. In some embodiments, the agonist of the
human GPR84 is an agonist of SEQ ID NO:20.
[0220] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0221] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0222] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0223] In some embodiments, the agonist is a partial agonist.
[0224] In some embodiments, the modulator is orally active.
[0225] In a third aspect, the invention features a pharmaceutical
composition comprising a modulator of a mammalian GPR84 and a
pharmaceutically acceptable carrier. The invention also relates to
a composition comprising a modulator of a mammalian GPR84.
[0226] The invention also relates to a pharmaceutical composition
comprising a modulator of atherogenesis or a modulator of MCP-1
expression or a modulator of ABCA1 expression and a
pharmaceutically acceptable carrier or to a composition comprising
the modulator of atherogenesis or the modulator of MCP-1 expression
or the modulator of ABCA1 expression, wherein said modulator of
atherogenesis or said modulator of MCP-1 expression or said
modulator of ABCA1 expression is an agonist, partial agonist,
inverse agonist or antagonist of a mammalian GPR84.
[0227] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0228] In some embodiments, the modulator is according to the
second aspect.
[0229] In some embodiments, the modulator of the mammalian GPR84 is
an agonist, partial agonist, inverse agonist, or antagonist of the
mammalian GPR84.
[0230] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0231] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0232] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0233] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0234] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0235] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0236] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0237] In some embodiments, the agonist is a partial agonist.
[0238] In some embodiments, the modulator is orally active.
[0239] In a fourth aspect, the invention features a method of
preparing a pharmaceutical composition comprising admixing a
modulator of a mammalian GPR84 and a pharmaceutically acceptable
carrier.
[0240] The invention also relates to a method of preparing a
pharmaceutical composition comprising admixing a modulator of
atherogenesis or a modulator of MCP-1 expression or a modulator of
ABCA1 expression and a pharmaceutically acceptable carrier, wherein
said modulator of atherogenesis or said modulator of MCP-1
expression or said modulator of ABCA1 expression is an agonist,
partial agonist, inverse agonist or antagonist of a mammalian
GPR84.
[0241] In some embodiments, the modulator is according to the
second aspect.
[0242] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0243] In some embodiments, the modulator of the mammalian GPR84 is
an agonist, partial agonist, inverse agonist, or antagonist of the
mammalian GPR84.
[0244] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0245] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0246] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0247] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0248] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0249] In some embodiments, the method comprises admixing the
modulator of a mammalian GPR84 or the modulator of atherogenesis or
the modulator of MCP-1 expression or the modulator of ABCA1
expression and the pharmaceutically acceptable carrier and a
compound selected from the group consisting of an HMG-CoA reductase
inhibitor (i.e. a statin), an agonist or partial agonist of the
nicotinic acid receptor GPR109A (e.g. niacin), adiponectin or an
orally active analog thereof (including orally active agonists or
partial agonists of adiponectin receptor AdipoR1 or AdipoR2),
methotrexate, a phosphodiesterase (PDE) inhibitor (inclusive of an
inhibitor selective for type 4 cAMP-specific PDE (PDE4), e.g.,
roflumilast, or an inhibitor selective for PDE4B, or an inhibitor
selective for PDE4B2), a biologic agent for neutralizing tumor
necrosis factor alpha (TNF.alpha.) activity (such as etanercept and
infliximab), and CTLA4-Ig.
[0250] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0251] In some embodiments, the agonist is a partial agonist.
[0252] In some embodiments, the modulator is orally active.
[0253] In a fifth aspect, the invention features a method of
inhibiting atherogenesis comprising administering to a mammal in
need thereof a therapeutically effective amount of a modulator of
the mammalian GPR84 or a pharmaceutical composition comprising the
modulator and a pharmaceutically acceptable carrier or a
composition comprising the modulator.
[0254] In some embodiments, the modulator is according to the
second aspect. In some embodiments, the modulator of the mammalian
GPR84 is a modulator of a human GPR84. In some embodiments, the
modulator of the human GPR84 is a modulator of SEQ ID NO:2. In some
embodiments, the modulator of the human GPR84 is a modulator of SEQ
ID NO:20.
[0255] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0256] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0257] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0258] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0259] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not. Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0260] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0261] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0262] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0263] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0264] In some embodiments, the mammal is a human.
[0265] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0266] In some embodiments, the agonist is a partial agonist.
[0267] In some embodiments, the modulator or the agonist is orally
active.
[0268] In some embodiments, said administering is oral.
[0269] In a sixth aspect, the invention features use of a modulator
of a mammalian GPR84 or of a pharmaceutical composition comprising
the modulator and a pharmaceutically acceptable carrier or of a
composition comprising the modulator to inhibit atherogenesis in
the human or animal body by therapy.
[0270] In some embodiments, the modulator is according to the
second aspect.
In some embodiments, the modulator of the mammalian GPR84 is a
modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0271] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0272] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some, embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0273] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0274] In certain, embodiments, the modulator of the mammalian
GPR84 is an agonist of the mammalian GPR84. In some embodiments,
the agonist of the mammalian GPR84 is an agonist of a human GPR84.
In some embodiments, the agonist of the human GPR84 is an agonist
of SEQ ID NO:2. In some embodiments, the agonist of the human GPR84
is an agonist of SEQ ID NO:20.
[0275] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0276] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0277] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0278] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0279] In some embodiments, the pharmaceutical composition or the
composition further comprise a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0280] In some embodiments, the animal is a non-human mammal.
[0281] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In, some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0282] In some embodiments, the agonist is a partial agonist.
[0283] In some embodiments, the modulator or the agonist is orally
active.
[0284] In a seventh aspect, the invention features a method of
preventing or treating atherosclerosis or an atherosclerotic
disease comprising administering to a mammal in need thereof a
therapeutically effective amount of a modulator of the mammalian
GPR84 or of a pharmaceutical composition comprising the modulator
and a pharmaceutically acceptable carrier or of a composition
comprising the modulator.
[0285] In certain embodiments, the method of preventing or treating
atherosclerosis or an atherosclerotic disease is a method of
preventing or treating atherosclerosis. In certain embodiments, the
method of preventing or treating atherosclerosis or an
atherosclerotic disease is a method of preventing or treating an
atherosclerotic disease.
[0286] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral arterial disease, and ischemic stroke.
[0287] In some embodiments, the modulator is according to the
second aspect.
[0288] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0289] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0290] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0291] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0292] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0293] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0294] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0295] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0296] In some embodiments; the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0297] In some embodiments, the pharmaceutical composition further
comprises a compound selected from the group consisting of an
HMG-CoA reductase inhibitor (i.e. a statin), an agonist or partial
agonist of the nicotinic acid receptor GPR109A (e.g. niacin),
adiponectin or an orally active analog thereof (including orally
active agonists or partial agonists of adiponectin receptor AdipoR1
or AdipoR2), methotrexate, a phosphodiesterase (PDE) inhibitor
(inclusive of an inhibitor selective for type 4 cAMP-specific PDE
(PDE4), e.g. roflumilast, or an inhibitor selective for PDE4B, or
an inhibitor selective for PDE4B2), a biologic agent for
neutralizing tumor necrosis factor alpha (TNF.alpha.) activity
(such as etanercept and infliximab), and CTLA4-Ig.
[0298] In some embodiments, the mammal is a human.
[0299] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0300] In some embodiments, the agonist is a partial agonist.
[0301] In some embodiments, the modulator or the agonist is orally
active.
[0302] In some embodiments, said administering is oral.
[0303] In an eighth aspect, the invention features a use of a
modulator of a mammalian GPR84 or of a pharmaceutical composition
comprising the modulator and a pharmaceutically acceptable carrier
or of a composition comprising the modulator to prevent or treat
atherosclerosis or an atherosclerotic disease in the human or
animal body by therapy.
[0304] In certain embodiments, to prevent or treat atherosclerosis
or an atherosclerotic disease is to prevent or treat
atherosclerosis. In certain embodiments, to prevent or treat
atherosclerosis or an atherosclerotic disease is to prevent or
treat an atherosclerotic disease.
[0305] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral arterial disease, and ischemic stroke.
[0306] In some embodiments, the modulator is according to the
second aspect.
In some embodiments, the modulator of the mammalian GPR84 is a
modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0307] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0308] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0309] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0310] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0311] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0312] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0313] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0314] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0315] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0316] In some embodiments, the animal is a non-human mammal.
[0317] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human. GPR84. In some
embodiments, the modulator is an agonist with an EC.sub.50 of less
than a value selected from the interval of about 10 nM to 10 .mu.M.
In some embodiments, modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 1
.mu.M. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than a value selected from the interval of about
10 nM to 100 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0318] In some embodiments, the agonist is a partial agonist.
[0319] In some embodiments, the modulator or agonist is orally
active.
[0320] In a ninth aspect, the invention features a method of
preventing or treating a condition related to MCP-1 expression
comprising administering to a mammal in need thereof a
therapeutically effective amount of a modulator of the mammalian
GPR84 or of a pharmaceutical composition comprising the modulator
and a pharmaceutically acceptable carrier or of a composition
comprising the modulator.
[0321] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis.
[0322] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, insulin
resistance, Type 1 diabetes, Type 2 diabetes, metabolic syndrome,
obesity, lower than normal HDL-cholesterol, hypertension,
hyperlipidemia, ischemic heart disease, congestive heart failure,
osteoporosis, restenosis, septic shock, ischemia/reperfusion
injury, disseminated intravascular coagulation, psoriasis, allergic
inflammation, asthma, systemic lupus erythematosus, acute
transplant rejection, chronic hepatitis, interstitial lung disease,
idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome,
interstitial nephritis, hepatic steatosis, chronic obstructive
pulmonary disease, higher than normal osteoclastogenesis, multiple
sclerosis, ischemic stroke, Parkinson's disease, prion-associated
disease, excitotoxic injury, mild cognitive impairment (MCI) and
Alzheimer's disease.
[0323] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder.
[0324] In some embodiments, the MCP-1 expression comprises MCP-1
expression in a monocyte/macrophage.
[0325] In some embodiments, the MCP-1 expression is MCP-1
expression in a monocyte/macrophage.
[0326] In some embodiments, the modulator is according to the
second aspect.
[0327] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0328] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0329] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0330] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0331] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0332] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0333] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0334] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0335] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0336] In some embodiments, the mammal is a human.
[0337] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0338] In some embodiments, the agonist is a partial agonist.
[0339] In some embodiments, the modulator or the agonist is orally
active.
[0340] In some embodiments, said administering is oral.
[0341] In a tenth aspect, the invention features use of a modulator
of a mammalian GPR84 or of a pharmaceutical composition comprising
the modulator and a pharmaceutically acceptable carrier or of a
composition comprising the modulator to prevent or treat a
condition related to MCP-1 expression in a human or animal body by
therapy.
[0342] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis.
[0343] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, insulin
resistance, Type 1 diabetes, Type 2 diabetes, metabolic syndrome,
obesity, lower than normal HDL-cholesterol, hypertension,
hyperlipidemia, ischemic heart disease, congestive heart failure,
osteoporosis, restenosis, septic shock, ischemia/reperfusion
injury, disseminated intravascular coagulation, psoriasis, allergic
inflammation, asthma, systemic lupus erythematosus, acute
transplant rejection, chronic hepatitis, interstitial lung disease,
idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome,
interstitial nephritis, hepatic steatosis, chronic obstructive
pulmonary disease, higher than normal osteoclastogenesis, multiple
sclerosis, ischemic stroke, Parkinson's disease, prion-associated
disease, excitotoxic injury, mild cognitive impairment (MCI) and
Alzheimer's disease.
[0344] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder.
[0345] In some embodiments, the MCP-1 expression comprises MCP-1
expression in a monocyte/macrophage.
[0346] In some embodiments, the MCP-1 expression is MCP-1
expression in a monocyte/macrophage.
[0347] In some embodiments, the modulator is according to the
second aspect.
[0348] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0349] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0350] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0351] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0352] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0353] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0354] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0355] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0356] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0357] In some embodiments, the animal is a non-human mammal.
[0358] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM at human,
mouse or rat GPR84, preferably at human GPR84. In some embodiments,
the modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 10 .mu.M. In some
embodiments, modulator is an agonist with an EC.sub.50 of less than
a value selected from the interval of about 10 nM to 1 .mu.M. In
some embodiments, the modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 100
nM. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0359] In some embodiments, the agonist is a partial agonist.
[0360] In some embodiments, the modulator or the agonist is orally
active.
[0361] In an eleventh aspect, the invention features a use of a
modulator of a mammalian GPR84 or of a pharmaceutical composition
comprising the modulator and a pharmaceutically acceptable carrier
or of a composition comprising the modulator in the manufacture of
a medicament for inhibiting atherogenesis in the mammal.
[0362] In some embodiments, the modulator is according to the
second aspect.
[0363] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0364] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0365] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound I or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0366] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0367] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0368] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0369] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0370] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0371] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0372] In some embodiments, the mammal is a human.
[0373] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0374] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM. In some
embodiments, the modulator is an agonist with an EC.sub.50 of less
than a value selected from the interval of about 10 nM to 10 .mu.M.
In some embodiments, modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 1
.mu.M. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than a value selected from the interval of about
10 nM to 100 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0375] In some embodiments, the agonist is a partial agonist.
[0376] In some embodiments, the modulator or the agonist is orally
active.
[0377] In a twelfth aspect, the invention features use of a
modulator of a mammalian GPR84 or of a pharmaceutical composition
comprising the modulator and a pharmaceutically acceptable carrier
or of a composition comprising the modulator in the manufacture of
a medicament for preventing or treating atherosclerosis or an
atherosclerotic disease in the mammal.
[0378] In certain embodiments, the medicament for preventing or
treating atherosclerosis or an atherosclerotic disease is a
medicament for preventing or treating atherosclerosis. In certain
embodiments, the medicament for preventing or treating
atherosclerosis or an atherosclerotic disease is a medicament for
preventing or treating an atherosclerotic disease.
[0379] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral arterial disease, and ischemic stroke.
[0380] In some embodiments, the modulator is according to the
second aspect.
[0381] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0382] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0383] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0384] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0385] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0386] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0387] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0388] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0389] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist increases ABCA1 expression. In some
embodiments, the agonist increases ABCA1 expression in a
monocyte/macrophage.
[0390] In some embodiments, the mammal is a human.
[0391] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0392] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM. In some
embodiments, the modulator is an agonist with an EC.sub.50 of less
than a value selected from the interval of about 10 nM to 10 .mu.M.
In some embodiments, modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 1
.mu.M. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than a value selected from the interval of about
10 nM to 100 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M., of less than about
1 .mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0393] In some embodiments, the agonist is a partial agonist.
[0394] In some embodiments, the modulator is orally active.
[0395] In a thirteenth aspect, the invention features use of a
modulator of a mammalian GPR84 or of a pharmaceutical composition
comprising the modulator and a pharmaceutically acceptable carrier
or of a composition comprising the modulator in the manufacture of
a medicament for preventing or treating a condition related to
MCP-1 expression in the mammal.
[0396] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis.
[0397] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, insulin
resistance, Type 1 diabetes, Type 2 diabetes, metabolic syndrome,
obesity, lower than normal HDL-cholesterol, hypertension,
hyperlipidemia, ischemic heart disease, congestive heart failure,
osteoporosis, restenosis, septic shock, ischemia/reperfusion
injury, disseminated intravascular coagulation; psoriasis, allergic
inflammation, asthma, systemic lupus erythematosus, acute
transplant rejection, chronic hepatitis, interstitial lung disease,
idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome,
interstitial nephritis, hepatic steatosis, chronic obstructive
pulmonary disease, higher than normal osteoclastogenesis, multiple
sclerosis, ischemic stroke, Parkinson's disease, prion-associated
disease, excitotoxic injury, mild cognitive impairment (MCI) and
Alzheimer's disease.
[0398] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder.
[0399] In some embodiments, the MCP-1 expression comprises MCP-1
expression in a monocyte/macrophage.
[0400] In some embodiments, the MCP-1 expression is in a
monocyte/macrophage.
[0401] In some embodiments, the modulator is according to the
second aspect.
[0402] In some embodiments, the modulator of the mammalian GPR84 is
a modulator of a human GPR84. In some embodiments, the modulator of
the human GPR84 is a modulator of SEQ ID NO:2. In some embodiments,
the modulator of the human GPR84 is a modulator of SEQ ID
NO:20.
[0403] In some embodiments, the modulator is an agonist, partial
agonist, inverse agonist, or antagonist of the mammalian GPR84.
[0404] In some embodiments, the modulator is a small molecule. In
some embodiments, the modulator is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the
modulator is not a polypeptide. In some embodiments, the modulator
is not a lipid. In certain embodiments, the modulator is not
identical to a compound in Table 1. In some embodiments, the
modulator is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the modulator is not Compound 1. In some
embodiments, the modulator is not Compound 2. In some embodiments,
the modulator is not Compound 3. In some embodiments, the modulator
is not Compound 1 or Compound 2. In some embodiments, the modulator
is not Compound 1 or Compound 3. In some embodiments, the modulator
is not Compound 2 or Compound 3. In certain embodiments, the
modulator is non-endogenous. In certain embodiments, the modulator
is not endogenous. In some embodiments, the modulator is not
material that a prokaryote or eukaryote naturally produces. In some
embodiments, the modulator is not material that a prokaryote
naturally produces. In some embodiments, the modulator is not
material that a eukaryote naturally produces.
[0405] In some embodiments, the modulator is a compound selected
from Table 1. In some embodiments, the modulator is Compound 1.
[0406] In certain embodiments, the modulator of the mammalian GPR84
is an agonist of the mammalian GPR84. In some embodiments, the
agonist of the mammalian GPR84 is an agonist of a human GPR84. In
some embodiments, the agonist of the human GPR84 is an agonist of
SEQ ID NO:2. In some embodiments, the agonist of the human GPR84 is
an agonist of SEQ ID NO:20.
[0407] In some embodiments, the agonist is a small molecule. In
some embodiments, the agonist is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the agonist
is not a polypeptide. In some embodiments, the agonist is not a
lipid. In certain embodiments, the agonist is not identical to a
compound in Table 1. In some embodiments, the agonist is not
Compound 1, not Compound 2, and not Compound 3. In some
embodiments, the agonist is not Compound 1. In some embodiments,
the agonist is not Compound 2. In some embodiments, the agonist is
not Compound 3. In some embodiments, the agonist is not Compound 1
or Compound 2. In some embodiments, the agonist is not Compound 1
or Compound 3. In some embodiments, the agonist is not Compound 2
or Compound 3. In certain embodiments, the agonist is
non-endogenous. In certain embodiments, the agonist is not
endogenous. In some embodiments, the agonist is not material that a
prokaryote or eukaryote naturally produces. In some embodiments,
the agonist is not material that a prokaryote naturally produces.
In some embodiments, the agonist is not material that a eukaryote
naturally produces.
[0408] In some embodiments, the agonist is a compound selected from
Table 1. In some embodiments, the agonist is Compound 1.
[0409] In some embodiments, the modulator is an agonist. In some
embodiments, the agonist decreases MCP-1 expression. In some
embodiments, the agonist decreases MCP-1 expression in a
monocyte/macrophage.
[0410] In some embodiments, the mammal is a human.
[0411] In some embodiments, the pharmaceutical composition or the
composition further comprises a compound selected from the group
consisting of an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab),
and CTLA4-Ig.
[0412] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, or of less than about 10 nM. In some
embodiments, the modulator is an agonist with an EC.sub.50 of less
than a value selected from the interval of about 10 nM to 10 .mu.M.
In some embodiments, modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 10 nM to 1
.mu.M. In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than a value selected from the interval of about
10 nM to 100 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
GTP.gamma.S binding assay carried out with membrane from
transfected CHO cells, or in pigment aggregation assay carried out
in transfected melanophores, or in cAMP assay carried out in
transfected 293 cells, or in IP.sub.3 assay carried out in
transfected 293 cells comprising Gq(del)/Gi chimeric G protein,
wherein the transfected CHO cells or the transfected melanophore
cells or the transfected 293 cells express a recombinant GPR84
having an amino acid sequence selected from SEQ ID NO:20, SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the
recombinant GPR84 has the amino acid sequence of SEQ ID NO:20. In
some embodiments, the recombinant GPR84 has the amino acid sequence
of SEQ ID NO:2. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than about 10 .mu.M, of less than about 1
.mu.M, of less than about 100 nM, or of less than about 10 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM.
[0413] In some embodiments, the agonist is a partial agonist.
[0414] In some embodiments, the modulator or the agonist is orally
active.
[0415] In a fourteenth aspect, the invention features use of a GPCR
to screen candidate compounds as pharmaceutical agents for
inhibiting atherogenesis, as pharmaceutical agents for
atherosclerosis or an atherosclerotic disease, or as pharmaceutical
agents for a condition related to MCP-1 expression, wherein the
GPCR comprises an amino acid sequence selected from the group
consisting of: [0416] (a) the amino acid sequence of SEQ ID NO:2;
[0417] (b) amino acids-2-396 of SEQ ID NO:2; [0418] (c) amino acids
2-396 of SEQ ID NO:2, wherein the GPCR does not comprise amino
acids 1-396 of SEQ ID NO:2; [0419] (d) the amino acid sequence of
SEQ ID NO:20; [0420] (e) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0421]
(f) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1; [0422] (g) the amino acid sequence of an
endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:2; [0423] (h) the amino acid sequence of SEQ ID NO:4; [0424]
(i) amino acids 2-396 of SEQ ID NO:4; [0425] (j) amino acids 2-396
of SEQ ID NO:4 wherein the GPCR does not comprise amino acids 1-396
of SEQ ID NO:4; [0426] (k) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:3; [0427]
(l) the amino acid sequence of an endogenous G protein-coupled
receptor having at least about 75% identity, at least about 80%
identity, at least about 85% identity, at least about 90% identity,
or at least about 95% identity to SEQ ID NO:4; [0428] (m) the amino
acid sequence of SEQ ID NO:6; [0429] (n) amino acids 2-396 of SEQ
ID NO:6; [0430] (o) amino acids 2-396 of SEQ ID NO:6, wherein the
GPCR does not comprise amino acids 1-396 of SEQ ID NO:6; [0431] (p)
the amino acid sequence of a G protein-coupled receptor encoded by
a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0432] (q) the amino acid sequence
of an endogenous G protein-coupled receptor having at least about
75% identity, at least about 80% identity, at least about 85%
identity, at least about 90% identity, or at least about 95%
identity to SEQ ID NO:6; [0433] or a variant or biologically active
fragment thereof.
[0434] In certain embodiments, pharmaceutical agents for
atherosclerosis or an atherosclerotic disease are pharmaceutical
agents for atherosclerosis. In certain embodiments, pharmaceutical
agents for atherosclerosis or an atherosclerotic disease are
pharmaceutical agents for an atherosclerotic disease.
[0435] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral arterial disease, and ischemic stroke.
[0436] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis.
[0437] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, insulin
resistance, Type 1 diabetes, Type 2 diabetes, metabolic syndrome,
obesity, lower than normal HDL-cholesterol, hypertension,
hyperlipidemia, ischemic heart disease, congestive heart failure,
osteoporosis, restenosis, septic shock, ischemia/reperfusion
injury, disseminated intravascular coagulation, psoriasis, allergic
inflammation, asthma, systemic lupus erythematosus, acute
transplant rejection, chronic hepatitis, interstitial lung disease,
idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome,
interstitial nephritis, hepatic steatosis, chronic obstructive
pulmonary disease, higher than normal osteoclastogenesis, multiple
sclerosis, ischemic stroke, Parkinson's disease, prion-associated
disease, excitotoxic injury, mild cognitive impairment (MCI) and
Alzheimer's disease.
[0438] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder.
[0439] In a fifteenth aspect, the invention features an isolated
polynucleotide encoding a G protein-coupled receptor, wherein the
polynucleotide comprises a nucleic acid sequence selected from the
group consisting of: [0440] (a) a nucleic acid sequence encoding
the amino acid sequence of SEQ ID NO:6; [0441] (b) the nucleic acid
sequence of SEQ ID NO:5; [0442] (c) nucleotides 4-1191 of SEQ ID
NO:5; [0443] (d) nucleotides 1-1188 of SEQ ID NO:5; [0444] (e)
nucleotides 4-1188 of SEQ ID NO:5; [0445] (f) a nucleic acid
sequence hybridizing under stringent conditions to the complement
of SEQ ID NO:5; [0446] (g) a nucleic acid sequence encoding an
endogenous G protein-coupled receptor having at least about 95%
identity to SEQ ID NO:6; and [0447] (h) a nucleic acid encoding a
variant or biologically active fragment of SEQ ID NO:6; or the
complement thereof.
[0448] In some embodiments, the nucleic acid sequence encodes an
endogenous rat GPCR. In some embodiments, the nucleic acid encodes
an endogenous rat GPR84. In some embodiments, the nucleic acid
encodes a non-endogenous version of an endogenous rat GPCR.
[0449] In a sixteenth aspect, the invention features a recombinant
vector comprising an isolated polynucleotide according to the
fifteenth aspect.
[0450] In some embodiments, the vector is an expression vector. In
some embodiments, the expression vector is a eukaryotic expression
vector. In some embodiments, the vector is an expression vector
wherein the isolated polynucleotide is operably linked to a
promoter.
[0451] In a seventeenth aspect, the invention features a host cell
comprising a recombinant vector according to the sixteenth
aspect.
[0452] In some embodiments, the host cell comprises an expression
vector according to the twelfth aspect.
[0453] In some embodiments, the host cell is a eukaryotic cell. In
some embodiments, the host cell is a melanophore cell. In some
embodiments, the host cell is a mammalian cell. In some
embodiments, the mammalian cell is a 293 cell, a 293T cell, a CHO
cell or a COS-7 cell. In some embodiments, the host cell is a yeast
cell.
[0454] In an eighteenth aspect, the invention features an isolated
or recombinant GPCR polypeptide comprising an amino acid sequence
selected from the group consisting of: [0455] (a) the amino acid
sequence of SEQ ID NO:6; [0456] (b) amino acids 2-396 of SEQ ID
NO:6; [0457] (c) amino acids 2-396 of SEQ ID NO:6, wherein the GPCR
does not comprise amino acids 1-396 of SEQ ID NO:6; [0458] (d) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0459] (e) the amino acid sequence
of an endogenous GPCR having at least 95% identity to SEQ ID NO:6;
[0460] or a variant or biologically active fragment thereof.
[0461] In some embodiments, the GPCR is an endogenous rat GPCR. In
some embodiments, the GPCR is an endogenous rat GPR84. In some
embodiments, the GPCR is non-endogenous. In some embodiments, the
GPCR is a non-endogenous version of an endogenous rat GPCR.
[0462] In a nineteenth aspect, the invention features a method of
making a recombinant host cell comprising the steps of: [0463] (a)
transfecting an expression vector according to the sixteenth aspect
into a suitable host cell; and [0464] (b) culturing the host cell
under conditions which allow expression of the G protein-coupled
receptor encoded by the expression vector.
[0465] In some embodiments, the host cell is a eukaryotic cell. In
some embodiments, the host cell is a melanophore cell. In some
embodiments, the host cell is a mammalian cell. In some
embodiments, the mammalian cell is a 293 cell, a 293T cell, a CHO
cell or a COS-7 cell. In some embodiments, the host cell is a yeast
cell.
[0466] In a twentieth aspect, the invention features a method of
identifying a candidate compound as a ligand of a GPCR, wherein the
GPCR comprises an amino acid sequence selected from the group
consisting of: [0467] (a) the amino acid sequence of SEQ ID NO:2;
[0468] (b) amino acids 2-396 of SEQ ID NO:2; [0469] (h) amino acids
2-396 of SEQ ID NO:2, wherein the GPCR does not comprise amino
acids 1-396 of SEQ ID NO:2; [0470] (i) the amino acid sequence of
SEQ ID NO:20; [0471] (j) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide that is
amplifiable by polymerase chain reaction (PCR) on a human DNA
sample using specific primers SEQ ID NO:7 and SEQ ID NO:8; [0472]
(k) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1; [0473] (l) the amino acid sequence of an
endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:2; [0474] (h) the amino acid sequence of SEQ ID NO:4; [0475]
(i) amino acids 2-396 of SEQ ID NO:4; [0476] (j) amino acids 2-396
of SEQ ID NO:4 wherein the GPCR does not comprise amino acids 1-396
of SEQ ID NO:4; [0477] (k) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:3; [0478]
(l) the amino acid sequence of an endogenous G protein-coupled
receptor having at least about 75% identity, at least about 80%
identity, at least about 85% identity, at least about 90% identity,
or at least about 95% identity to SEQ ID NO:4; [0479] (m) the amino
acid sequence of SEQ ID NO:6; [0480] (n) amino acids 2-396 of SEQ
ID NO:6; [0481] (o) amino acids 2-396 of SEQ ID NO:6, wherein the
GPCR does not comprise amino acids 1-396 of SEQ ID NO:6; [0482] (p)
the amino acid sequence of a G protein-coupled receptor encoded by
a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0483] (q) the amino acid sequence
of an endogenous G protein-coupled receptor having at least about
75% identity, at least about 80% identity, at least about 85%
identity, at least about 90% identity, or at least about 95%
identity to SEQ ID NO:6; [0484] or a variant or biologically active
fragment thereof; comprising the steps of: [0485] (a') contacting
said GPCR with an optionally labeled known ligand to the GPCR in
the presence or absence of the candidate compound; [0486] (b')
detecting the complex between the known ligand and said GPCR; and
[0487] (c') determining whether less of said complex is formed in
the presence of the candidate compound than in the absence of the
candidate compound, wherein said determination is indicative of the
candidate compound being a ligand of said receptor.
[0488] In some embodiments, said contacting comprises contacting
with a host cell comprising the GPCR or with a host cell membrane
that comprises the GPCR, wherein said host cell comprises an
expression vector comprising a polynucleotide encoding the GPCR. In
certain embodiments, the host cell is a eukaryotic host cell. In
some embodiments, the eukaryotic host cell is a mammalian host
cell. In some embodiments, the eukaryotic host cell is a
melanophore host cell. In some embodiments, the eukaryotic host
cell is a yeast host cell. In some embodiments, the mammalian host
cell is a non-myeloid host cell. In some embodiments, the mammalian
host cell is not identical to a monocyte or to a cell derived from
a monocyte. In some embodiments, the mammalian host cell is a
myeloid host cell. In some embodiments, the mammalian host cell is
identical a monocyte or to a cell derived from a monocyte.
[0489] In some embodiments, the known ligand to the GPCR is a known
ligand to a mammalian GPR84. In some embodiments, the known ligand
to the GPCR is a known ligand to a human GPR84.
[0490] In some embodiments, the known ligand to the GPCR is a known
ligand to SEQ ID NO:2. In some embodiments, the known ligand to the
GPCR is a known ligand to SEQ ID NO:20.
[0491] In some embodiments, the known ligand to the GPCR is a
compound selected from Table 1. In some embodiments, the known
ligand to the GPCR is Compound 1.
[0492] In some embodiments, the known ligand to the GPCR is a small
molecule. In some embodiments, the known ligand to the GPCR is not
an antibody or an antigen-binding fragment thereof. In some
embodiments, the known ligand to the GPCR is not a polypeptide. In
some embodiments, the known ligand to the GPCR is not a lipid. In
certain embodiments, the known ligand to the GPCR is not identical
to a compound in Table 1. In some embodiments, the known ligand to
the GPCR is not Compound 1, not Compound 2, and not Compound 3. In
some embodiments, the known ligand to the GPCR is not Compound 1.
In some embodiments, the known ligand to the GPCR is not Compound
2. In some embodiments, the known ligand to the GPCR is not
Compound 3. In some embodiments, the known ligand to the GPCR is
not Compound 1 or Compound 2. In some embodiments, the known ligand
to the GPCR is not Compound 1 or Compound 3. In some embodiments,
the known ligand to the GPCR is not Compound 2 or Compound 3. In
certain embodiments, the known ligand to the GPCR is
non-endogenous. In certain embodiments, the known ligand to the
GPCR is not endogenous. In some embodiments, the known ligand to
the GPCR is not material that a prokaryote or eukaryote naturally
produces. In some embodiments, the known ligand to the GPCR is not
material that a prokaryote naturally produces. In some embodiments,
the known ligand to the GPCR is not material that a eukaryote
naturally produces.
[0493] The invention also features a method of identifying a ligand
of a GPCR, wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: [0494] (a) the amino acid
sequence of SEQ ID NO:2; [0495] (b) amino acids 2-396 of SEQ ID
NO:2; [0496] (c) amino acids 2-396 of SEQ ID NO:2, wherein the GPCR
does not comprise amino acids 1-396 of SEQ ID NO:2; [0497] (d) the
amino acid sequence of SEQ ID NO:20; [0498] (e) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction
(PCR) on a human DNA sample using specific primers SEQ ID NO:7 and
SEQ ID NO:8; [0499] (f) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1;
[0500] (g) the amino acid sequence of an endogenous G
protein-coupled receptor having at least about 75% identity, at
least about 80% identity, at least about 85% identity, at least
about 90% identity, or at least about 95% identity to SEQ ID NO:2;
[0501] (h) the amino acid sequence of SEQ ID NO:4; [0502] (i) amino
acids 2-396 of SEQ ID NO:4; [0503] (j) amino acids 2-396 of SEQ ID
NO:4 wherein the GPCR does not comprise amino acids 1-396 of SEQ ID
NO:4; [0504] (k) the amino acid sequence of a G protein-coupled
receptor encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO:3; [0505] (l) the amino
acid sequence of an endogenous G protein-coupled receptor having at
least about 75% identity, at least about 80% identity, at least
about 85% identity, at least about 90% identity, or at least about
95% identity to SEQ ID NO:4; [0506] (m) the amino acid sequence of
SEQ ID NO:6; [0507] (n) amino acids 2-396 of SEQ ID NO:6; [0508]
(o) amino acids 2-396 of SEQ ID NO:6, wherein the GPCR does not
comprise amino acids 1-396 of SEQ ID NO:6; [0509] (p) the amino
acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:5; and [0510] (q) the amino acid sequence
of an endogenous G protein-coupled receptor having at least about
75% identity, at least about 80% identity, at least about 85%
identity, at least about 90% identity, or at least about 95%
identity to SEQ ID NO:6; [0511] or a variant or biologically active
fragment thereof; comprising the steps of: [0512] (a') contacting a
test ligand with a host cell comprising said GPCR or with a host
cell membrane that comprises said GPCR, under conditions which
permit interaction between said receptor and said test ligand; and
[0513] (b') detecting a ligand bound to said GPCR.
[0514] In some embodiments, the G protein-coupled receptor encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 is an
endogenous GPCR. In some embodiments, the G protein-coupled
receptor encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:5 can decrease a level of intracellular cAMP in response to a
compound selected from Table 1. In some embodiments, the G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1, SEQ
ID NO:3 or SEQ ID NO:5 can increase a level of intracellular
IP.sub.3 accumulation in a cell comprising Gq(del)/Gi chimeric G
protein in response to a compound selected from Table 1. In some
embodiments, the G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 can cause
melanophore cells to undergo pigment aggregation in response to a
compound selected from Table 1. In some embodiments, the G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1, SEQ
ID NO:3 or SEQ ID NO:5 which can decrease a level of intracellular
cAMP, which can increase a level of intracellular IP.sub.3
accumulation in a cell comprising Gq(del)/Gi chimeric G protein, or
which can cause melanophore cells to undergo pigment aggregation in
response to a compound selected from Table 1 exhibits a detectable
level of constitutive activity. In some embodiments, the compound
selected from Table 1 is Compound 1.
[0515] In a twenty-first aspect, the invention features a process
for screening candidate compounds as compounds selected from the
group consisting of: [0516] (a) modulators of atherogenesis; [0517]
(b) modulators of MCP-1 expression; [0518] (c) modulators of ABCA1
expression; [0519] (d) compounds for inhibiting atherogenesis;
[0520] (e) compounds for the prevention or treatment of
atherosclerosis or an atherosclerotic disease; and [0521] (f)
compounds for the prevention or treatment of a condition related to
MCP-1 expression; wherein said process comprises performing a
method according to the twentieth aspect.
[0522] In certain embodiments, compounds for the prevention or
treatment of atherosclerosis or an atherosclerotic disease are
compounds for the prevention or treatment of atherosclerosis. In
certain embodiments, compounds for the prevention or treatment of
atherosclerosis or an atherosclerotic disease are compounds for the
prevention or treatment of an atherosclerotic disease.
[0523] In certain embodiments, the process further comprises
further identifying the identified ligand as a modulator of a
mammalian GPR84. In some embodiments, the modulator of the
mammalian GPR84 is an agonist, partial agonist, inverse agonist, or
antagonist of the mammalian GPR84. In some embodiments, the
modulator of the mammalian GPR84 is an agonist or partial agonist
of the mammalian GPR84. In certain embodiments, the mammalian GPR84
is a human GPR84.
[0524] Applicant reserves the right to exclude any one or more
candidate compounds from any of the embodiments of the invention.
Applicant reserves the right to exclude any one or more modulators
from any of the embodiments of the invention. By way of example and
not limitation, Applicant reserves the right to exclude any one or
more agonists from any of the embodiments of the invention. By way
of example and not limitation, Applicant reserves the right to
exclude Compound 1, Compound 2 and Compound 3 individually or in
any combination from any of the embodiments of the invention.
Applicant reserves the right to exclude any polynucleotide or
polypeptide from any of the embodiments of the invention. Applicant
additionally reserves the right to exclude any atherosclerotic
disease or any condition related to MCP-1 expression from any of
the embodiments of the invention. It is also expressly contemplated
that atherosclerotic diseases of the invention can be included in
an embodiment either individually or in any combination. It is also
expressly contemplated that conditions related to MCP-1 expression
of the invention can be included in an embodiment either
individually or in any combination.
[0525] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, practice the
present invention to its fullest extent. The foregoing detailed
description is given for clearness of understanding only, and no
unnecessary limitation should be understood therefrom, as
modifications within the scope of the invention may become apparent
to those skilled in the art.
[0526] Throughout this application, various publications, patents
and published patent applications are cited. The disclosures of
these publications, patents and published patent applications
referenced in this application are herein incorporated by reference
in their entirety into the present disclosure. Citation herein by
Applicant of a publication, patent, or published patent application
is not an admission by Applicant of said publication, patent, or
published patent application as prior art.
[0527] This application claims the benefit of priority from the
following provisional patent application, filed via U.S. Express
mail with the United States Patent and Trademark Office on the
indicated date: U.S. Provisional Patent Application No. 60/714,137,
filed Sep. 2, 2005. The disclosure of the foregoing provisional
patent application is herein incorporated by reference in its
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0528] FIG. 1. By way of illustration and not limitation, FIG. 1
depicts results from a primary screen of candidate compounds
against a "target receptor" which is a Gs.alpha. Fusion Protein
construct of an endogenous, constitutively active Gs-coupled GPCR
unrelated to GPR84. Results for "Compound A" are provided in well
A2. Results for "Compound "B" are provided in well G9. (See,
Example 6.)
[0529] FIG. 2. A. Microarray analysis of GPR84 expression. B.
Expression of GPR84 in human macrophage. C. Expression of GPR84 in
mouse macrophage. (See, Example 10.)
[0530] FIG. 3. A. Upregulation of GPR84 mRNA in human monocytes by
TNF.alpha.. B. Upregulation of GPR84 mRNA in human monocytes by
IFN-.gamma.. (See, Example 11.)
[0531] FIG. 4. Analysis of GPR84 mRNA expression in rheumatoid
arthritis synovium by in situ hybridization. (See, Example 12.)
[0532] FIG. 5. A. Identification of Compound 1 as an agonist of
GPR84. B. Identification of Compound 2 as an agonist of GPR84. C.
Identification of Compound 3 as an agonist of GPR84. (See, Example
13.)
[0533] FIG. 6. A. Agonist to GPR84 stimulates GTP.gamma.S binding
to membrane. B. Agonist to GPR84 decreases intracellular cAMP. C.
Agonist to GPR84 increases IP.sub.3 accumulation in the presence of
Gq(del)/Gi chimeric G protein. (See, Example 14.)
[0534] FIG. 7. A. Agonist to GPR84 increases steady-state ABCA1
mRNA level in human macrophage. B. Agonist to GPR84 selectively
modulates steady-state chemokine mRNA level in human macrophage. C.
Agonist to GPR84 does not increase TNF.alpha. secretion in human
macrophage. D. Agonist to GPR84 does not increase TNF.alpha.
secretion in mouse macrophage. (See, Example 15.)
DETAILED DESCRIPTION
Definitions
[0535] AGONIST shall mean an agent (e.g., ligand, candidate
compound) that by virtue of binding to a GPCR activates the GPCR so
as to elicit an intracellular response mediated by the GPCR. By way
of example and not limitation, an agonist may be endogenous or
non-endogenous.
[0536] AMINO ACID ABBREVIATIONS used herein are set out in Table
A:
TABLE-US-00002 TABLE A ALANINE ALA A ARGININE ARG R ASPARAGINE ASN
N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE
GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L
LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P
SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE
VAL V
[0537] ANTAGONIST shall mean an agent (e.g., ligand, candidate
compound) that binds, and preferably binds competitively, to a GPCR
at about the same site as an agonist or partial agonist but which
does not activate an intracellular response initiated by the active
form of the GPCR, and can thereby inhibit the intracellular
response by agonist or partial agonist. An antagonist typically
does not diminish the baseline intracellular response in the
absence of an agonist or partial agonist. By way of example and not
limitation, an antagonist may be endogenous or non-endogenous.
[0538] ANTIBODY is intended herein to encompass monoclonal antibody
and polyclonal antibody. Antibodies of the present invention may be
prepared by any suitable method known in the art.
[0539] ATHEROGENESIS as used herein refers to the formation of
atheromatous plaques containing cholesterol and lipids on the
innermost layer of the walls of large and medium-sized arteries. In
some embodiments, a compound which reduces an atherosclerotic
lesion area is an inhibitor of atherogenesis. In some embodiments,
a compound which reduces a diameter of lipid droplets in fatty
streaks is an inhibitor of atherogenesis.
[0540] ATHEROSCLEROSIS as used herein refers to a form of vascular
disease characterized by the deposition of atheromatous plaques
containing cholesterol and lipids on the innermost layer of the
walls of large and medium-sized arteries. Atherosclerosis
encompasses vascular diseases and conditions that are recognized
and understood by physicians practicing in the relevant fields of
medicine. Atherosclerotic cardiovascular disease, including
restenosis following revascularization procedures, coronary artery
disease (also known as coronary heart disease or ischemic heart
disease), cerebrovascular disease including multi-infarct dementia,
and peripheral vessel disease including erectile dysfunction, are
all clinical manifestations of atherosclerosis and are therefore
encompassed by the terms "atherosclerosis" and "atherosclerotic
disease."
[0541] ATHEROSCLEROTIC DISEASE as used herein shall refer to
vascular diseases and conditions that are recognized and understood
by physicians practicing in the relevant fields of medicine,
including but not limited to coronary artery disease, myocardial
infarction, peripheral artery disease, and ischemic, stroke.
Atherosclerotic diseases may be included in embodiments of the
invention individually or in any combination.
[0542] ATHEROSCLEROTIC LESION as used herein shall refer to an
atheromatous plaque containing cholesterol and lipids on the
innermost layer of the wall of a large or medium-sized artery.
[0543] BIOLOGICALLY ACTIVE FRAGMENT of a GPCR polypeptide or amino
acid sequence shall mean a fragment of the polypeptide or amino
acid sequence having structural and biochemical functions of a
naturally occurring GPCR. In certain embodiments, the biologically
active fragment couples to a G protein. In certain embodiments, the
biologically active fragment binds to a ligand.
[0544] CANDIDATE COMPOUND shall mean a molecule (for example, and
not limitation, a chemical compound) that is amenable to a
screening technique and is used interchangeably herein with TEST
COMPOUND. By way of example and not limitation, a candidate
compound may be a compound not known to be a ligand or a modulator
of a GPCR. By way of example and not limitation, a candidate
compound may be endogenous or non-endogenous.
[0545] CODON shall mean a grouping of three nucleotides (or
equivalents to nucleotides) which generally comprise a nucleoside
[adenosine (A), guanosine (G), cytidine (C), uridine (U) and
thymidine (T)] coupled to a phosphate group and which, when
translated, encodes an amino acid.
[0546] COMPOSITION means a material comprising at least one
component.
[0547] COMPOUND EFFICACY or EFFICACY shall mean the ability of a
compound to inhibit or stimulate one or more GPCR functions, e.g.
by measurement of cAMP level, IP.sub.3 level or Ca.sup.2+ level in
the presence or absence of a candidate compound. Exemplary means of
measuring compound efficacy are disclosed in the Examples section
of this patent document.
[0548] CONDITION RELATED TO MCP-1 EXPRESSION as used herein shall
refer to diseases and conditions that are recognized and understood
by physicians practicing in the relevant fields of medicine,
including but not limited to atherosclerosis, an atherosclerotic
disease, and rheumatoid arthritis. In certain embodiments, the
condition related to MCP-1 expression is an inflammation-related
disease or disorder. In certain embodiments, the condition related
to MCP-1 expression is characterized by elevated MCP-1 expression.
In some embodiments, the MCP-1 expression comprises MCP-1
expression in monocytes/macrophages. In some embodiments, the MCP-1
expression is MCP-1 expression in monocytes/macrophages. In some
embodiments, the condition is related adversely to MCP-1
expression. Conditions related to MCP-1 expression may be included
in embodiments of the invention individually or in any combination.
In certain embodiments, the condition related to MCP-1 expression
is selected from atherosclerosis, an atherosclerotic disease,
rheumatoid arthritis, Crohn's disease, and multiple sclerosis.
[0549] CONSTITUTIVELY ACTIVE RECEPTOR shall mean a receptor
stabilized in an active state by means other than through binding
of the receptor to its ligand or a chemical equivalent thereof. A
constitutively active receptor may be endogenous or
non-endogenous.
[0550] CONSTITUTIVELY ACTIVATED RECEPTOR shall mean an endogenous
receptor that has been modified so as to be constitutively active
or to be more constitutively active.
[0551] CONSTITUTIVE RECEPTOR ACTIVATION shall mean activation of a
receptor in the absence of binding to its ligand or a chemical
equivalent thereof.
[0552] CONTACT or CONTACTING shall mean bringing at least two
moieties together, whether in an in vitro system or an in vivo
system.
[0553] DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship
to the phrase "candidate compound" or "test compound", shall mean
the screening of a compound against a G protein-coupled receptor in
the absence of a known ligand (e.g., a known agonist) to the G
protein-coupled receptor.
[0554] ENDOGENOUS shall mean a material that a mammal naturally
produces. Endogenous in reference to, for example and not
limitation, the term "receptor," shall mean that which is naturally
produced by a mammal (for example, and not limitation, a human).
Endogenous shall be understood to encompass allelic variants of a
gene as well as the allelic polypeptide variants so encoded. As
used herein, "endogenous GPCR" and "native GPCR" are used
interchangeably.
[0555] By contrast, the term NON-ENDOGENOUS in this context shall
mean that which is not naturally produced by a mammal (for example,
and not limitation, a human).
[0556] EXPRESSION VECTOR shall mean a DNA sequence that is required
for the transcription of cloned DNA and translation of the
transcribed mRNA in an appropriate host cell recombinant for the
expression vector. An appropriately constructed expression vector
should contain an origin of replication for autonomous replication
in host cells, selectable markers, a limited number of useful
restriction enzyme sites, a potential for high copy number, and
active promoters. The cloned DNA to be transcribed is operably
linked to a constitutively or conditionally active promoter within
the expression vector.
[0557] G PROTEIN-COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION
PROTEIN, in the context of the invention disclosed herein, each
mean a non-endogenous protein comprising an endogenous,
constitutively active GPCR or a non-endogenous, constitutively
activated GPCR fused to at least one G protein, most preferably the
alpha (.alpha.) subunit of such G protein (this being the subunit
that binds GTP), with the G protein preferably being of the same
type as the G protein that naturally couples with endogenous GPCR.
In the preferred form, the G protein can be fused directly to the
C-terminus of the GPCR or there may be spacers between the two.
[0558] HOST CELL shall mean a cell capable of having a vector
incorporated therein. In the present context, the vector will
typically contain nucleic acid encoding a GPCR or GPCR fusion
protein in operable connection with a suitable promoter sequence to
permit expression of the GPCR or GPCR fusion protein to occur. In
particular embodiment, the host cell is a eukaryotic host cell. In
certain embodiments, the eukaryotic host cell is a mammalian host
cell. In certain embodiments, the eukaryotic host cell is a
melanophore host cell. In certain embodiments, the eukaryotic host
cell is a yeast host cell.
[0559] IN NEED OF PREVENTION OR TREATMENT as used herein refers to
a judgement made by a caregiver (e.g. physician, nurse, nurse
practitioner, etc. in the case of humans; veterinarian in the case
of animals, including non-human mammals) that a subject or animal
requires or will benefit from treatment. This judgement is made
based on a variety of factors that are in the realm of a
caregiver's expertise, but that include the knowledge that the
subject or animal is ill, or will be ill, as the result of a
condition that is treatable by the compounds of the invention.
[0560] INHIBIT or INHIBITING, in relationship to the term
"response" shall mean that a response is decreased or prevented in
the presence of a compound as opposed to in the absence of the
compound.
[0561] INVERSE AGONIST shall mean an agent (e.g., ligand, candidate
compound) which binds to either the endogenous form of a GPCR or to
the constitutively activated form of a GPCR and which inhibits the
baseline intracellular response initiated by the active form of the
receptor below the normal base level activity which is observed in
the absence of an agonist or partial agonist. By way of example and
not limitation, an inverse agonist may be endogenous or
non-endogenous.
[0562] LIGAND as used herein shall mean a molecule that
specifically binds to a GPCR. An endogenous ligand is an endogenous
molecule that binds to a native GPCR. By way of example and not
limitation, a ligand may be endogenous or non-endogenous. A ligand
of a GPCR may be, but is not limited to, an agonist, a partial
agonist, an inverse agonist or an antagonist of the GPCR.
[0563] MCP-1 EXPRESSION as used herein shall refer to a cellular
level of steady-state MCP-1 mRNA or to a level of cell secreted
MCP-1. In some embodiments, the cell is a monocyte/macrophage.
[0564] As used herein, the terms MODULATE or MODIFY are meant to
refer to an increase or decrease in the amount, quality, or effect
of a particular activity, function or molecule.
[0565] MODULATOR shall be understood to encompass agonist, partial
agonist, inverse agonist and antagonist as hereinbefore
defined.
[0566] MONOCYTE/MACROPHAGE as used herein is intended to encompass
a monocyte, a monocyte which has differentiated toward a
macrophage, and a macrophage, which is understood to include a
monocyte-derived macrophage and a macrophage-derived foam cell. By
way of example and not limitation, a monocyte, a monocyte-derived
macrophage and a macrophage-derived foam cell may be included in
embodiments of the invention relating to a MONOCYTE/MACROPHAGE
individually or in any combination. A monocyte may undergo
differentiation toward a macrophage either in vitro or in vivo.
[0567] PARTIAL AGONIST shall mean an agent (e.g., ligand, candidate
compound) that by virtue of binding to a GPCR activates the GPCR so
as to elicit an intracellular response mediated by the GPCR, albeit
to a lesser exent or degree than does a full agonist. By way of
example and not limitation, a partial agonist may be endogenous or
non-endogenous.
[0568] PHARMACEUTICAL COMPOSITION shall mean a composition
comprising at least one active ingredient, whereby the composition
is amenable to investigation for a specified, efficacious outcome
in a mammal (for example, and not limited to a human). Those of
ordinary skill in the art will understand and appreciate the
techniques appropriate for determining whether an active ingredient
has a desired efficacious outcome e.g., based upon the needs of the
artisan.
[0569] POLYNUCLEOTIDE shall refer to RNA, DNA, or RNA/DNA hybrid
sequence of more than one nucleotide in either single chain or
duplex form. The polynucleotides of the invention may be prepared
by any known method, including synthetic, recombinant, ex vivo
generation, or a combination thereof, as well as utilizing any
purification methods known in the art.
[0570] POLYPEPTIDE shall refer to a polymer of amino acids without
regard to the length of the polymer. Thus, PEPTIDES, oligopeptides,
and proteins are included within the definition of polypeptide.
This term also does not specify or exclude post-expression
modifications of polypeptides. For example, polypeptides that
include the covalent attachment of glycosyl groups, acetyl groups,
phosphate groups, lipid groups and the like are expressly
encompassed by the term polypeptide.
[0571] PRIMER is used herein to denote a specific oligonucleotide
sequence which is complementary to a target nucleotide sequence and
used to hybridize to the target nucleotide sequence. A primer
serves as an initiation point for nucleotide polymerization
catalyzed by DNA polymerase, RNA polymerase, or reverse
transcriptase.
[0572] RECEPTOR FUNCTIONALITY shall refer to the normal operation
of a receptor to receive a stimulus and moderate an effect in the
cell, including, but not limited to regulating gene transcription,
regulating the influx or efflux of ions, effecting a catalytic
reaction, and/or modulating activity through G-proteins, such as
eliciting a second messenger response.
[0573] SECOND MESSENGER shall mean an intracellular response
produced as a result of receptor activation. A second messenger can
include, for example, inositol 1,4,5-triphosphate (IP.sub.3),
diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP
kinase activity, MAPK/ERK kinase kinase-1 (MEKK1) activity, and
Ca.sup.2+. Second messenger response can be measured for a
determination of receptor activation. In addition, second messenger
response can be measured for the identification of candidate
compounds as, for example, inverse agonists, partial agonists,
agonists, and antagonists of the receptor.
[0574] SMALL MOLECULE shall be taken to mean a compound having a
molecular weight of less than about 10,000 grams per mole,
including a peptide, peptidomimetic, amino acid, amino acid
analogue, polynucleotide, polynucleotide analogue, nucleotide,
nucleotide analogue, organic compound or inorganic compound (i.e.
including a heterorganic compound or organometallic compound), and
salts, esters and other pharmaceutically acceptable forms thereof.
In certain preferred embodiments, small molecules are organic or
inorganic compounds having a molecular weight of less than about
5,000 grams per mole. In certain preferred embodiments, small
molecules are organic or inorganic compounds having molecular
weight of less than about 1,000 grams per mole. In certain
preferred embodiments, small molecules are organic or inorganic
compounds having a molecular weight of less than about 500 grams
per mole.
[0575] STIMULATE or STIMULATING, in relationship to the term
"response" shall mean that a response is increased in the presence
of a compound as opposed to in the absence of the compound.
[0576] SUBJECT as used herein is a mammal. The mammal may be a
human or it may be a non-human mammal. Non-human mammal is intended
to include, but not be limited to, horse, cow, sheep, pig, cat,
dog, rabbit, mouse, rat, and non-human primate. The non-human
mammal may be a laboratory animal (such as a mouse, a rat, a
hamster, a pig, a dog, a rabbit, etc.), a farm animal (such as a
cow, a sheep, a goat, a horse, a pig, etc.), a human companion
animal (such as a dog, a cat, etc.) or an exotic animal (such as an
animal found in a zoo, etc.). Non-human mammals may be included in
embodiments individually or in any combination. In certain
embodiments, the subject is a human.
[0577] THERAPEUTICALLY EFFECTIVE AMOUNT as used herein refers to
the amount of active compound or pharmaceutical agent that elicits
the biological or medicinal response in a tissue, system, animal,
subject or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes one
or more of the following: [0578] (1) Preventing the disease; for
example, preventing a disease, condition or disorder in a subject
that may be predisposed to the disease, condition or disorder but
does not yet experience or display the pathology or symptomatology
of the disease, [0579] (2) Inhibiting the disease; for example,
inhibiting a disease, condition or disorder in a subject that is
experiencing or displaying the pathology or symptomatology of the
disease, condition or disorder (i.e., arresting further development
of the pathology and/or symptomatology), and [0580] (3)
Ameliorating the disease; for example, ameliorating a disease,
condition or disorder in a subject that is experiencing or
displaying the pathology or symptomatology of the disease,
condition or disorder (i.e., reversing the pathology and/or
symptomatology).
[0581] VARIANT as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
A typical variant of a polypeptide differs in amino acid sequence
from another, reference polypeptide. A variant and reference
polypeptide may differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant
of a polynucleotide or polypeptide may be a naturally occurring one
such as an ALLELIC VARIANT, or it may be a variant that is not
known to occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
A. Introduction
[0582] The order of the following sections is set forth for
presentational efficiency and is not intended, nor should be
construed, as a limitation on the disclosure or the claims to
follow.
B. Receptor Expression
[0583] 1. GPCR Polypeptides of Interest
[0584] A GPCR of the invention may comprise an amino acid sequence
selected from the group consisting of: [0585] (a) the amino acid
sequence of SEQ ID NO:2; [0586] (b) amino acids 2-396 of SEQ ID
NO:2; [0587] (c) amino acids 2-396 of SEQ ID NO:2, wherein the GPCR
does not comprise amino acids 1-396 of SEQ ID NO:2; [0588] (d) the
amino acid sequence of SEQ ID NO:20; [0589] (e) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction
(PCR) on a human DNA sample using specific primers SEQ ID NO:7 and
SEQ ID NO:8; [0590] (f) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:1; [0591]
(g) the amino acid sequence of an endogenous G protein-coupled
receptor having at least about 75% identity, at least about 80%
identity, at least about 85% identity, at least about 90% identity,
or at least about 95% identity to SEQ ID NO:2; [0592] (h) the amino
acid sequence of SEQ ID NO:4; [0593] (i) amino acids 2-396 of SEQ
ID NO:4; [0594] (j) amino acids 2-396 of SEQ ID NO:4 wherein the
GPCR does not comprise amino acids 1-396 of SEQ ID NO:4; [0595] (k)
the amino acid sequence of a G protein-coupled receptor encoded by
a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:3; [0596] (l) the amino acid sequence of an
endogenous G protein-coupled receptor having at least about 75%
identity, at least about 80% identity, at least about 85% identity,
at least about 90% identity, or at least about 95% identity to SEQ
ID NO:4; [0597] (m) the amino acid sequence of SEQ ID NO:6; [0598]
(n) amino acids 2-396 of SEQ ID NO:6; [0599] (o) amino acids 2-396
of SEQ ID NO:6, wherein the GPCR does not comprise amino acids
1-396 of SEQ ID NO:6; [0600] (p) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:5; and
[0601] (q) the amino acid sequence of an endogenous G
protein-coupled receptor having at least about 75% identity, at
least about 80% identity, at least about 85% identity, at least
about 90% identity, or at least about 95% identity to SEQ ID NO:6;
[0602] or a variant or biologically active fragment thereof.
[0603] In some embodiments, the GPCR comprising amino acids 2-396
of SEQ ID NO:2, wherein the GPCR does not comprise amino acids
1-396 of SEQ ID NO:2, is SEQ ID NO:20.
[0604] In some embodiments, the human DNA is human genomic DNA. In
some embodiments, the human DNA is human cDNA derived from a tissue
or cell type that expresses GPR84. In some embodiments, the human
cDNA is derived from a leukocyte. In some embodiments, the human
cDNA is derived from a monocyte/macrophage. In some embodiments,
the human cDNA is derived from a granulocyte. In some embodiments
the G protein-coupled receptor encoded by the polynucleotide that
is amplifiable by polymerase chain reaction is an endogenous G
protein-coupled receptor. In some embodiments the G protein-coupled
receptor encoded by the polynucleotide that is amplifiable by
polymerase chain reaction is a mammalian endogenous G
protein-coupled receptor. In some embodiments the G protein-coupled
receptor encoded by the polynucleotide that is amplifiable by
polymerase chain reaction is an endogenous G protein-coupled
receptor for which Compound 1 is an agonist having an EC.sub.50
value at said receptor in melanophore assay according to Example 9,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 100 nM, less than about 50 nM,
less than about 25 nM, less than about 10 nM, or less than about 5
nM. In some embodiments the G protein-coupled receptor encoded by
the polynucleotide that is amplifiable by polymerase chain reaction
is an endogenous G protein-coupled receptor for which Compound 2 is
an agonist having an EC.sub.50 value at said receptor in
melanophore assay according to Example 9, infra, of less than about
10 .mu.M, less than about 5 .mu.M, less than about 1 .mu.M, less
than about 100 nM, less than about 50 nM, less than about 25 nM,
less than about 10 nM, or less than about 5 nM. In some embodiments
the G protein-coupled receptor encoded by the polynucleotide that
is amplifiable by polymerase chain reaction is an endogenous G
protein-coupled receptor for which Compound 3 is an agonist having
an EC.sub.50 value at said receptor in melanophore assay according
to Example 9, infra, of less than about 10 .mu.M, less than about 5
.mu.M, less than about 1 .mu.M, less than about 100 nM, less than
about 50 nM, less than about 25 nM, less than about 10 nM, or less
than about 5 nM.
[0605] In some embodiments, a GPCR of the invention is recombinant.
In some embodiments, the recombinant GPCR is recombinant human
GPR84. In some embodiments, recombinant human GPR84 has SEQ ID
NO:2. In some embodiments, the recombinant GPCR is a non-endogenous
version of a human GPR84, wherein the non-endogenous version has
SEQ ID NO:20.
[0606] In some embodiments, a GPCR of the invention is
endogenous.
[0607] In some embodiments, a GPCR of the invention is a mammalian
GPR84.
[0608] In some embodiments, a GPCR of the invention is
constitutively active. In some embodiments, an endogenous GPCR of
the invention is constitutively active. In some embodiments, a
mammalian GPR84 of the invention is constitutively active. In some
embodiments, the mammalian GPR84 is human GPR84. In some
embodiments, the human GPR84 is SEQ ID NO:2 or an allele
thereof.
[0609] In some embodiments, a GPCR of the invention exhibits a
detectable level of constitutive activity. In some embodiments, an
endogenous GPCR of the invention exhibits a detectable level of
constitutive activity. In some embodiments, a mammalian GPR84 of
the invention exhibits a detectable level of constitutive activity.
In some embodiments, the mammalian GPR84 is human GPR84. In some
embodiments, the human GPR84 is SEQ ID NO:2 or an allele
thereof.
[0610] By way of illustration and not limitation, deletion of an
N-terminal methionine residue or an N-terminal signal peptide is
envisioned to provide a biologically active fragment that may be
used in the subject invention. In some embodiments, a biologically
active fragment of the invention is a fragment optionally fused at
its N-terminus to a peptide comprising an N-terminal methionine
residue and an HA epitope tag (from hemagglutinin influenza virus).
In some embodiments, a biologically active fragment of the
invention is a fragment that can decrease a level of intracellular
cAMP in response to a compound selected from Table 1. In some
embodiments, a biologically active fragment of the invention is a
fragment that can increase a level of intracellular IP.sub.3
accumulation in a cell comprising Gq(del)/Gi chimeric G protein in
response to a compound selected from Table 1. In some embodiments,
a biologically active fragment of the invention is a fragment that
can cause melanophore cells to undergo pigment aggregation in
response to a compound selected from Table 1. In some embodiments,
the compound selected from Table 1 is Compound 1. In some
embodiments, a biologically active fragment is a fragment
optionally fused at its N-terminus to a peptide comprising an
N-terminal methionine residue and an HA epitope tag (from
hemagglutinin influenza virus) for which Compound 1 is an agonist
having an EC.sub.50 value at said fragment optionally fused at its
N-terminus to said peptide in melanophore assay according to
Example 9, infra, of less than about 10 .mu.M, less than about 5
.mu.M, less than about 1 .mu.M, less than about 100 nM, less than
about 50 nM, less than about 25 nM, less than about 10 nM, or less
than about 5 nM. In some embodiments, a biologically active
fragment is a fragment optionally fused at its N-terminus to a
peptide comprising an N-terminal methionine residue and an HA
epitope tag (from hemagglutinin influenza virus) for which Compound
2 is an agonist having an EC.sub.50 value at said fragment
optionally fused at its N-terminus to said peptide in melanophore
assay according to Example 9, infra, of less than about 10 .mu.M,
less than about 5 .mu.M, less than about 1 .mu.M, less than about
100 nM, less than about 50 nM, less than about 25 nM, less than
about 10 nM, or less than about 5 nM. In some embodiments, a
biologically active fragment is a fragment optionally fused at its
N-terminus to a peptide comprising an N-terminal methionine residue
and an HA epitope tag (from hemagglutinin influenza virus) for
which Compound 3 is an agonist having an EC.sub.50 value at said
fragment optionally fused at its N-terminus to said peptide in
melanophore assay according to Example 9, infra, of less than about
10 .mu.M, less than about 5 .mu.M, less than about 1 .mu.M, less
than about 100 nM, less than about 50 nM; less than about 25 nM,
less than about 10 nM, or less than about 5 nM.
[0611] An allelic variant of human GPR84 of SEQ ID NO:2, of mouse
GPR84 of SEQ ID NO:4, or of rat GPR84 of SEQ ID NO:6 is envisioned
to be within the scope of the invention. In some embodiments, a
GPCR that may be used in the subject methods may comprise an
allelic variant of SEQ ID NO:2.
[0612] A variant which is a mammalian ortholog of human GPR84 of
SEQ ID NO:2 is envisioned to be within the scope of the invention.
By way of illustration and not limitation, additional to mouse
GPR84 and rat GPR84, chimpanzee GPR84 (GenBank.RTM. Accession No.
XP.sub.--522412) and cow GPR84 (GenBank.RTM. Accession No.
AAX31354) are envisioned to be within the scope of the
invention.
[0613] By way of example and not limitation, a variant of SEQ ID
NO:2, SEQ ID NO:4 or SEQ ID NO:6 may be a polypeptide having any
number of amino acid substitutions, amino acid deletions, or amino
acid additions at any position in SEQ ID NO:2, SEQ ID NO:4 or SEQ
ID NO:6 (e.g., the C- or N-terminus, or at internal positions).
[0614] A variant of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 having
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, at least about 99%, at least about
99.1%, at least about 99.2%, at least about 99.3%, at least about
99.4%, at least about 99.5%, at least about 99.6%, at least about
99.7%, at least about 99.8%, or at least about 99.9% identity to
SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, respectively, is
envisioned to be within the scope of the invention. In some
embodiments, said variant is a variant of SEQ ID NO:2. In some
embodiments, the variant which is a variant of SEQ ID NO:2, SEQ ID
NO:4 or SEQ ID NO:6 is an endogenous GPCR. In some embodiments, the
variant can decrease a level of intracellular cAMP in response to a
compound selected from Table 1. In some embodiments, the variant
can increase a level of intracellular IP.sub.3 accumulation in a
cell comprising Gq(del)/Gi chimeric G protein in response to a
compound selected from Table 1. In some embodiments, the variant
can cause melanophore cells to undergo pigment aggregation in
response to a compound selected from Table 1. In some embodiments,
the variant which can decrease a level of intracellular cAMP, which
can increase a level of intracellular IP.sub.3 accumulation in a
cell comprising Gq(del)/Gi chimeric G protein, or which can cause
melanophore cells to undergo pigment aggregation in response to a
compound selected from Table 1 exhibits a detectable level of
constitutive activity. In some embodiments, the compound selected
from Table 1 is Compound 1. In some embodiments, the variant is a
GPCR for which Compound 1 is an agonist having an EC.sub.50 value
at said receptor in melanophore assay according to Example 9,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 100 nM, less than about 50 nM,
less than about 25 nM, less than about 10 nM, or less than about 5
nM. In some embodiments, the variant is a GPCR for which Compound 2
is an agonist having an EC.sub.50 value at said receptor in
melanophore assay according to Example 9, infra, of less than about
10 .mu.M, less than about 5 .mu.M, less than about 1 .mu.M, less
than about 100 nM, less than about 50 nM, less than about 25 nM,
less than about 10 nM, or less than about 5 nM. In some
embodiments, the variant is a GPCR for which Compound 3 is an
agonist having an EC.sub.50 value at said receptor in melanophore
assay according to Example 9, infra, of less than about 10 .mu.M,
less than about 5 .mu.M, less than about 1 .mu.M, less than about
100 nM, less than about 50 nM, less than about 25 nM, less than
about 10 nM, or less than about 5 nM. Percent identity can be
determined conventionally using known computer programs.
[0615] In certain embodiments, a variant GPCR that may be used in
the subject methods has an amino acid sequence having at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, of: at least about 96%, at least about
97%, at least about 98%, at least about 99%, at least about 99.1%,
at least about 99.2%, at least about 99.3%, at least about 99.4%,
at least about 99.5%, at least about 99.6%, at least about 99.7%,
at least about 99.8%, or at least about 99.9% identity to SEQ ID
NO:2. By a variant GPCR having, for example, 95% "identity" to SEQ
ID NO:2 is meant that the amino acid sequence of the variant is
identical to amino acids 1-396 of SEQ ID NO:2 except that it may
include up to five amino acid alterations per each 100 amino acids
of amino acids SEQ ID NO:2. Thus, to obtain for example an amino
acid sequence having at least 95% identity to the amino acid
sequence of SEQ ID NO:2, up to 5% (5 of 100) of the amino acid
residues in the sequence may be inserted, deleted, or substituted
with another amino acid compared with amino acids 1-396 of SEQ ID
NO:2. These alternations may occur at the amino or carboxy termini
or anywhere between those terminal positions, interspersed either
subjectly among residues in the sequence or in one or more
contiguous groups within the sequence.
[0616] In some embodiments, a variant GPCR that may be used in the
subject methods is a GPCR encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:1, SEQ ID
NO:3 or SEQ ID NO:5. In some embodiments, the polynucleotide
hybridizes under stringent conditions to the complement of SEQ ID
NO:1. In some embodiments, the variant is an endogenous GPCR. In
some embodiments, the variant can decrease a level of intracellular
cAMP in response to a compound selected from Table 1. In some
embodiments, the variant can increase a level of intracellular
IP.sub.3 accumulation in a cell comprising Gq(del)/Gi chimeric G
protein in response to a compound selected from Table 1. In some
embodiments, the variant can cause melanophore cells to undergo
pigment aggregation in response to a compound selected from Table
1. In some embodiments, the variant which can decrease a level of
intracellular cAMP, which can increase a level of intracellular
IP.sub.3 accumulation in a cell comprising Gq(del)/Gi chimeric G
protein, or which can cause melanophore cells to undergo pigment
aggregation in response to a compound selected from Table 1
exhibits a detectable level of constitutive activity. In some
embodiments, the compound selected from Table 1 is Compound 1. In
some embodiments, the variant is a GPCR for which Compound 1 is an
agonist having an EC.sub.50 value at said receptor in melanophore
assay according to Example 9, infra, of less than about 10 .mu.M,
less than about 54 .mu.M, less than about 1 .mu.M, less than about
100 nM, less than about 50 nM, less than about 25 nM, less than
about 10 nM, or less than about 5 nM. In some embodiments, the
variant is a GPCR for which Compound 2 is an agonist having an
EC.sub.50 value at said receptor in melanophore assay according to
Example 9, infra, of less than about 10 .mu.M, less than about 5
.mu.M, less than about 1 .mu.M, less than about 100 nM, less than
about 50 nM, less than about 25 nM, less than about 10 nM, or less
than about 5 nM. In some embodiments, the variant is a GPCR for
which Compound 3 is an agonist having an EC.sub.50 value at said
receptor in melanophore assay according to Example 9, infra, of
less than about 10 .mu.M, less than about 5 .mu.M, less than about
1 .mu.M, less than about 100 nM, less than about 50 nM, less than
about 25 nM, less than about 10 nM, or less than about 5 nM.
Hybridization techniques are well known to the skilled artisan. In
some embodiments, stringent hybridization conditions include
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (1.times.SSC=150 mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's
solution, 10% dextran sulfate, and 20 .mu.g/ml denatured, sheared
salmon sperm DNA; followed by washing the filter in 0.1.times.SSC
at about 50.degree. C., at about 55.degree. C., at about 60.degree.
C. or at about 65.degree. C.
[0617] a. Sequence Identity
[0618] A preferred method for determining the best overall match
between a query sequence (e.g., the amino acid sequence of SEQ ID
NO:2) and a sequence to be interrogated, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. [Comp App
Biosci (1990) 6:237-245; the disclosure of which is herein
incorporated by reference in its entirety]. In a sequence alignment
the query and interrogated sequences are both amino acid sequences.
The results of said global sequence alignment is in percent
identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group=25, Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=247 or the length of the interrogated amino acid
sequence, whichever is shorter.
[0619] If the interrogated sequence is shorter than the query
sequence due to N- or C-terminal deletions, not because of internal
deletions, the results, in percent identity, must be manually
corrected because the FASTDB program does not account for N- and
C-terminal truncations of the interrogated sequence when
calculating global percent identity. For interrogated sequences
truncated at the N- and C-termini, relative to the query sequence,
the percent identity is corrected by calculating the number of
residues of the query sequence that are N- and C-terminal of the
interrogated sequence, that are not matched/aligned with a
corresponding interrogated sequence residue, as a percent of the
total bses of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the interrogated sequence, which are not matched/aligned with the
query sequence, are considered for the purposes of manually
adjusting the percent identity score. That is, only query amino
acid residues outside the farthest N- and C-terminal residues of
the interrogated sequence.
[0620] For example, a 90 amino acid residue interrogated sequence
is aligned with a 100-residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the interrogated
sequence and therefore, the FASTDB alignment does not match/align
with the first residues at the N-terminus. The 10 unpaired residues
represent 10% of the sequence (number of residues at the N- and
C-termini not matched/total number of residues in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched, the final percent identity would be 90%.
[0621] In another example, a 90-residue interrogated sequence is
compared with a 100-residue query sequence. This time the deletions
are internal so there are no residues at the N- or C-termini of the
interrogated sequence, which are not matched/aligned with the
query. In this case, the percent identity calculated by FASTDB is
not manually corrected. Once again, only residue positions outside
the N- and C-terminal ends of the subject sequence, as displayed in
the FASTDB alignment, which are not matched/aligned with the query
sequence are manually corrected. No other corrections are made for
the purposes of the present invention.
[0622] b. Fusion Proteins
[0623] In certain embodiments, a polypeptide of interest is a
fusion protein, and may contain, for example, an affinity tag
domain or a reporter domain. Suitable affinity tags include any
amino acid sequence that may be specifically bound to another
moiety, usually another polypeptide, most usually an antibody.
Suitable affinity tags include epitope tags, for example, the V5
tag, the FLAG tag, the HA tag (from hemagglutinin influenza virus),
the myc tag, and the like, as is known in the art. Suitable
affinity tags also include domains for which, binding substrates
are known, e.g., HIS, GST and MBP tags, as is known in the art, and
domains from other proteins for which specific binding partners,
e.g., antibodies, particularly monoclonal antibodies, are
available. Suitable affinity tags also include any protein-protein
interaction domain, such as a IgG Fc region, which may be
specifically bound and detected using a suitable binding partner,
e.g. the IgG Fc receptor. It is expressly contemplated that such a
fusion protein may contain a heterologous N-terminal domain (e.g.,
an epitope tag) fused in-frame with a GPCR that has had its
N-terminal methionine residue either deleted or substituted with an
alternative amino acid.
[0624] Suitable reporter domains include any domain that can report
the presence of a polypeptide. While it is recognized that an
affinity tag may be used to report the presence of a polypeptide
using, e.g., a labeled antibody that specifically binds to the tag,
light emitting reporter domains are more usually used. Suitable
light emitting reporter domains include luciferase (from, e.g.,
firefly, Vargula, Renilla reniformis or Renilla muelleri), or light
emitting variants thereof. Other suitable reporter domains include
fluorescent proteins, (from e.g., jellyfish, corals and other
coelenterates as such those from Aequoria, Renilla, Ptilosarcus,
Stylatula species), or light emitting variants thereof. Light
emitting variants of these reporter proteins are very well known in
the art and may be brighter, dimmer, or have different excitation
and/or emission spectra, as compared to a native reporter protein.
For example, some variants are altered such that they no longer
appear green, and may appear blue, cyan, yellow, enhanced yellow
red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other
emission spectra, as is known in the art. Other suitable reporter
domains include domains that can report the presence of a
polypeptide through a biochemical or color change, such as
.beta.-galactosidase, .beta.-glucuronidase, chloramphenicol acetyl
transferase, and secreted embryonic alkaline phosphatase. [0625]
Also as is known in the art, an affinity tags or a reporter domain
may be present at any position in a polypeptide of interest.
However, in most embodiments, they are present at the C- or
N-terminal end of a polypeptide of interest.
[0626] 2. Nucleic Acids Encoding GPCR Polypeptides of Interest
[0627] Since the genetic code and recombinant techniques for
manipulating nucleic acid are known, and the amino acid sequences
of GPCR polypeptides of interest described as above, the design and
production of nucleic acids encoding a GPCR polypeptide of interest
is well within the skill of an artisan. In certain embodiments,
standard recombinant DNA technology (Ausubel, et al, Short
Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995;
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. For
example, GPCR coding sequences may be isolated from a library of
GPCR coding sequence using any one or a combination of a variety of
recombinant methods that do not need to be described herein.
Subsequent substitution, deletion, and/or addition of nucleotides
in the nucleic acid sequence encoding a protein may also be done
using standard recombinant DNA techniques. By way of example and
not limitation, a variant of SEQ ID NO:5 may be a polynucleotide
having any number of nucleotide substitutions, nucleotide
deletions, or nucleotide additions at any position in SEQ ID NO:5
(e.g., the 5- or 3-terminus, or at internal positions).
[0628] For example, site directed mutagenesis and subcloning may be
used to introduce/delete/substitute nucleic acid residues in a
polynucleotide encoding a polypeptide of interest. In other
embodiments, PCR may be used. Nucleic acids encoding a polypeptide
of interest may also be made by chemical synthesis entirely from
oligonucleotides (e.g., Cello et al., Science (2002)
297:1016-8).
[0629] In some embodiments, the codons of the nucleic acids
encoding polypeptides of interest are optimized for expression in,
cells of a particular species, particularly a mammalian, e.g.,
mouse, rat, hamster, non-human primate, or human, species. In some
embodiments, the codons of the nucleic acids encoding polypeptides
of interest are optimized for expression in cells of a particular
species, particularly an amphibian species.
[0630] a. Vectors
[0631] The invention further provides vectors (also referred to as
"constructs") comprising a subject nucleic acid. In many
embodiments of the invention, the subject nucleic acid sequences
will be expressed in a host after the sequences have been operably
linked to an expression control sequence, including, e.g. a
promoter. The subject nucleic acids are also typically placed in an
expression vector that can replicate in a host cell either as an
episome or as an integral part of the host chromosomal DNA.
Commonly, expression vectors will contain selection markers, e.g.,
tetracycline or neomycin, to permit detection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362, which is incorporated herein by reference). Vectors,
including single and dual expression cassette vectors are well
known in the art (Ausubel, et al, Short Protocols in Molecular
Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold
Spring Harbor, N.Y.). Suitable vectors include viral vectors,
plasmids, cosmids, artificial chromosomes (human artificial
chromosomes, bacterial artificial chromosomes, yeast artificial
chromosomes, etc.), mini-chromosomes, and the like. Retroviral,
adenoviral and adeno-associated viral vectors may be used.
[0632] A variety of expression vectors are available to those in
the art for purposes of producing a polypeptide of interest in a
cell. One suitable vector is pCMV, which is used in certain
embodiments. This vector was deposited with the American Type
Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,
Manassas, Va. 20110-2209 USA) under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure. The DNA was
tested by the ATCC and determined to be viable. The ATCC has
assigned the following deposit number to pCMV: ATCC #203351. The
expression vector may be adenoviral. An exemplary adenoviral vector
may be purchased as AdEasy.TM. from Qbiogene (Carlsbad, Calif.) [He
T C et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and U.S.
Pat. No. 5,922,576; the disclosure of each of which is herein
incorporated by reference in its entirety]. Other suitable
expression vectors will be readily apparent to those of ordinary
skill in the art.
[0633] The subject nucleic acids usually comprise an single open
reading frame encoding a subject polypeptide of interest, however,
in certain embodiments, since the host cell for expression of the
polypeptide of interest may be a eukaryotic cell, e.g., a mammalian
cell, such as a human cell, the open reading frame may be
interrupted by introns. Subject nucleic acid are typically part of
a transcriptional unit which may contain, in addition to the
subject nucleic acid 3' and 5' untranslated regions (UTRs) which
may direct RNA stability, translational efficiency, etc. The
subject nucleic acid may also be part of an expression cassette
which contains, in addition to the subject nucleic acid a promoter,
which directs the transcription and expression of a polypeptide of
interest, and a transcriptional terminator.
[0634] Eukaryotic promoters can be any promoter that is functional
in a eukaryotic host cell, including viral promoters and promoters
derived from eukaryotic genes. Exemplary eukaryotic promoters
include, but are not limited to, the following: the promoter of the
mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl.
Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight,
Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al.,
Nature (London) 290:304-310, 1981); the yeast gall gene sequence
promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)
79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA)
81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter,
Ecdysone-responsive promoter(s), tetracycline-responsive promoter,
and the like. Viral promoters may be of particular interest as they
are generally particularly strong promoters. In certain
embodiments, a promoter is used that is a promoter of the target
pathogen. Promoters for use in the present invention are selected
such that they are functional in the cell type (and/or animal) into
which they are being introduced. In certain embodiments, the
promoter is a CMV promoter.
[0635] In certain embodiments, a subject vector may also provide
for expression of a selectable marker. Suitable vectors and
selectable markers are well known in the art and discussed in
Ausubel, et al, (Short Protocols if Molecular Biology, 3rd ed.,
Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A
Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.).
A variety of different genes have been employed as selectable
markers, and the particular gene employed in the subject vectors as
a selectable marker is chosen primarily as a matter of convenience.
Known selectable marker genes include: the thymidine kinase gene,
the dihydrofolate reductase gene, the xanthine-guanine
phosphoribosyl transferase gene, CAD, the adenosine deaminase gene,
the asparagine synthetase gene, the antibiotic resistance genes,
e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside
phosphotransferase genes), the hygromycin B phosphotransferase
gene, and the like.
[0636] As mentioned above, polypeptides of interest may be fusion
proteins that contain an affinity domain and/or a reporter domain.
Methods for making fusions between a reporter or tag and a GPCR,
for example, at the C- or N-terminus of the GPCR, are well within
the skill of one of skill in the art (e.g. McLean et al, Mol.
Pharma. Mol. Pharmacol. 1999 56:1182-91; Ramsay et al., Br. J.
Pharmacology, 2001, 315-323) and will not be described any further.
It is expressly contemplated that such a fusion protein may contain
a heterologous N-terminal domain (e.g., an epitope tag) fused
in-frame with a GPCR that has had its N-terminal methionine residue
either deleted or substituted with an alternative amino acid. It is
appreciated that a polypeptide of interest may first be made from a
native polypeptide and then operably linked to a suitable
reporter/tag as described above.
[0637] The subject nucleic acids may also contain restriction
sites, multiple cloning sites, primer binding sites, ligatable
ends, recombination sites etc., usually in order to facilitate the
construction of a nucleic acid encoding a polypeptide of
interest.
[0638] b. Host Cells
[0639] The invention further provides host cells comprising a
vector comprising a subject nucleic acid. Suitable host cells
include prokaryotic, e.g., bacterial cells (for example E. coli),
as well as eukaryotic cells e.g. an animal cell (for example an
insect, mammal, fish, amphibian, bird or reptile cell), a plant
cell (for example a maize or Arabidopsis cell), or a fungal cell
(for example, a yeast cell, a S. cerevisiae cell). In certain
embodiments, any cell suitable for expression of a polypeptide of
interest-encoding nucleic acid may be used as a host cell. Usually,
an animal host cell line is used, examples of which are as follows:
monkey kidney cells (COS cells), monkey kidney CV1 cells
transformed by SV40 (COS-7, ATCC CRL 165 l); human embryonic kidney
cells (HEK-293 ["293"], Graham et al. J. Gen Virol. 36:59 (1977));
HEK-293T ["293T"] cells; baby hamster kidney cells (BHK, ATCC CCL
10); chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc.
Natl. Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells
MCB3901 (ATCC CRL-9595); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);
african green monkey kidney cells (VERO-76, ATCC CRL-1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI
cells (Mather et al., Annals N.Y. Acad. Sci 383:44-68 (1982));
NH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
[0640] In certain embodiments, melanophores are used. Melanophores
are skin cells found in lower vertebrates. Relevant materials and
methods will be followed according to the disclosure of U.S. Pat.
No. 5,462,856 and U.S. Pat. No. 6,051,386. These patent disclosures
are herein incorporated by reference in their entirety.
[0641] Additional cell lines will become apparent to those of
ordinary skill in the art, and a wide variety of cell lines are
available from the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209.
C. Screening of Candidate Compounds
[0642] 1. Generic GPCR Screening Assay Techniques
[0643] When a G protein receptor becomes active, it binds to a G
protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of
GTP to the G protein. The G protein then acts as a GTPase and
slowly hydrolyzes the GTP to GDP, whereby the receptor, under
normal conditions, becomes deactivated. However, activated
receptors continue to exchange GDP to GTP. A non-hydrolyzable
analog of GTP, [.sup.35S]GTP.gamma.S, can be used to monitor
enhanced binding to membranes which express activated receptors. It
is reported that [.sup.35S]GTP.gamma.S can be used to monitor G
protein coupling to membranes in the absence and presence of
ligand. An example of this monitoring, among other examples
well-known and available to those in the art, was reported by
Traynor and Nahorski in 1995. A preferred use of this assay system
is for initial screening of candidate compounds because the system
is generically applicable to all G protein-coupled receptors
regardless of the particular G protein that interacts with the
intracellular domain of the receptor.
[0644] 2. Specific GPCR Screening Assay Techniques
[0645] Once candidate compounds are identified using the "generic"
G protein-coupled receptor assay (i.e., an assay to select
compounds that are agonists or inverse agonists), in some
embodiments further screening to confirm that the compounds have
interacted at the receptor site is preferred. For example, a
compound identified by the "generic" assay may not bind to the
receptor, but may instead merely "uncouple" the G protein from the
intracellular domain.
[0646] a. Gs, Gz and Gi.
[0647] Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and
Go), on the other hand, inhibit adenylyl cyclase. Adenylyl cyclase
catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that
couple the Gs protein are associated with increased cellular levels
of cAMP. On the other hand, activated GPCRs that couple Gi (or Gz,
Go) protein are associated with decreased cellular levels of cAMP.
See, generally, "Indirect Mechanisms of Synaptic Transmission,"
Chpt. 8, From Neuron To Brain (3.sup.rd Ed.) Nichols, J. G. et al
eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP
can be utilized to determine if a candidate compound is, e.g., an
inverse agonist to the receptor (i.e., such a compound would
decrease the levels of cAMP). A variety of approaches known in the
art for measuring cAMP can be utilized; in some embodiments a
preferred approach relies upon the use of anti-cAMP antibodies in
an ELISA-based format. Another type of assay that can be utilized
is a whole cell second messenger reporter system assay. Promoters
on genes drive the expression of the proteins that a particular
gene encodes. Cyclic AMP drives gene expression by promoting the
binding of a cAMP-responsive DNA binding protein or transcription
factor (CREB) that then binds to the promoter at specific sites
called cAMP response elements and drives the expression of the
gene. Reporter systems can be constructed which have a promoter
containing multiple cAMP response elements before the reporter
gene, e.g., .beta.-galactosidase or luciferase. Thus, an activated
Gs-linked receptor causes the accumulation of cAMP that then
activates the gene and expression of the reporter protein. The
reporter protein such as .beta.-galactosidase or luciferase can
then be detected using standard biochemical assays (Chen et al.
1995).
[0648] b. Go and Gq.
[0649] Gq and Go are associated with activation of the enzyme
phospholipase C, which in turn hydrolyzes the phospholipid
PIP.sub.2, releasing two intracellular messengers: diacyclglycerol
(DAG) and inositol 1,4,5-triphosphate (IP.sub.3). Increased
accumulation of IP.sub.3 is associated with activation of Gq- and
Go-associated receptors. See, generally, "Indirect Mechanisms of
Synaptic Transmission," Chpt. 8, From Neuron To Brain (3.sup.rd
Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992).
Assays that detect IP.sub.3 accumulation can be utilized to
determine if a candidate compound is, e.g., an inverse agonist to a
Gq- or Go-associated receptor (i.e., such a compound would decrease
the levels of IP.sub.3). Gq-associated receptors can also been
examined using an AP1 reporter assay in that Gq-dependent
phospholipase C causes activation of genes containing AP1 elements;
thus, activated Gq-associated receptors will evidence an increase
in the expression of such genes, whereby inverse agonists thereto
will evidence a decrease in such expression, and agonists will
evidence an increase in such expression. Commercially available
assays for such detection are available.
[0650] 3. GPCR Fusion Protein
[0651] The use of an endogenous, constitutively active GPCR or a
non-endogenous, constitutively activated GPCR, for use in screening
of candidate compounds for the direct identification of inverse
agonists or agonists provides an interesting screening challenge in
that, by definition, the receptor is active even in the absence of
an endogenous ligand bound thereto. Thus, in order to differentiate
between, e.g., the non-endogenous receptor in the presence of a
candidate compound and the non-endogenous receptor in the absence
of that compound, with an aim of such a differentiation to allow
for an understanding as to whether such compound may be an inverse
agonist or agonist or have no affect on such a receptor, in some
embodiments it is preferred that an approach be utilized that can
enhance such differentiation. In some embodiments, a preferred
approach is the use of a GPCR Fusion Protein.
[0652] Generally, once it is determined that a non-endogenous GPCR
has been constitutively activated using the assay techniques set
forth above (as well as others known to the art-skilled), it is
possible to determine the predominant G protein that couples with
the endogenous GPCR. Coupling of the G protein to the GPCR provides
a signaling pathway that can be assessed. In some embodiments it is
preferred that screening take place using a mammalian or a
melanophore expression system, as such a system will be expected to
have endogenous G protein therein. Thus, by definition, in such a
system, the non-endogenous, constitutively activated GPCR will
continuously signal. In some embodiments it is preferred that this
signal be enhanced such that in the presence of, e.g., an inverse
agonist to the receptor, it is more likely that it will be able to
more readily differentiate, particularly in the context of
screening, between the receptor when it is contacted with the
inverse agonist.
[0653] The GPCR Fusion Protein is intended to enhance the efficacy
of G protein coupling with the GPCR. The GPCR Fusion Protein may be
preferred for screening with either an endogenous, constitutively
active GPCR or a non-endogenous, constitutively activated GPCR
because such an approach increases the signal that is generated in
such screening techniques. This is important in facilitating a
significant "signal to noise" ratio; such a significant ratio is
preferred for the screening of candidate compounds as disclosed
herein.
[0654] The construction of a construct useful for expression of a
GPCR Fusion Protein is within the purview of those having ordinary
skill in the art. Commercially available expression vectors and
systems offer a variety of approaches that can fit the particular
needs of an investigator. Important criteria in the construction of
such a GPCR Fusion Protein construct include but are not limited
to, that the GPCR sequence and the G protein sequence both be
in-frame (preferably, the sequence for the endogenous GPCR is
upstream of the G protein sequence), and that the "stop" codon of
the GPCR be deleted or replaced such that upon expression of the
GPCR, the G protein can also be expressed. The GPCR can be linked
directly to the G protein, or there can be spacer residues between
the two (preferably, no more than about 12, although this number
can be readily ascertained by one of ordinary skill in the art).
Based upon convenience, it is preferred to use a spacer. In some
embodiments, it is preferred that the G protein that couples to the
non-endogenous GPCR will have been identified prior to the creation
of the GPCR Fusion Protein construct. Because there are only a few
G proteins that have been identified, it is preferred that a
construct comprising the sequence of the G protein (i.e., a
universal G protein construct, see Example 4(a) below) be available
for insertion of a GPCR sequence therein; this provides for further
efficiency in the context of large-scale screening of a variety of
different GPCRs having different sequences.
[0655] As noted above, activated GPCRs that couple to Gi, Gz and Go
are expected to inhibit the formation of cAMP making assays based
upon these types of GPCRs challenging [i.e., the cAMP signal
decreases upon activation, thus making the direct identification
of, e.g., agonists (which would further decrease this signal)
challenging]. As will be disclosed herein, it has been ascertained
that for these types of receptors, it is possible to create a GPCR
Fusion Protein that is not based upon the GPCR's endogenous G
protein, in an effort to establish a viable cyclase-based assay.
Thus, for example, an endogenous Gi coupled receptor can be fused
to a Gs protein--such a fusion construct, upon expression, "drives"
or "forces" the endogenous GPCR to couple with, e.g., Gs rather
than the "natural" Gi protein, such that a cyclase-based assay can
be established. Thus, for Gi, Gz and Go coupled receptors, in some
embodiments it is preferred that when a GPCR Fusion Protein is used
and the assay is based upon detection of adenylyl cyclase activity,
that the fusion construct be established with Gs (or an equivalent
G protein that stimulates the formation of the enzyme adenylyl
cyclase).
TABLE-US-00003 TABLE B Effect on IP.sub.3 Effect on cAMP
Accumulation Effect on Production upon cAMP upon Activation
Activation Production of GPCR (i.e., of GPCR (i.e., upon Effect on
IP.sub.3 constitutive constitutive contact Accumulation activation
or activation or with an upon contact G agonist agonist Inverse
with an protein binding) binding) Agonist Inverse Agonist Gs
Increase N/A Decrease N/A Gi Decrease N/A Increase N/A Gz Decrease
N/A Increase N/A Go Decrease Increase Increase Decrease Gq N/A
Increase N/A Decrease
[0656] Equally effective is a G Protein Fusion construct that
utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. In
some embodiments a preferred fusion construct can be accomplished
with a Gq Protein wherein the first six (6) amino acids of the
G-protein .alpha.-subunit ("G.alpha.q") is deleted and the last
five (5) amino acids at the C-terminal end of G.alpha.q is replaced
with the corresponding amino acids of the G.alpha. of the G protein
of interest. For example, a fusion construct can have a Gq (6 amino
acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi
Fusion Construct". This fusion construct will force the endogenous
Gi coupled receptor to couple to its non-endogenous G protein, Gq,
such that the second messenger, for example, inositol triphosphate
or diacylgycerol, can be measured in lieu of cAMP production.
[0657] 4. Co-Transfection of a Target Gi Coupled GPCR with a
Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
[0658] A Gi coupled receptor is known to inhibit adenylyl cyclase,
and, therefore, decreases the level of cAMP production, which can
make the assessment of cAMP levels challenging. In certain
embodiments, an effective technique in measuring the decrease in
production of cAMP as an indication of activation of a receptor
that predominantly couples Gi upon activation can be accomplished
by co-transfecting a signal enhancer, e.g., a non-endogenous,
constitutively activated receptor that predominantly couples with
Gs upon activation (e.g., TSHR-A623I; see infra), with the Gi
linked GPCR. As is apparent, activation of a Gs coupled receptor
can be determined based upon an increase in production of cAMP.
Activation of a Gi coupled receptor leads to a decrease in
production cAMP. Thus, the co-transfection approach is intended to
advantageously exploit these "opposite" affects. For example,
co-transfection of a non-endogenous, constitutively activated Gs
coupled receptor (the "signal enhancer") with expression vector
alone provides a baseline cAMP signal (i.e., although the Gi
coupled receptor will decrease cAMP levels, this "decrease" will be
relative to the substantial increase in cAMP levels established by
constitutively activated Gs coupled signal enhancer). By then
co-transfecting the signal enhancer with the "target receptor", an
inverse agonist of the Gi coupled target receptor will increase the
measured cAMP signal, while an agonist of the Gi coupled target
receptor will decrease this signal.
[0659] Candidate compounds that are directly identified using this
approach should be assessed independently to ensure that these do
not target the signal enhancing receptor (this can be done prior to
or after screening against the co-transfected receptors).
D. Medicinal Chemistry
[0660] Candidate Compounds
[0661] Any molecule known in the art can be tested for its ability
to modulate (increase or decrease) the activity of a GPCR of the
present invention. For identifying a compound that modulates
activity, candidate compounds can be directly provided to a cell
expressing the receptor.
[0662] This embodiment of the invention is well suited to screen
chemical libraries for molecules which modulate, e.g., inhibit,
antagonize, or agonize, the amount of, or activity of, a receptor.
The chemical libraries can be peptide libraries, peptidomimetic
libraries, chemically synthesized libraries, recombinant, e.g.,
phage display libraries, and in vitro translation-based libraries,
other non-peptide synthetic organic libraries, etc. This embodiment
of the invention is also well suited to screen endogenous candidate
compounds comprising biological materials, including but not
limited to plasma- and, tissue extracts, and to screen libraries of
endogenous compounds known to have biological activity.
[0663] In some embodiments, direct identification of candidate
compounds is conducted in conjunction with compounds generated via
combinatorial chemistry techniques, whereby thousands of compounds
are randomly prepared for such analysis. The candidate compound may
be a member of a chemical library. This may comprise any convenient
number of subject members, for example tens to hundreds to thousand
to millions of suitable compounds, for example peptides, peptoids
and other oligomeric compounds (cyclic or linear), and
template-based smaller molecules, for example benzodiazepines,
hydantoins, biaryls, carbocyclic and polycyclic compounds (e.g.,
naphthalenes, phenothiazines, acridines, steroids etc.),
carbohydrate and amino acid derivatives, dihydropyridines,
benzhydryls and heterocycles (e.g., trizines, indoles,
thiazolidines etc.). The numbers quoted and the types of compounds
listed are illustrative, but not limiting. Preferred chemical
libraries comprise chemical compounds of low molecular weight and
potential therapeutic agents.
[0664] Exemplary chemical libraries are commercially available from
several sources (ArQule, Tripos/PanLabs, ChemDesign,
Pharmacopoeia). In some cases, these chemical libraries are
generated using combinatorial strategies that encode the identity
of each member of the library on a substrate to which the member
compound is attached, thus allowing direct and immediate
identification of a molecule that is an effective modulator. Thus,
in many combinatorial approaches, the position on a plate of a
compound specifies that compound's composition. Also, in one
example, a single plate position may have from 1-20 chemicals that
can be screened by administration to a well containing the
interactions of interest. Thus, if modulation is detected, smaller
and smaller pools of interacting pairs can be assayed for the
modulation activity. By such methods, many candidate molecules can
be screened.
[0665] Many diversity libraries suitable for use are known in the
art and can be used to provide compounds to be tested according to
the present invention. Alternatively, libraries can be constructed
using standard methods. Further, more general, structurally
constrained, organic diversity (e.g., nonpeptide) libraries, can
also be used. By way of example, a benzodiazepine library (see
e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)
may be used.
[0666] In another embodiment of the present invention,
combinatorial chemistry can be used to identify modulators of the
GPCRs of the present invention. Combinatorial chemistry is capable
of creating libraries containing hundreds of thousands of
compounds, many of which may be structurally, similar. While high
throughput screening programs are capable of screening these vast
libraries for affinity for known targets, new approaches have been
developed that achieve libraries of smaller dimension but which
provide maximum chemical diversity. (See e.g., Matter, 1997,
Journal of Medicinal Chemistry 40:1219-1229).
[0667] One method of combinatorial chemistry, affinity
fingerprinting, has previously been used to test a discrete library
of small molecules for binding affinities for a defined panel of
proteins. The fingerprints obtained by the screen are used to
predict the affinity of the subject library members for other
proteins or receptors of interest (in the instant invention, the
receptors of the present invention). The fingerprints are compared
with fingerprints obtained from other compounds known to react with
the protein of interest to predict whether the library compound
might similarly react. For example, rather than testing every
ligand in a large library for interaction with a complex or protein
component, only those ligands having a fingerprint similar to other
compounds known to have that activity could be tested. (See, e.g.,
Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995,
Affinity fingerprinting, Pharmaceutical Manufacturing
International. 8:25-28; and Kauvar, Toxic-Chemical Detection by
Pattern Recognition in New Frontiers in Agrochemical Immunoassay,
D. Kurtz. L. Stanker and J. H. Skerritt. Editors, 1995, AOAC:
Washington, D.C., 305-312).
[0668] In some embodiments, the candidate compound is a
polypeptide. In some preferred embodiments, the candidate compound
is a small molecule.
[0669] Candidate Compounds Identified as Modulators
[0670] Generally, the results of such screening will be compounds
having unique core structures; thereafter, these compounds may be
subjected to additional chemical modification around a preferred
core structure(s) to further enhance the medicinal properties
thereof. Such techniques are known to those in the art and will not
be addressed in detail in this patent document.
[0671] In certain embodiments, a modulator of the invention is
orally active. A number of computational approaches available to
those of ordinary skill in the art have been developed for
prediction of oral bioavailability of a drug [Ooms et al., Biochim
Biophys Acta (2002) 1587:118-25; Clark & Grootenhuis, Curr
OpinDrug Discov Devel (2002) 5:382-90; Cheng et al., J Comput Chem
(2002) 23:172-83; Norinder & Haeberlein, Adv Drug Deliv Rev
(2002) 54:291-313; Matter et al., Comb Chem High Throughput Screen
(2001) 4:453-75; Podlogar & Muegge, Curr Top Med Chem (2001)
1:257-75; the disclosure of each of which is herein incorporated by
reference in its entirety). Furthermore, positron emission
tomography (PET) has been successfully used by a number of groups
to obtain direct measurements of drug distribution, including an
assessment of oral bioavailability, in the mammalian body following
oral administration of the drug, including non-human primate and
human body [Noda et al., J Nucl Med (2003) 44:105-8; Gulyas et al.,
Eur J Nucl Med Mol Imaging (2002) 29:1031-8; Kanerva et al.,
Psychopharmacology (1999) 145:76-81; the disclosure of each of
which is herein incorporated by reference in its entirety]. In some
embodiments, a modulator of the invention is orally active.
[0672] In certain embodiments, modulator of the invention which is
orally active is not able to cross the blood-brain barrier. In
certain embodiments, modulator of the invention which is orally
active is able to cross the blood-brain barrier. A number of
computational approaches available to those of ordinary skill in
the art have been developed for prediction of the permeation of the
blood-brain barrier [Ooms et al., Biochim Biophys Acta (2002)
1587:118-25; Clark & Grootenhuis, Curr OpinDrug Discov Devel
(2002) 5:382-90; Cheng et al., J Comput Chem (2002) 23:172-83;
Norinder & Haeberlein, Adv Drug Deliv Rev (2002) 54:291-313;
Matter et al., Comb Chem High Throughput Screen (2001) 4:453-75;
Podlogar & Muegge, Curr Top Med Chem (2001) 1:257-75; the
disclosure of each of which is herein incorporated by reference in
its entirety). A number of in vitro methods have been developed to
predict blood-brain barrier permeability of drugs [Lohmann et al.,
J Drug Target (2002) 10:263-76; Hansen et al., J Pharm Biomed Anal
(2002) 27:945-58; Otis et al., J Pharmocol Toxicol Methods (2001)
45:71-7; Dehouck et al, J Neurochem (1990) 54:1798-801; the
disclosure of each of which is herein incorporated by reference in
its entirety]. Furthermore, a number of strategies have been
developed to enhance drug delivery across the blood-brain barrier
[Scherrmann, Vascul Pharmacol (2002) 38:349-54; Pardridge, Arch
Neurol (2002) 59:35-40; Pardridge, Neuron (2002) 36:555-8; the
disclosure of each of which is hereby incorporated by reference in
its entirety]. Finally, positron emission tomography (PET) has been
successfully used by a number of groups to obtain direct
measurements of drug distribution, including that within brain, in
the mammalian body, including non-human primate and human body
[Noda et al., J Nucl Med (2003) 44:105-8; Gulyas et al., Eur J Nucl
Med Mol Imaging (2002) 29:1031-8; Kanerva et al.,
Psychopharmacology (1999) 145:76-81; the disclosure of each of
which is herein incorporated by reference in its entirety].
[0673] In some embodiments, said modulator is selective for GPR84,
wherein a modulator selective for GPR84 is understood to refer to a
modulator having selectivity for GPR84 over one or more closely
related receptors, such as GPR88 (GenBank.RTM. Accession No.
NP.sub.--071332). In certain embodiments, a GPR84 selective
modulator is a GPR84 selective agonist having a selectivity for
GPR84 over GPR88 of at least about 100-fold. In some preferred
embodiments, GPR84 is human GPR84.
[0674] In some embodiments, the modulator is an agonist with an
EC.sub.50 of less than about 10 .mu.M, of less than about 1 .mu.M,
of less than about 100 nM, of less than about 10 nM, or of less
than about 1 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than a value selected from the interval
of about 10 nM to 10 .mu.M. In some embodiments, modulator is an
agonist with an EC.sub.50 of less than a value selected from the
interval of about 10 nM to 1 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 of less than a value
selected from the interval of about 10 nM to 100 nM. In some
embodiments, the modulator is an agonist with an EC.sub.50 of less
than a value selected from the interval of about 1 nM to 10 .mu.M.
In some embodiments, modulator is an agonist with an EC.sub.50 of
less than a value selected from the interval of about 1 nM to 1
.mu.M. In some embodiments; the modulator is an agonist with an
EC.sub.50 of less than a value selected from the interval of about
1 nM to 100 nM. In some embodiments, the modulator is an agonist
with an EC.sub.50 of less than a value selected from the interval
of about 1 nM to 10 nM. In some embodiments, the modulator is an
agonist with an EC.sub.50 of less than about 10 .mu.M, of less than
about 1 .mu.M, of less than about 100 nM, of less than about 10 nM,
or of less than about 1 nM in GTP.gamma.S binding assay carried out
with membrane from transfected CHO cells, or in pigment aggregation
assay carried out in transfected melanophores, or in cAMP assay
carried out in transfected 293 cells, or in IP.sub.3 assay carried
out in transfected 293 cells comprising Gq(del)/Gi chimeric G
protein, wherein the transfected CHO cells or the transfected
melanophore cells or the transfected 293 cells express a
recombinant GPR84 having an amino acid sequence selected from SEQ.
ID NO:20, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. In some
embodiments, the recombinant GPR84 has the amino acid sequence of
SEQ ID NO:20. In some embodiments, the recombinant GPR84 has the
amino acid sequence of SEQ ID NO:2. In some embodiments, the
modulator is an agonist with an EC.sub.50 of less than about 10
.mu.M, of less than about 1 .mu.M, of less than about 100 nM, of
less than about 10 nM, or of less than about 1 nM in said assay. In
some embodiments, said modulator is an agonist with an EC.sub.50 of
less than 10 .mu.M in said assay, of less than 9 .mu.M in said
assay, of less than 8 .mu.M in said assay, of less than 7 .mu.M in
said assay, of less than 6 .mu.M in said assay, of less than 5
.mu.M in said assay, of less than 4 .mu.M in said assay, of less
than 3 .mu.M in said assay, of less than 2 .mu.M in said assay, of
less than 1 .mu.M in said assay, of less than 900 nM in said assay,
of less than 800 nM in said assay, of less than 700 nM in said
assay, of less than 600 nM in said assay, of less than 500 nM in
said assay, of less than 400 nM in said assay, of less than 300 nM
in said assay, of less than 200 nM in said assay, of less than 100
nM in said assay, of less than 90 nM in said assay, of less than 80
nM in said assay, of less than 70 nM in said assay, of less than 60
nM in said assay, of less than 50 nM in said assay, of less than 40
nM n said assay, of less than 30 nM in said assay, of less than 20
nM in said assay, of less than 10 nM, of less than 9 nM in said
assay, of less than 8 nM in said assay, of less than 7 nM in said
assay, of less than 6 nM in said assay, of less than 5 nM in said
assay, of less than 4 nM n said assay, of less than 3 nM in said
assay, of less than 2 nM in said assay, or of less than 1 nM in
said assay. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 10 nM to 10 .mu.M. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 10 nM to 1 .mu.M.
In some embodiments, the modulator is an agonist with an EC.sub.50
in said assay of less than a value selected from the interval of
about 10 nM to 100 nM. In some embodiments, the modulator is an
agonist with an EC.sub.50 in said assay of less than a value
selected from the interval of about 1 nM to 10 .mu.M. In some
embodiments, the modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of about 1 nM
to 1 .mu.M. In some embodiments, the modulator is an agonist with
an EC.sub.50 in said assay of less than a value selected from the
interval of about 1 nM to 100 nM. In some embodiments, the
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of about 1 nM to 10 nM.
E. Compounds of the Invention
[0675] Certain aspects of the present invention pertain to a
compound selected from Table 1. The compounds in Table 1 can be
obtained from commercial sources or can be prepared by one of skill
in the art by synthetic methods.
TABLE-US-00004 TABLE 1 Cmpd No. Chemical Structure Chemical Name 1
##STR00001## 2,5-Dihydroxy-3- undecyl- [1,4]benzoquinone 2
##STR00002## Icosa-5,8,11,14- tetraynoic acid 3 ##STR00003##
5S,6R-Dihydroxy- icosa-7,9,11,14- tetraenoic acid
[0676] Additionally, compounds of the invention, including those
illustrated in Table 1, encompass all pharmaceutically acceptable
salts, solvates, and hydrates thereof. See, e.g., Berge et al,
Journal of Pharmaceutical Sciences (1977) 66:1-19; and Polymorphism
in Pharmaceutical Solids (1999) Brittain, ed., Marcel Dekker, Inc.;
the disclosure of each of which is herein incorporated by reference
in its entirety.
F. Pharmaceutical Compositions
[0677] Compounds of the invention can be formulated into
pharmaceutical compositions using techniques well known in the art.
By way of example and not limitation, compounds of the invention
include but are not limited to modulators of a mammalian GPR84
(including but not limited to agonists, partial agonists,
antagonists and inverse agonists of a mammalian GPR84) and ligands
of a mammalian GPR84.
[0678] The invention provides methods of treatment (and prevention)
by administration to a subject in need of said treatment (or
prevention) a therapeutically effect amount of a modulator of the
invention [also see, e.g., PCT Application Number PCT/IB02/01461
published as WO 02/066505 on 29 Aug. 2002; the disclosure of each
of which is herein incorporated by reference in its entirety]. In a
preferred aspect, the modulator is an agonist. In a preferred
aspect, the modulator is substantially purified. The subject is a
mammal including, but not limited to cows, pigs, horses, chickens,
non-human primates, cats, dogs, rabbits, rats, mice, etc., and is
preferably a human.
[0679] Modulators of the invention can be administered to non-human
mammals [see Examples, infra] and/or humans, alone or in
pharmaceutical compositions where they are mixed with suitable
carriers or excipient(s) using techniques well known to those in
the art. Suitable pharmaceutically-acceptable carriers are
available to those in the art; for example, see Remington's
Pharmaceutical Sciences, 16.sup.th Edition, 1980, Mack Publishing
Co., (Oslo et al., eds.).
[0680] The pharmaceutical composition is then provided at a
therapeutically effective dose. A therapeutically effective dose
refers to that amount of a modulator sufficient to result in
prevention or amelioration of symptoms or physiological status of a
disorder as determined illustratively and not by limitation by the
methods described herein, wherein the prevention or amelioration of
symptoms or physiological status of a disorder includes but is not
limited to inhibiting atherogenesis, treating or preventing
atherosclerois or an atherosclerotic disease, and treating or
preventing a condition related to MCP-1 expression.
[0681] It is expressly considered that the modulators of the
invention may be provided alone or in combination with other
pharmaceutically or physiologically acceptable compounds. Other
compounds for the treatment of disorders of the invention, wherein
the treatment of disorders of the invention includes but is not
limited to inhibiting atherogenesis, treating or preventing
atherosclerois or an atherosclerotic disease, and treating or
preventing a condition related to MCP-1 expression.
[0682] Combination Therapy
[0683] While the compounds of the invention can be administered as
the sole active pharmaceutical agent (i.e., mono-therapy),
compounds of the invention can also be used in combination with
other pharmaceutical agents (i.e., combination-therapy) for the
treatment of the diseases/conditions/disorders described herein.
Therefore, another aspect of the present invention includes methods
of treatment comprising administering to a subject in need of
treatment a therapeutically effective amount of an agonist of the
present invention in combination with one or more additional
pharmaceutical agent as described herein.
[0684] It will be understood that the scope of combination-therapy
of the compounds of the present invention with other pharmaceutical
agents is not limited to those listed herein, supra or infra, but
includes in principle any combination with any pharmaceutical agent
or pharmaceutical composition useful for the treatment diseases,
conditions or disorders of the present invention in a subject.
[0685] Some embodiments of the present invention include methods of
treatment of a disease, disorder or condition as described herein
comprising administering to a subject in need of such treatment a
therapeutically effect amount or dose of a compound of the present
invention in combination with at least one pharmaceutical agent
selected from the group consisting of: an HMG-CoA reductase
inhibitor, cholesterol-lowering drugs (for example, fibrates that
include: fenofibrate, bezafibrate, gemfibrozil, clofibrate and the
like; bile acid sequestrants which include: cholestyramine,
colestipol and the like; and niacin and analogs thereof or other
such compound having agonist or partial agonist activity at the
nicotinic acid receptor GPR109A), anti-lipolytic agents (for
example, niacin and analogs thereof or other such compound having
agonist or partial agonist activity at the nicotinic acid receptor
GPR109A), and inflammation-lowering drugs. Compounds having agonist
or partial agonist activity at the nicotinic acid receptor GPR109A
are known in the art and include compounds disclosed in
PCT/US2003/032174 (published as WO 2004/032938), PCT/US2004/018389
(published as WO 2005/011677), PCT/US2004/038920 (published as WO
2005/051937), and PCT/US2005/046599 (published as WO 2006/069242).
In certain embodiments, the compounds having agonist or partial
agonist activity at the nicotinic acid receptor GPR109A are orally
active. In some embodiments, methods of the present invention
include compounds of the present invention and the pharmaceutical
agents are administered separately. In further embodiments,
compounds of the present invention and the pharmaceutical agents
are administered together.
[0686] Suitable pharmaceutical agents that can be used in
conjunction with compounds of the present invention include the
HMG-CoA reductase inhibitors. In certain embodiments, the HMG-CoA
reductase inhibitor is orally active. The HMG-CoA reductase
inhibitors are agents also referred to as Statin compounds that
belong to a class of drugs that lower blood cholesterol levels by
inhibiting hydroxymethylglutalyl CoA (HMG-CoA) reductase. HMG-CoA
reductase is the rate-limiting enzyme in cholesterol biosynthesis.
The statins lower plasma LDL-cholesterol concentrations by
upregulating the activity of LDL receptors and are responsible for
clearing LDL from the blood. Some representative examples the
statin compounds include rosuvastatin, pravastatin and its sodium
salt, simvastatin, lovastatin, atorvastatin, fluvastatin,
cerivastatin, rosuvastatin, pitavastatin, BMS's "superstatin", and
HMG-CoA reductase inhibitors known in the art.
[0687] Other treatments for one or more of the diseases cited
herein include the use of pharmaceutical agents known in the art
belonging to the classes of drugs referred to, but not limited to,
the following: acyl CoA cholesterol acetyltransferase inhibitors
(for example, Ezetimibe, eflucimibe, and like compounds),
cholesterol absorption inhibitors (for example, ezetimibe,
pamaqueside and like compounds), cholesterol ester transfer protein
inhibitors (for example, CP-529414, JTT-705, CETi-1, and like
compounds), microsomal triglyceride transfer protein inhibitors
(for example, implitapide, and like compounds), cholesterol
modulators (for example, NO-1886, and like compounds), bile acid
modulators (for example, GT103-279 and like compounds) and squalene
synthase inhibitors.
[0688] Additional suitable pharmaceutical agent that can be used in
conjunction with compounds of the present invention comprises the
combination of ezetimibe and simvastatin.
[0689] Squalene synthesis inhibitors belong to a class of drugs
that lower blood cholesterol levels by inhibiting synthesis of
squalene. Examples of the squalene synthesis inhibitors include
(S)-.alpha.-[Bis[2,2-dimethyl-1-oxopropoxy)methoxy]phosphinyl]-3-phenoxyb-
enzenebutanesulfonic acid, mono potassium salt (BMS-188494) and
squalene synthesis inhibitors known in the art.
[0690] Other suitable pharmaceutical agents that can be used in
conjunction with compounds of the present invention include
nonsteroidal anti-inflammatory drug (e.g., celecoxib, rofecoxib),
aminosalicylate (e.g., sulfasalazine, mesalamine, azodisalicylate,
balsalazide), hydroxychoroquine, aurothioglucose,
sodium-aurothiomalate, auranofin, penicillamine, leflunomide,
corticosteroid (e.g., prednisone, prednisolone, budesonide,
hydrocortisone, methylprednisolone), immunosuppressant (e.g.,
azathioprine, cyclosporine, methotrexate, 6-mercaptopurine), and
biologic agent (e.g., etanercept, infliximab, or other such
biologic agent for neutralizing tumor necrosis factor alpha
(TNF.alpha.) activity; e.g., adiponectin or an analog thereof, or
other such biologic agent having agonist or partial agonist
activity at adiponectin receptor AdipoR1 or AdipoR2; e.g.,
interleukin-10 (IL-10); e.g., CTLA4-Ig (inclusive of abatacept)).
Additionally, it is expressly contemplated that the modulators of
the invention, e.g. agonists and partial agonists of the invention,
may be provided alone or in combination with a phosphodiesterase
(PDE) inhibitor (inclusive of an inhibitor selective for type 4
cAMP-specific PDE (PDE4), e.g. roflumilast; an inhibitor selective
for PDE4B; and an inhibitor selective for PDE4B2). PDE4 inhibitors
are known in the art and include compounds disclosed in U.S. Pat.
Nos. 7,087,634, 7,087,625, 7,022,696, 6,953,810, 6,919,353 and
6,894,041.
[0691] In particular embodiment, a compound of the invention (e.g.
an agonist or partial agonist of a mammalian GPR84) is used in
combination with an HMG-CoA reductase inhibitor (i.e. a statin), an
agonist or partial agonist of the nicotinic acid receptor GPR109A
(e.g. niacin), adiponectin or an orally active analog thereof
(including orally active agonists or partial agonists of
adiponectin receptor AdipoR1 or AdipoR2), methotrexate, a
phosphodiesterase (PDE) inhibitor (inclusive of an inhibitor
selective for type 4 cAMP-specific PDE (PDE4), e.g. roflumilast, or
an inhibitor selective for PDE4B, or an inhibitor selective for
PDE4B2), a biologic agent for neutralizing tumor necrosis factor
alpha (TNF.alpha.) activity (such as etanercept and infliximab), or
CTLA4-Ig.
[0692] In accordance to an aspect of the present invention, a
compound of the present invention can be used in combination with a
pharmaceutical agent or agents belonging to one or more of the
classes of drugs cited herein. In certain embodiments, it is
expressly contemplated that the pharmaceutical agent belonging to
one or more of the classes of drugs cited herein is orally
active.
[0693] Routes of Administration
[0694] Suitable routes of administration include oral, nasal,
rectal, transmucosal, transdermal, or intestinal administration,
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal,
intrapulmonary (inhaled) or intraocular injections using methods
known in the art. Other particularly preferred routes of
administration are aerosol and depot formulation. Sustained release
formulations, particularly depot, of the invented medicaments are
expressly contemplated. In certain embodiments, route of
administration is oral.
[0695] Composition/Formulation
[0696] Pharmaceutical or physiologically acceptable compositions
and medicaments for use in accordance with the present invention
may be formulated in a conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries. Proper formulation is dependent upon the route of
administration chosen.
[0697] Certain of the medicaments described herein will include a
pharmaceutically or physiologically acceptable carrier and at least
one modulator of the invention. For injection, the agents of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiological saline buffer such as a
phosphate or bicarbonate buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0698] Pharmaceutical or physiologically acceptable preparations
that can be taken orally include push-fit capsules made of gelatin,
as well as soft, sealed capsules made of gelatin and a plasticizer,
such as glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with fillers such as lactose,
binders such as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers may be added. All
formulations for oral administration should be in dosages suitable
for such administration.
[0699] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0700] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs
for a nebulizer, with the use of a suitable gaseous propellant,
e.g., carbon dioxide. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, e.g., gelatin, for use
in an inhaler or insufflator, may be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or
starch.
[0701] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage for, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspension, solutions or emulsions in aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0702] Pharmaceutical or physiologically acceptable formulations
for parenteral administration include aqueous solutions of the
active compounds in water-soluble form. Aqueous suspension may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0703] Alternatively, the active ingredient may be in powder or
lyophilized form for constitution with a suitable vehicle, such as
sterile pyrogen-free water, before use.
[0704] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0705] In a particular embodiment, the compounds can be delivered
via a controlled release system. In one embodiment, a pump may be
used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201-240; Buchwald et al., 1980, Surgery 88:507-516; Saudek et
al., 1989, N. Engl. J. Med. 321:574-579). In another embodiment,
polymeric materials can be used (Medical Applications of Controlled
Release, Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974;
Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball, eds., Wiley, New York, 1984; Ranger
and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy
et al., 1985, Science 228:190-192; During et al., 1989, Ann.
Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).
Other controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533).
[0706] Additionally, the compounds may be delivered using a
sustained-release system, such as semipermeable matrices of solid
hydrophobic polymers containing the therapeutic agent. Various
sustained release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the compounds for a few
weeks up to over 100 days.
[0707] Depending on the chemical nature and the biological
stability of the therapeutic reagent, additional strategies for
modulator stabilization may be employed.
[0708] The pharmaceutical or physiologically acceptable
compositions also may comprise suitable solid or gel phase carriers
or excipients. Examples of such carriers or escipients include but
are not limited to calcium carbonate, calcium phosphate, various
sugars, starches, cellulose derivatives, gelatin, and polymers such
as polyethylene glycols.
[0709] Effective Dosage
[0710] Pharmaceutical or physiologically acceptable compositions
suitable for use in the present invention include compositions
wherein the active ingredients are contained in an effective amount
to achieve their intended purpose. More specifically, a
therapeutically effective amount (of a compound of the invention,
e.g. an agonist or partial agonist of a mammalian GPR84, or of a
combination comprising said compound) means an amount effective to
prevent development of or to alleviate the existing symptoms of the
subject being treated. Determination of the effective amounts is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
[0711] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
or encompasses a concentration point or range shown to decrease
MCP-1 expression or to increase ABCA1 expression in
monocyte/macrophage assay. Such information can be used to more
accurately determine useful doses in humans.
[0712] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms in a patient.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the test population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the test population). The
dose ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio between LD.sub.50 and
ED.sub.50. Compounds that exhibit high therapeutic indices are
preferred.
[0713] The data obtained from these cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50, with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the subject physician in
view of the patient's condition. (See, e.g., Fingl et al., 1975, in
"The Pharmacological Basis of Therapeutics", Ch. 1).
[0714] Dosage amount and interval may be adjusted subjectly to
provide plasma levels of the active compound which are sufficient
to prevent or treat a disorder of the invention, depending on the
particular situation. Dosages necessary to achieve these effects
will depend on subject characteristics and route of
administration.
[0715] Dosage intervals can also be determined using the value for
the minimum effective concentration. Compounds should be
administered using a regimen that maintains plasma levels above the
minimum effective concentration for 10-90% of the time, preferably
between 30-99%, and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0716] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration, and
the judgement of the prescribing physician.
[0717] A preferred dosage range for the amount of a modulator of
the invention, which can be administered on a daily or regular
basis to achieve desired results is 0.1-100 mg/kg body mass. Other
preferred dosage range is 0.1-30 mg/kg body mass. Other preferred
dosage range is 0.1-10 mg/kg body mass. Other preferred dosage
range is 0.1-3.0 mg/kg body mass. Of course, these daily dosages
can be delivered or administered in small amounts periodically
during the course of a day. It is noted that these dosage ranges
are only preferred ranges and are not meant to be limiting to the
invention. Said desired results include, but are not limited to,
inhibiting atherogenesis, treating or preventing atherosclerois or
an atherosclerotic disease, and treating or preventing a condition
related to MCP-1 expression.
[0718] G. Methods of Treatment
[0719] The invention is drawn inter alia to methods including, but
not limited to, methods of inhibiting atherogenesis, of treating or
preventing atherosclerois or an atherosclerotic disease, and of
treating or preventing a condition related to MCP-1 expression,
comprising administering to a subject in need of said inhibiting,
treating or preventing with a modulator of the invention. In some
embodiments, the preventing or treating atherosclerosis or an
atherosclerotic disease is preventing or treating atherosclerosis.
In some embodiments, the preventing or treating atherosclerosis or
an atherosclerotic disease is preventing or treating an
atherosclerotic disease. In some embodiments, the modulator is an
agonist. In some embodiments, the agonist is a partial agonist. In
some embodiments, said modulator is orally active. In some
embodiments, said orally active modulator is further able to cross
the blood-brain barrier. In some embodiments, said orally active
modulator is not able to cross the blood-brain barrier. In some
embodiments, the modulator is administered to the subject in a
pharmaceutical composition. In some embodiments, the modulator is
provided to the subject in a pharmaceutical composition. In some
embodiments, the modulator is provided to the subject in a
pharmaceutical composition that is taken orally. In some
embodiments, the subject is a non-human mammal. In some
embodiments, the subject is a mammal. In certain embodiments, the
mammal is a non-human primate. In certain preferred embodiments,
the subject or mammal is a human.
[0720] In some embodiments, the subject is in need of having
atherogenesis inhibited. In some embodiments, the subject is in
need of preventing or treating atherosclerois or an atherosclerotic
disease. In some embodiments, the subject is in need of preventing
or treating a condition related to MCP-1 expression.
[0721] In some embodiments, the atherosclerotic disease is selected
from the group consisting of coronary artery disease, myocardial
infarction, peripheral artery disease, and ischemic stroke.
Atherosclerotic diseases may be included in embodiments of the
invention individually or in any combination.
[0722] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, an atherosclerotic disease, rheumatoid arthritis,
Crohn's disease, and multiple sclerosis. Conditions related to
MCP-1 expression may be included in embodiments of the invention
individually or in any combination.
[0723] In some embodiments, the condition related to MCP-1
expression is selected from the group consisting of
atherosclerosis, arthritis (inclusive of rheumatoid arthritis,
psoriatic arthritis and osteoarthritis), inflammatory bowel disease
(inclusive of ulcerative colitis and Crohn's disease), a
metabolic-related disorder (inclusive of insulin resistance,
diabetes (inclusive of Type 1 diabetes and Type 2 diabetes),
metabolic syndrome, obesity, lower than normal HDL-cholesterol,
hypertension and hyperlipidemia), ischemic heart disease,
congestive heart failure, osteoporosis, restenosis, septic shock,
ischemia/reperfusion injury, disseminated intravascular
coagulation, psoriasis, allergic inflammation, asthma, systemic
lupus erythematosus, acute transplant rejection, chronic hepatitis,
interstitial lung disease, idiopathic pulmonary fibrosis,
bronchiolitis obliterans syndrome, interstitial nephritis, hepatic
steatosis, chronic obstructive pulmonary disease, higher than
normal osteoclastogenesis, and a neuroinflammatory disorder
(inclusive of multiple sclerosis, ischemic stroke, Parkinson's
disease, prion-associated disease, excitotoxic injury, mild
cognitive impairment (MCI) and Alzheimer's disease). Conditions
related to MCP-1 expression may be included in embodiments of the
invention individually or in any combination.
[0724] In some embodiments, the condition related to MCP-1
expression is an inflammation-related disease or disorder. In some
embodiments, the condition related to MCP-1 is an
inflammation-related disease or disorder selected from the group
consisting of atherosclerosis, arthritis (inclusive of rheumatoid
arthritis, psoriatic arthritis and osteoarthritis), inflammatory
bowel disease (inclusive of ulcerative colitis and Crohn's
disease), a metabolic-related disorder (inclusive of insulin
resistance, diabetes (inclusive of Type 1 diabetes and Type 2
diabetes), metabolic syndrome, obesity, lower than normal
HDL-cholesterol, hypertension, and hyperlipidemia), ischemic heart
disease, congestive heart failure, osteoporosis, restenosis, septic
shock, ischemia/reperfusion injury, disseminated intravascular
coagulation, psoriasis, allergic inflammation, asthma, systemic
lupis erythematosus, acute transplant rejection, chronic hepatitis,
interstitial lung disease, idiopathic pulmonary fibrosis,
bronchiolitis obliterans syndrome, interstitial nephritis, hepatic
steatosis, chronic obstructive pulmonary disease, higher than
normal osteoclastogenesis, and a neuroinflammatory disorder
(inclusive of multiple sclerosis, ischemic stroke, Parkinson's
disease, prion-associated disease, excitotoxic injury, mild
cognitive impairment (MCI) and Alzheimer's disease). Conditions
related to MCP-1 expression that are inflammation-related disorders
may be included in embodiments of the invention individually or in
any combination.
[0725] I. Other Utility
[0726] Agents that modulate (i.e., increase, decrease, or block)
receptor functionality of a GPCR of the invention such as a
mammalian GPR84 may be identified by contacting a candidate
compound with the GPCR and determining the effect of the candidate
compound on receptor functionality. The selectivity of a compound
that modulates the functionality of a mammalian GPR84 such as human
GPR84 can be evaluated by comparing its effects on GPR84 to its
effects on one or more other G protein-coupled receptors. In
certain embodiments, a GPR84 selective compound is a GPR84
selective agonist having a selectivity for GPR84 over GPR88
(GenBank.RTM. Accession No. NP.sub.--071332) of at least about
100-fold. Following identification of compounds that modulate GPR84
functionality, such candidate compounds may be further tested in
other assays including, but not limited to, in vivo models, in
order to confirm or quantitate their activity. Modulators of GPR84
functionality are therapeutically useful, e.g., in treatment of
diseases and physiological conditions in which normal or aberrant
GPR84 functionality is involved.
[0727] Agents that are ligands of a GPCR of the invention such as a
mammalian GPR84 may be identified by contacting a candidate
compound with the GPCR (or with a recombinant host cell that
comprises the mammalian GPR84 or with a host cell membrane that
comprises the mammalian GPCR) and determining whether the candidate
compound binds to the receptor. The selectivity of a compound that
binds to a mammalian GPR84 such as human GPR84 can be evaluated by
comparing its binding to GPR84 to its binding to one or more other
G protein-coupled receptors. Ligands that are modulators of GPR84
receptor functionality are therapeutically useful in treatment of
diseases and physiological conditions in which normal or aberrant
GPR84 functionality is involved.
[0728] Antagonists and inverse agonists of the invention (e.g., an
antagonist or inverse agonist of a mammalian GPR84) are envisioned
to be useful as adjuvants for increasing the immunogenicity of
vaccine compositions for the purpose of eliciting a specific immune
response. The specific immune response may be an antibody response
and/or a cytotoxic T cell response. The present invention expressly
contemplates vaccine compositions comprising an antagonist and/or
inverse agonist of the invention and the use thereof in a subject
for elicitation of a specific immune response. An antagonist or
inverse agonist of the invention can be shown to have adjuvant
activity using methods well known in the art, including methods
disclosed in U.S. Pat. Nos. 6,780,421 and 6,630,161.
[0729] The present invention also relates to radioisotope-labeled
versions of compounds of the invention identified as modulators or
ligands of a GPCR of the invention such as a mammalian GPR84 that
would be useful not only in radio-imaging but also in assays, both
in vitro and in vivo, for localizing and quantitating GPR84 in
tissue samples, including human, and for identifying GPR84 ligands
in methods relating to inhibition of binding of a
radioisotope-labeled compound such as a known ligand of GPR84. It
is a further object of this invention to develop novel assays
relating to a GPCR of the invention such as a mammalian GPR84, such
as human GPR84, which comprise such radioisotope-labeled
compounds.
[0730] The present invention embraces radioisotope-labeled versions
of compounds of the invention identified as modulators or ligands
of a GPCR of the invention such as a mammalian GPR84, such as human
GPR84.
[0731] The present invention also relates to radioisotope-labeled
versions of test ligands that are useful for detecting a ligand
bound to a GPCR of the invention such as a mammalian GPR84, such as
human GPR84. In some embodiments, the present invention expressly
contemplates a library of said radiolabeled test ligands useful for
detecting a ligand bound to a GPCR of the invention such as a
mammalian GPR84, such as human GPR84. In certain embodiments, said
library comprises at least about 10, at least about 10.sup.2, at
least about 10.sup.3, at least about 10.sup.5, or at least about
10.sup.6 said radiolabeled test compounds. It is a further object
of this invention to develop novel assays relating to a GPCR of the
invention such as a mammalian GPR84, such as human GPR84, which
comprise such radioisotope-labeled test ligands.
[0732] In some embodiments, a radioisotope-labeled version of a
compound is identical to the compound, but for the fact that one or
more atoms are replaced or substituted by an atom having an atomic
mass or mass number different from the atomic mass or mass number
typically found in nature (i.e., naturally occurring). Suitable
radionuclides that may be incorporated in compounds of the present
invention include but are not limited to .sup.2H (deuterium),
.sup.3H (tritium), .sup.11C, .sup.13C, .sup.14C, .sup.13N,
.sup.15N, .sup.15O, .sup.17O, .sup.18O, .sup.18F, .sup.35S,
.sup.36Cl, .sup.82Br, .sup.75Br, .sup.76Br, .sup.77Br, .sup.123I,
.sup.124I, .sup.125I and .sup.131I. The radionuclide that is
incorporated in the instant radio-labeled compound will depend on
the specific application of that radio-labeled compound. For
example, for in vitro RUP40 receptor labeling and competition
assays, compounds that incorporate .sup.3H, .sup.14C, .sup.82Br,
.sup.125I, .sup.131I, .sup.35S or will generally be most useful.
For radio-imaging applications .sup.11C, .sup.18F, .sup.125I,
.sup.123I, .sup.124I, .sup.131I, .sup.75Br, .sup.76Br or .sup.77Br
will generally be most useful. In some embodiments, the
radionuclide is selected from the group consisting of .sup.3H,
.sup.11C, .sup.18F, .sup.14C, .sup.125I, .sup.124I, .sup.131I,
.sup.35S and .sup.82Br.
[0733] Synthetic methods for incorporating radio-isotopes into
organic compounds are applicable to compounds of the invention and
are well known in the art. These synthetic methods, for example,
incorporating activity levels of tritium into target molecules, are
as follows:
[0734] A. Catalytic Reduction with Tritium Gas--This procedure
normally yields high specific activity products and requires
halogenated or unsaturated precursors.
[0735] B. Reduction with Sodium Borohydride [.sup.3H]--This
procedure is rather inexpensive and requires precursors containing
reducible functional groups such as aldehydes, ketones, lactones,
esters, and the like.
[0736] C. Reduction with Lithium Aluminum Hydride [.sup.3H]--This
procedure offers products at almost theoretical specific
activities. It also requires precursors containing reducible
functional groups such as aldehydes, ketones, lactones, esters, and
the like.
[0737] D. Tritium Gas Exposure Labeling--This procedure involves
exposing precursors containing exchangeable protons to tritium gas
in the presence of a suitable catalyst.
[0738] E. N-Methylation using Methyl Iodide [.sup.3H]--This
procedure is usually employed to prepare O-methyl or N-methyl
(.sup.3H) products by treating appropriate precursors with high
specific activity methyl iodide (.sup.3H). This method in general
allows for higher specific activity, such as for example, about
70-90 Ci/mmol.
Synthetic methods for incorporating activity levels of .sup.125I
into target molecules include:
[0739] A. Sandmeyer and like reactions--This procedure transforms
an aryl or heteroaryl amine into a diazonium salt, such as a
tetrafluoroborate salt, and subsequently to .sup.125I labeled
compound using Na.sup.125I. A represented procedure was reported by
Zhu, D.-G. and co-workers in J. Org. Chem. 2002, 67, 943-948.
[0740] B. Ortho .sup.125Iodination of phenols--This procedure
allows for the incorporation of .sup.125I at the ortho position of
a phenol as reported by Collier, T. L. and co-workers in J. Labeled
Compd Radiopharm. 1999, 42, S264-S266.
[0741] C. Aryl and heteroaryl bromide exchange with .sup.125I--This
method is generally a two step process. The first step is the
conversion of the aryl or heteroaryl bromide to the corresponding
tri-alkyltin intermediate using for example, a Pd catalyzed
reaction [i.e. Pd(Ph.sub.3P).sub.4] or through an aryl or
heteroaryl lithium, in the presence of a tri-alkyltinhalide or
hexaalkylditin [e.g., (CH.sub.3).sub.3SnSn(CH.sub.3).sub.3]. A
represented procedure was reported by Bas, M.-D. and co-workers in
J. Labeled Compd Radiopharm. 2001, 44, S280-S282.
[0742] In some embodiments, a radioisotope-labeled version of a
compound is identical to the compound, but for the addition of one
or more substituents comprising a radionuclide. In some further
embodiments, the compound is a polypeptide. In some further
embodiments, the compound is an antibody or an antigen-binding
fragment thereof. In some further embodiments, said antibody is
monoclonal. Suitable said radionuclide includes but is not limited
to .sup.2H (deuterium), .sup.3H (tritium), .sup.11C, .sup.13C,
.sup.14C, .sup.13N, .sup.15N, .sup.17O, .sup.18O, .sup.18F,
.sup.35S, .sup.36Cl, .sup.82Br, .sup.75Br, .sup.76Br, .sup.77Br,
.sup.123I, .sup.124I, .sup.125I and .sup.131I. The radionuclide
that is incorporated in the instant radio-labeled compound will
depend on the specific application of that radio-labeled compound.
For example, for in vitro RUP40 receptor labeling and competition
assays, compounds that incorporate .sup.3H, .sup.14C, .sup.82Br,
.sup.125I, .sup.131I, .sup.35S or will generally be most useful.
For radio-imaging applications .sup.11C, .sup.18F, .sup.125I,
.sup.123I, .sup.124I, .sup.131I, .sup.75Br, .sup.76Br or .sup.77Br
will generally be most useful. In some embodiments, the
radionuclide is selected from the group consisting of .sup.3H,
.sup.11C, .sup.18F, .sup.14C, .sup.125I, .sup.124I, .sup.131I,
.sup.35S and .sup.82Br.
[0743] Methods for adding one or more substituents comprising a
radionuclide are within the purview of the skilled artisan and
include, but are not limited to, addition of radioisotopic iodine
by enzymatic method [Marchalonic J J, Biochemical Journal (1969)
113:299-305; Thorell J I and Johansson B G, Biochimica et
Biophysica Acta (1969) 251:363-9; the disclosure of each of which
is herein incorporated by reference in its entirety] and or by
Chloramine-T/Iodogen/Iodobead methods [Hunter W M and Greenwood F
C, Nature (1962) 194:495-6; Greenwood F C et al., Biochemical
Journal (1963) 89:114-23; the disclosure of each of which is herein
incorporated by reference in its entirety].
[0744] Other uses of the disclosed receptors and methods will
become apparent to those in the art based upon, inter alia, a
review of this patent document.
EXAMPLES
[0745] The following examples are presented for purposes of
elucidation, and not limitation, of the present invention. While
specific nucleic acid and amino acid sequences are disclosed
herein, those of ordinary skill in the art are credited with the
ability to make minor modifications to these sequences while
achieving the same or substantially similar results reported below.
Such modified approaches are considered within the purview of this
disclosure. Without further elaboration, it is believed that one
skilled in the art can, using the preceding description, practice
the present invention to its fullest extent. The following detailed
examples are to be construed as merely illustrative, and not
limitations of the preceding disclosure in any way whatsoever.
Those skilled in the art will promptly recognize appropriate
variations from the procedures.
[0746] Recombinant DNA techniques relating to the subject matter of
the present invention and well known to those of ordinary skill in
the art can be found, e.g., in Maniatis T et al., Molecular
Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory;
U.S. Pat. No. 6,399,373; and PCT Application Number PCT/IB02/01461
published as WO 02/066505 on 29 Aug. 2002; the disclosure of each
of which is herein incorporated by reference in its entirety.
Example 1
Full-Length Cloning of Endogenous Human GPR84
[0747] Polynucleotide encoding endogenous human GPR84 can be cloned
by PCR using the GPR84 specific primers
5'-ACTAAGCTTCTATCATGTGGAACAGC-3' (SEQ ID NO:7; sense with HindIII
site, ATG as initiation codon) and 5'-AGGAGACAGTCCTGAATT-3' (SEQ ID
NO:8; antisense with the last 5 nucleotides being part of the
endogenous EcoRI site in the 3' untranslated region) and human
genomic DNA as template. Cloned pfu DNA polymerase (Stratagene) can
be used for amplification by the following cycle with step 2 to
step 4 repeated 25 times: 94.degree. C., 3 minutes; 94.degree. C.,
1 minute; 52.degree. C., 1 minute; 72.degree. C., 2 minute;
72.degree. C., 10 minutes.
[0748] A 1.24 Kb PCR fragment of predicted size can be isolated,
digested with HindIII and EcoRI, and cloned into the pCMV
expression vector and sequenced using the T7 DNA sequenase kit
(Amersham). See, e.g., SEQ ID NO: 1 for nucleic acid sequence
encoding human GPR84 and SEQ ID NO:2 for the encoded human GPR84
amino acid sequence.
[0749] It is expressly contemplated that human cDNA derived from a
tissue or cell type that expresses GPR84 can alternatively be used
as template.
Example 2
Receptor Expression
[0750] Although a variety of cells are available to the art for the
expression of proteins, it is most preferred that mammalian cells
or melanophores be utilized. The primary reason for this is
predicated upon practicalities, i.e., utilization of, e.g., yeast
cells for the expression of a GPCR, while possible, introduces into
the protocol a non-mammalian cell which may not (indeed, in the
case of yeast, does not) include the receptor-coupling,
genetic-mechanism and secretary pathways that have evolved for
mammalian systems--thus, results obtained in non-mammalian cells,
while of potential use, are not as preferred as that obtained from
mammalian cells or melanophores. Of the mammalian cells, CHO,
COS-7, MCB3901, 293 and 293T cells are particularly preferred,
although the specific mammalian cell utilized can be predicated
upon the particular needs of the artisan. See infra as relates to
melanophores, including Example 9.
[0751] a. Transient Transfection
[0752] On day one, 4.times.10.sup.6 293 cells per 10 cm dish are
plated out. On day two, two reaction tubes are prepared (the
proportions to follow for each tube are per plate): tube A is
prepared by mixing 4 .mu.g DNA (e.g., pCMV vector; pCMV vector
comprising polynucleotide encoding a GPCR of the invention, etc.)
in 0.5 ml serum free DMEM (Gibco BRL); tube B is prepared by mixing
24 .mu.l lipofectamine (Gibco BRL) in 0.5 ml serum free DMEM. Tubes
A and B are admixed by inversions (several times), followed by
incubation at room temperature for 30-45 min. The admixture is
referred to as the "transfection mixture". Plated 293 cells are
washed with 1.times.PBS, followed by addition of 5 ml serum free
DMEM. 1 ml of the transfection mixture is added to the cells,
followed by incubation for 4 hrs at 37.degree. C./5% CO.sub.2. The
transfection mixture is removed by aspiration, followed by the
addition of 10 ml of DMEM/10% Fetal Bovine Serum. Cells are
incubated at 37.degree. C./5% CO.sub.2. After 48 hr incubation,
cells are harvested and utilized for analysis.
[0753] b. Stable Cell Lines
[0754] Approximately 12.times.10.sup.6 293 cells are plated on a 15
cm tissue culture plate. Grown in DME High Glucose Medium
containing ten percent fetal bovine serum and one percent sodium
pyruvate, L-glutamine, and antibiotics. Twenty-four hours following
plating of 293 cells (or to .about.80% confluency), the cells are
transfected using 12 .mu.g of DNA (e.g., pCMV-neo.sup.r vector
comprising polynucleotide encoding a GPCR of the invention). The 12
.mu.g of DNA is combined with 60 .mu.l of lipofectamine and 2 ml of
DME High Glucose Medium without serum. The medium is aspirated from
the plates and the cells are washed once with medium without serum.
The DNA, lipofectamine, and medium mixture are added to the plate
along with 10 ml of medium without serum. Following incubation at
37.degree. C. for four to five hours, the medium is aspirated and
25 ml of medium containing serum is added. Twenty-four hours
following transfection, the medium is aspirated again, and fresh
medium with serum is added. Forty-eight hours following
transfection, the medium is aspirated and medium with serum is
added containing geneticin (G418 drug) at a final concentration of
500 .mu.g/ml. The transfected cells now undergo selection for
positively transfected cells containing the G418 resistance gene.
The medium is replaced every four to five days as selection occurs.
During selection, cells are grown to create stable pools, or split
for stable clonal selection.
Example 3
Assays for Determination of GPCR Activation (e.g., Screening
Assays)
[0755] A variety of approaches are available for assessing
activation of a GPCR of interest, or "target" GPCR. The following
are illustrative; those of ordinary skill in the art are credited
with the ability to determine those techniques that are
preferentially beneficial for the needs of the artisan.
[0756] 1. Membrane Binding Assays: [.sup.35S]GTP.gamma.S Assay
[0757] When a G protein-coupled receptor is in its active state,
either as a result of ligand binding or constitutive activation,
the receptor couples to a G protein and stimulates the release of
GDP and subsequent binding of GTP to the G protein. The alpha
subunit of the G protein-receptor complex acts as a GTPase and
slowly hydrolyzes the GTP to GDP, at which point the receptor
normally is deactivated. Activated receptors continue to exchange
GDP for GTP. The non-hydrolyzable GTP analog,
[.sup.35S]GTP.gamma.S, can be utilized to demonstrate enhanced
binding of [.sup.35S]GTP.gamma.S to membranes expressing activated
receptors. The advantage of using [.sup.35S]GTP.gamma.S binding to
measure activation is that: (a) it is generically applicable to all
G protein-coupled receptors; (b) it is proximal at the membrane
surface making it less likely to pick-up molecules which affect the
intracellular cascade.
[0758] The assay utilizes the ability of G protein coupled
receptors to stimulate [.sup.35S]GTP.gamma.S binding to membranes
expressing the relevant receptors. The assay can, therefore, be
used to screen candidate compounds as modulators of GPCRs. The
assay is generic and has application to drug discovery at all G
protein-coupled receptors.
[0759] The [.sup.35S]GTP.gamma.S assay is incubated in 20 mM HEPES
and between 1 and about 20 mM MgCl.sub.2 (this amount can be
adjusted for optimization of results, although 20 mM is preferred)
pH 7.4, binding buffer with between about 0.3 and about 1.2 nM
[.sup.35S]GTP.gamma.S (this amount can be adjusted for optimization
of results, although 1.2 is preferred) and 12.5 to 75 .mu.g
membrane protein (e.g., 293 cells expressing a GPCR of the
invention; this amount can be adjusted for optimization) and 10
.mu.M GDP (this amount can be changed for optimization) for 1 hour.
Wheatgerm agglutinin beads (25 .mu.l; Amersham) are then added and
the mixture incubated for another 30 minutes at room temperature.
The tubes are then centrifuged at 1500.times.g for 5 minutes at
room temperature and then counted in a scintillation counter.
[0760] 2. Adenylyl Cyclase
[0761] A Flash Plate.TM. Adenylyl Cyclase kit (New England Nuclear;
Cat. No. SMP004A) designed for cell-based assays can be modified
for use with crude plasma membranes. The Flash Plate wells can
contain a scintillant coating which also contains a specific
antibody recognizing cAMP. The cAMP generated in the wells can be
quantitated by a direct competition for binding of radioactive cAMP
tracer to the cAMP antibody. The following serves as a brief
protocol for the measurement of changes in cAMP levels in whole
cells that express the receptors.
[0762] Transfected cells are harvested approximately twenty-four to
forty-eight hours after transient transfection. Media is carefully
aspirated off and discarded. 10 ml of PBS is gently added to each
dish of cells followed by careful aspiration. 1 ml of Sigma cell
dissociation buffer and 3 ml of PBS are added to each plate. Cells
are pipetted off the plate and the cell suspension is collected
into a 50 ml conical centrifuge tube. Cells are then centrifuged at
room temperature at 1,100 rpm for 5 min. The cell pellet is
carefully re-suspended into an appropriate volume of PBS (about 3
ml/plate). The cells are then counted using a hemocytometer and
additional PBS is added to give the appropriate number of cells
(with a final volume of about 50 .mu.l/well).
[0763] cAMP standards and Detection Buffer (comprising 1 .mu.Ci of
tracer [.sup.125I]cAMP (50 .mu.l) to 11 ml Detection Buffer) is
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer is prepared fresh for screening and
contains 50 .mu.l of Stimulation Buffer, 3 ul of test compound (12
.mu.M final assay concentration) and 50 .mu.l cells. Assay Buffer
is stored on ice until utilized. The assay, preferably carried out
e.g. in a 96-well plate, is initiated by addition of 50 .mu.l of
cAMP standards to appropriate wells followed by addition of 50 ul
of PBSA to wells H-11 and H12. 50 .mu.l of Stimulation Buffer is
added to all wells. DMSO (or selected candidate compounds) is added
to appropriate wells using a pin tool capable of dispensing 3 .mu.l
of compound solution, with a final assay concentration of 12 .mu.M
test compound and 100 .mu.l total assay volume. The cells are then
added to the wells and incubated for 60 min at room temperature.
100 .mu.l of Detection Mix containing tracer cAMP is then added to
the wells. Plates are then incubated additional 2 hours followed by
counting in a Wallac MicroBeta scintillation counter. Values of
cAMP/well are then extrapolated from a standard cAMP curve which is
contained within each assay plate.
[0764] 3. Cell-Based cAMP Assay for Gi-Coupled Target GPCRs
[0765] TSHR is a Gs coupled GPCR that causes the accumulation of
cAMP upon activation. TSHR will be constitutively activated by
mutating amino acid residue 623 (i.e., changing an alanine residue
to an isoleucine residue). A Gi coupled receptor is expected to
inhibit adenylyl cyclase, and, therefore, decrease the level of
cAMP production, which can make assessment of cAMP levels
challenging. An effective technique for measuring the decrease in
production of cAMP as an indication of activation of a Gi coupled
receptor can be accomplished by co-transfecting, most preferably,
non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an
endogenous, constitutively active Gs coupled receptor) as a "signal
enhancer" with a Gi coupled Target GPCR to establish a baseline
level of cAMP. The Gi coupled receptor is co-transfected with the
signal enhancer, and it is this material that can be used for
screening. Such an approach can be utilized to effectively generate
a signal when a cAMP assay is used. In some embodiments, this
approach is preferably used in the identification of candidate
compounds against Gi coupled receptors. It is noted that for a Gi
coupled GPCR, when this approach is used, an inverse agonist of the
Target GPCR will increase the cAMP signal and an agonist will
decrease the cAMP signal.
[0766] On day one, 4.times.10.sup.6 293 cells per 10 cm dish will
be plated out. On day two, two reaction tubes will be prepared (the
proportions to follow for each tube are per plate): tube A will be
prepared by mixing 2 .mu.g DNA of each receptor transfected into
the mammalian cells, for a total of 4 .mu.g DNA (e.g., pCMV vector;
pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and the
Target GPCR, etc.) in 0.5 ml serum free DMEM (Irvine Scientific,
Irvine, Calif.); tube B will be prepared by mixing 24 .mu.l
lipofectamine (Gibco BRL) in 0.5 ml serum free DMEM. Tubes A and B
will then be admixed by inversions (several times), followed by
incubation at room temperature for 30-45 min. The admixture is
referred to as the "transfection mixture". Plated 293 cells will be
washed with 1.times.PBS, followed by addition of 5 ml serum free
DMEM. 1.0 ml of the transfection mixture will then be added to the
cells, followed by incubation for 4 hrs at 37.degree. C./5%
CO.sub.2. The transfection mixture will then be removed by
aspiration, followed by the addition of 10 ml of DMEM/10% Fetal
Bovine Serum. Cells will then be incubated at 37.degree. C./5%
CO.sub.2. After approximately 24-48 hr incubation, cells will then
be harvested and utilized for analysis.
[0767] A Flash Plate.TM. Adenylyl Cyclase kit (New England Nuclear;
Cat. No. SMP004A) is designed for cell-based assays, but can be
modified for use with crude plasma membranes depending on the need
of the skilled artisan. The Flash Plate wells will contain a
scintillant coating which also contains a specific antibody
recognizing cAMP. The cAMP generated in the wells can be
quantitated by a direct competition for binding of radioactive cAMP
tracer to the cAMP antibody. The following serves as a brief
protocol for the measurement of changes in cAMP levels in whole
cells that express the receptors.
[0768] Transfected cells will be harvested approximately
twenty-four to forty-eight hours after transient transfection.
Media will be carefully aspirated off and discarded. 10 ml of PBS
will be gently added to each dish of cells followed by careful
aspiration. 1 ml of Sigma cell dissociation buffer and 3 ml of PBS
will be added to each plate. Cells will be pipetted off the plate
and the cell suspension will be collected into a 50 ml conical
centrifuge tube. Cells will then be centrifuged at room temperature
at 1,100 rpm for 5 min. The cell pellet will be carefully
re-suspended into an appropriate volume of PBS (about 3 ml/plate).
The cells will then be counted using a hemocytometer and additional
PBS is added to give the appropriate number of cells (with a final
volume of about 50 .mu.l/well).
[0769] cAMP standards and Detection Buffer (comprising 1 .mu.Ci of
tracer [.sup.125I]cAMP (50 .mu.l) to 11 ml Detection Buffer) will
be prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer should be prepared fresh for screening
and contained 50 .mu.l of Stimulation Buffer, 3 .mu.l of test
compound (12 .mu.M final assay concentration) and 50 .mu.l cells,
Assay Buffer can be stored on ice until utilized. The assay can be
initiated by addition of 50 .mu.l of cAMP standards to appropriate
wells followed by addition of 50 .mu.l of PBSA to wells H-11 and
H12. Fifty .mu.l of Stimulation Buffer will be added to all wells.
Selected compounds (e.g., TSH) will be added to appropriate wells
using a pin tool capable of dispensing 3 .mu.l of compound
solution, with a final assay concentration of 12 .mu.M test
compound and 100 .mu.l total assay volume. The cells will then be
added to the wells and incubated for 60 min at room temperature.
100 .mu.l of Detection Mix containing tracer cAMP will then be
added to the wells. Plates were then incubated additional 2 hours
followed by counting in a Wallac MicroBeta scintillation counter.
Values of cAMP/well will then be extrapolated from a standard cAMP
curve which is contained within each assay plate.
[0770] 4. Reporter-Based Assays
[0771] a. CRE-LUC Reporter Assay (Gs-Associated Receptors)
[0772] 293 and 293T cells are plated-out on 96 well plates at a
density of 2.times.10.sup.4 cells per well and were transfected
using Lipofectamine Reagent (BRL) the following day according to
manufacturer instructions. A DNA/lipid mixture is prepared for each
6-well transfection as follows: 260 ng of plasmid DNA in 100 .mu.l
of DMEM is gently mixed with 2 .mu.l of lipid in 100 .mu.l of DMEM
(the 260 ng of plasmid DNA consists of 200 ng of a 8.times.CRE-Luc
reporter plasmid, 50 ng of pCMV comprising endogenous receptor or
non-endogenous receptor or pCMV alone, and 10 ng of a GPRS
expression plasmid (GPRS in pcDNA3 (Invitrogen)). The
8.times.CRE-Luc reporter plasmid was prepared as follows: vector
SRIF-.beta.-gal was obtained by cloning the rat somatostatin
promoter (-71/+51) at BglV-HindIII site in the p.beta.gal-Basic
Vector (Clontech). Eight (8) copies of cAMP response element were
obtained by PCR from an adenovirus template AdpCF126CCRE8 [see,
Suzuki et al., Hum Gene Ther (1996) 7:1883-1893; the disclosure of
which is herein incorporated by reference in its entirety) and
cloned into the SRIF-.beta.-gal vector at the Kpn-BglV site,
resulting in the 8.times.CRE-.beta.-gal reporter vector. The
8.times.CRE-Luc reporter plasmid was generated by replacing the
beta-galactosidase gene in the 8.times.CRE-.beta.-gal reporter
vector with the luciferase gene obtained from the pGL3-basic vector
(Promega) at the HindIII-BamHI site. Following 30 min. incubation
at room temperature, the DNA/lipid mixture is diluted with 400
.mu.l of DMEM and 100 .mu.l of the diluted mixture is added to each
well. 100 .mu.l of DMEM with 10% FCS are added to each well after a
4 hr incubation in a cell culture incubator. The following day the
transfected cells are changed with 200 .mu.l/well of DMEM with 10%
FCS. Eight (8) hours later, the wells are changed to 100 .mu.l/well
of DMEM without phenol red, after one wash with PBS. Luciferase
activity is measured the next day using the LucLite.TM. reporter
gene assay kit (Packard) following manufacturer instructions and
read on a 1450 .mu.MicroBeta.TM. scintillation and luminescence
counter (Wallac).
[0773] b. AP1 Reporter Assay (Gq-Associated Receptors)
[0774] A method to detect Gq stimulation depends on the known
property of Gq-dependent phospholipase C to cause the activation of
genes containing AP1 elements in their promoter. A Pathdetect.TM.
AP-1 cis-Reporting System (Stratagene, Catalogue #219073) can be
utilized following the protocol set forth above with respect to the
CREB reporter assay, except that the components of the calcium
phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor
expression plasmid, and 20 ng CMV-SEAP (secreted alkaline
phosphatase expression plasmid; alkaline phosphatase activity is
measured in the media of transfected cells to control for
variations in transfection efficiency between samples).
[0775] c. SRF-LUC Reporter Assay (Gq-Associated Receptors)
[0776] One method to detect Gq stimulation depends on the known
property of Gq-dependent phospholipase C to cause the activation of
genes containing serum response factors in their promoter. A
Pathdetect.TM. SRF-Luc-Reporting System (Stratagene) can be
utilized to assay for Gq coupled activity in, e.g., COS7 cells.
Cells are transfected with the plasmid components of the system and
the indicated expression plasmid encoding endogenous or
non-endogenous GPCR using a Mammalian Transfection.TM. Kit
(Stratagene, Catalogue #200285) according to the manufacturer's
instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor
expression plasmid and 20 ng CMV-SEAP are combined in a calcium
phosphate precipitate as per the manufacturer's instructions. Half
of the precipitate is equally distributed over 3 wells in a 96-well
plate, kept on the cells in a serum free media for 24 hours. The
last 5 hours the cells are incubated with, e.g. 1 .mu.M, test
compound. Cells are then lysed and assayed for luciferase activity
using a Luclite.TM. Kit (Packard, Cat. #6016911) and "Trilux 1450
.mu.Microbeta" liquid scintillation and luminescence counter
(Wallac) as per the manufacturer's instructions. The data can be
analyzed using GraphPad Prism.TM. 2.0a (GraphPad Software
Inc.).
[0777] d. Intracellular IP3 Accumulation Assay (Gq-Associated
Receptors)
[0778] On day 1, cells comprising the receptors (endogenous or
non-endogenous) can be plated onto 24 well plates, usually
1.times.10.sup.5 cells/well (although his number can be optimized.
On day 2 cells can be transfected by first mixing 0.25 .mu.g DNA in
50 .mu.l serum free DMEM/well and 2 .mu.l lipofectamine in 50 .mu.l
serum free DMEM/well. The solutions are gently mixed and incubated
for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS
and 400 .mu.l of serum free media is mixed with the transfection
media and added to the cells. The cells are then incubated for 3-4
hrs at 37.degree. C./5% CO.sub.2 and then the transfection media is
removed and replaced with 1 ml/well of regular growth media. On day
3 the cells are labeled with .sup.3H-myo-inositol. Briefly, the
media is removed and the cells are washed with 0.5 ml PBS. Then 0.5
ml inositol-free/serum free media (GIBCO BRL) is added/well with
0.25 .mu.Ci of .sup.3H-myo-inositol/well and the cells are
incubated for 16-18 hrs o/n at 37.degree. C./5% CO.sub.2. On Day 4
the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is
added containing inositol-free/serum free media 10 .mu.M pargyline
10 mM lithium chloride or 0.4 ml of assay medium and optionally 50
.mu.l of test compound to final concentration of 10 .mu.M. The
cells are then incubated for 30 min at 37.degree. C. The cells are
then washed with 0.5 ml PBS and 200 .mu.l of fresh/ice cold stop
solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The
solution is kept on ice for 5-10 min or until cells were lysed and
then neutralized by 200 .mu.l of fresh/ice cold neutralization sol.
(7.5% HCL). The lysate is then transferred into 1.5 ml eppendorf
tubes and 1 ml of chloroform/methanol (1:2) is added/tube. The
solution is vortexed for 15 sec and the upper phase is applied to a
Biorad AG1-X8.TM. anion exchange resin (100-200 mesh). Firstly, the
resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase
is loaded onto the column. The column is washed with 10 mls of 5 mM
myo-inositol and 10 ml of 5 mM Na-borate/60 mM Na-formate. The
inositol tris phosphates are eluted into scintillation vials
containing 10 ml of scintillation cocktail with 2 ml of 0.1 M
formic acid/1 M ammonium formate. The columns are regenerated by
washing with 10 ml of 0.1 M formic acid/3 .mu.M ammonium formate
and rinsed twice with dd H.sub.2O and stored at 4.degree. C. in
water.
Example 4
Fusion Protein Preparation
[0779] a. GPCR:Gs Fusion Construct
[0780] The design of the GPCR-G protein fusion construct can be
accomplished as follows: both the 5' and 3' ends of the rat G
protein Gs.alpha. (long form; Itoh, H. et al., 83 PNAS 3776 (1986))
are engineered to include a HindIII (5'-AAGCTT-3') sequence
thereon. Following confirmation of the correct sequence (including
the flanking HindIII sequences), the entire sequence is shuttled
into pcDNA3.1(-) (Invitrogen, cat. no. V795-20) by subcloning using
the HindIII restriction site of that vector. The correct
orientation for the Gs.alpha. sequence is determined after
subcloning into pcDNA3.1(-). The modified pcDNA3.1(-) containing
the rat Gs.alpha. gene at Hindu sequence is then verified; this
vector is now available as a "universal" Gs.alpha. protein vector.
The pcDNA3.1(-) vector contains a variety of well-known restriction
sites upstream of the HindIII site, thus beneficially providing the
ability to insert, upstream of the Gs protein, the coding sequence
of an endogenous, constitutively active GPCR. This same approach
can be utilized to create other "universal" G protein vectors, and,
of course, other commercially available or proprietary vectors
known to the artisan can be utilized--the important criteria is
that the sequence for the GPCR be upstream and in-frame with that
of the G protein.
[0781] b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct
[0782] A Gq(del)/Gi fusion construct is a chimeric G protein
whereby the first six (6) amino acids of the Gq-protein
.alpha.-subunit ("G.alpha.q") are deleted and the last five (5)
amino acids at the C-terminal end of G.alpha.q are replaced with
the corresponding amino acids of the G.alpha. subunit. A Gq(del)/Gi
fusion construct will force an endogenous Gi coupled receptor to
couple to its non-endogenous G protein, Gq (in the form of
Gq(del)/Gi), such that the second messenger, for example, inositol
triphosphate or diacylglycerol or Ca.sup.2+, can be measured in
lieu of cAMP production.
[0783] The Gq(del)/Gi fusion construct was designed as follows: the
N-terminal six (6) amino acids (amino acids 2 through 7, having the
sequence of TLESIM (SEQ ID NO: 9) of the G.alpha.q-subunit were
deleted and the C-terminal five (5) amino acids, having the
sequence EYNLV (SEQ ID NO: 10) were replaced with the corresponding
amino acids of the God Protein, having the sequence DCGLF (SEQ ID
NO: 11). This fusion construct was obtained by PCR using the
following primers:
5'-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3' (SEQ ID NO: 12) and
5'-gatcggatccTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3' (SEQ ID
NO: 13) and Plasmid 63313 (ATCC.RTM. Number 63313) which contains
the mouse G.alpha.q-wild-type version with a hemagglutinin tag as a
template. Nucleotides in lower case include cloning sites for
HindIII/BamHI and spacers.
[0784] TaqPlus Precision DNA polymerase (Stratagene) was utilized
for the amplification by the following cycles, with steps 2 through
4 repeated 35 times: 95.degree. C. for 2 min; 95.degree. C. for 20
sec; 56.degree. C. for 20 sec; 72.degree. C. for 2 min; and
72.degree. C. for 7 min. The PCR product was cloned into a
pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye
Terminator kit (P. E. Biosystems). Inserts from a TOPO clone
containing the sequence of the fusion construct was shuttled into
the expression vector pcDNA3.1(+) at the HindIII/BamHI site by a 2
step cloning process. See, SEQ ID NO:14 for the nucleic acid
sequence and SEQ ID NO:15 for the encoded amino acid sequence of
Gq(del)/Gi construct.
Example 5
[.sup.35S]GTP.gamma.S Assay
[0785] A. Membrane Preparation
[0786] In some embodiments membranes comprising a Target GPCR and
for use in the identification of candidate compounds as, e.g.,
inverse agonists, agonists, or antagonists, are preferably prepared
as follows:
[0787] a. Materials
[0788] "Membrane Scrape Buffer" is comprised of 20 mM HEPES and 10
mM EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES
and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20 mM
HEPES, 100 mM NaCl, and 10 mM MgCl.sub.2, pH 7.4.
[0789] b. Procedure
[0790] All materials will be kept on ice throughout the procedure.
Firstly, the media will be aspirated from a confluent monolayer of
cells, followed by rinse with 10 ml cold PBS, followed by
aspiration. Thereafter, 5 ml of Membrane Scrape Buffer will be
added to scrape cells; this will be followed by transfer of
cellular extract into 50 ml centrifuge tubes (centrifuged at 20,000
rpm for 17 minutes at 4.degree. C.). Thereafter, the supernatant
will be aspirated and the pellet will be resuspended in 30 ml
Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17
minutes at 4.degree. C. The supernatant will then be aspirated and
the pellet resuspended in Binding Buffer. This will then be
homogenized using a Brinkman Polytron.TM. homogenizer (15-20 second
bursts until the all material is in suspension). This is referred
to herein as "Membrane Protein".
[0791] Bradford Protein Assay
[0792] Following the homogenization, protein concentration of the
membranes will be determined using the Bradford Protein Assay
(protein can be diluted to about 1.5 mg/ml, aliquoted and frozen
(-80.degree. C.) for later use; when frozen, protocol for use will
be as follows: on the day of the assay, frozen Membrane Protein is
thawed at room temperature, followed by vortex and then homogenized
with a Polytron at about 12.times.1,000 rpm for about 5-10 seconds;
it is noted that for multiple preparations, the homogenizer should
be thoroughly cleaned between homogenization of different
preparations).
[0793] a. Materials
[0794] Binding Buffer (as per above); Bradford Dye Reagent;
Bradford Protein Standard will be utilized, following manufacturer
instructions (Biorad, cat. no. 500-0006).
[0795] b. Procedure
[0796] Duplicate tubes will be prepared, one including the
membrane, and one as a control "blank". Each contained 800 .mu.l
Binding Buffer. Thereafter, 10 .mu.l of Bradford Protein Standard
(1 mg/ml) will be added to each tube, and 10 .mu.l of membrane
Protein will then be added to just one tube (not the blank).
Thereafter, 200 .mu.l of Bradford Dye Reagent will be added to each
tube, followed by vortex of each. After five (5) minutes, the tubes
will be re-vortexed and the material therein will be transferred to
cuvettes. The cuvettes will then be read using a CECIL 3041
spectrophotometer, at wavelength 595 nm.
[0797] Identification Assay
[0798] a. Materials
[0799] GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP
(Sigma, cat. no. G-7127), followed by a series of dilutions in
Binding Buffer to obtain 0.2 .mu.M GDP (final concentration of GDP
in each well was 0.1 .mu.M GDP); each well comprising a candidate
compound, has a final volume of 200 .mu.l consisting of 100 .mu.l
GDP Buffer (final concentration, 0.1 .mu.M GDP), 50 .mu.l Membrane
Protein in Binding Buffer, and 50 .mu.l [.sup.35S]GTP.gamma.S (0.6
nM) in Binding Buffer (2.5 .mu.l [.sup.35S]GTP.gamma.S per 10 ml
Binding Buffer).
[0800] b. Procedure
[0801] Candidate compounds will be preferably screened using a
96-well plate format (these can be frozen at -80.degree. C.).
Membrane Protein (or membranes with expression vector excluding the
Target GPCR, as control), will be homogenized briefly until in
suspension. Protein concentration will then be determined using the
Bradford Protein Assay set forth above. Membrane Protein (and
control) will then be diluted to 0.25 mg/ml in Binding Buffer
(final assay concentration, 12.5 .mu.g/well). Thereafter, 100 .mu.l
GDP Buffer is added to each well of a Wallac Scintistrip.TM.
(Wallac). A 5 ul pin-tool will then be used to transfer 5 .mu.l of
a candidate compound into such well (i.e., 5 .mu.l in total assay
volume of 200 .mu.l is a 1:40 ratio such that the final screening
concentration of the candidate compound is 10 .mu.M). Again, to
avoid contamination, after each transfer step the pin tool should
be rinsed in three reservoirs comprising water (1.times.), ethanol
(1.times.) and water (2.times.)--excess liquid should be shaken
from the tool after each rinse and dried with paper and kimwipes.
Thereafter, 50 .mu.l of Membrane Protein will be added to each well
(a control well comprising membranes without the Target GPCR was
also utilized), and pre-incubated for 5-10 minutes at room
temperature. Thereafter, 50 .mu.l of [.sup.35S]GTP.gamma.S (0.6 nM)
in Binding Buffer will be added to each well, followed by
incubation on a shaker for 60 minutes at room temperature (again,
in this example, plates were covered with foil). The assay will
then be stopped by spinning of the plates at 4000 RPM for 15
minutes at 22.degree. C. The plates will then be aspirated with an
8 channel manifold and sealed with plate covers. The plates will
then be read on a Wallac 1450 using setting "Prot. #37" (as per
manufacturer's instructions).
Example 6
Cyclic Amp Assay
[0802] Another assay approach for identifying candidate compounds
as, e.g., inverse agonists, agonists, or antagonists, is
accomplished by utilizing a cyclase-based assay. In addition to so
identifying candidate compounds, this assay approach can be
utilized as an independent approach to provide confirmation of the
results from the [.sup.35S]GTP.gamma.S approach as set forth in
Example 5, supra.
[0803] A modified Flash Plate.TM. Adenylyl Cyclase kit (New England
Nuclear; Cat. No. SMP004A) is preferably utilized for
identification of candidate compounds as modulators of a Target
GPCR in accordance with the following protocol.
[0804] Cells transfected with the Target GPCR are harvested
approximately three days after transfection. Membranes are prepared
by homogenization of suspended cells in buffer containing 20 mM
HEPES, pH 7.4 and 10 mM MgCl.sub.2. Homogenization is performed on
ice using a Brinkman Polytron.TM. for approximately 10 seconds. The
resulting homogenate is centrifuged at 49,000.times.g for 15
minutes at 4.degree. C. The resulting pellet is then resuspended in
buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized
for 10 seconds, followed by centrifugation at 49,000.times.g for 15
minutes at 4.degree. C. The resulting pellet is then stored at
-80.degree. C. until utilized. On the day of direct identification
screening, the membrane pellet is slowly thawed at room
temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4
and 10 mM MgCl.sub.2, to yield a final protein concentration of
0.60 mg/ml (the resuspended membranes are placed on ice until
use).
[0805] cAMP standards and Detection Buffer (comprising 2 .mu.Ci of
tracer [.sup.125I]cAMP (100 .mu.l) to 11 ml Detection Buffer) are
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer is prepared fresh for screening and
contains 20 mM HEPES, pH 7.4, mM MgCl.sub.2, 20 mM phosphocreatine
(Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 .mu.M GTP
(Sigma), and 0.2 mM ATP (Sigma); Assay Buffer is then stored on ice
until utilized.
[0806] Candidate compounds are added, preferably, to e.g. 96-well
plate wells (3 .mu.l/well; 12 .mu.M final assay concentration),
together with 40 .mu.l Membrane Protein (30 .mu.g/well) and 50
.mu.l of Assay Buffer. This admixture was then incubated for 30
minutes at room temperature, with gentle shaking.
[0807] Following the incubation, 100 .mu.l of Detection Buffer is
added to each well, followed by incubation for 2-24 hours. Plates
are then counted in a Wallac MicroBeta.TM. plate reader using
"Prot. #31" (as per manufacturer's instructions).
[0808] By way of example and not limitation, an illustrative
screening assay plate (96 well format) result obtained is presented
in FIG. 1. Each bar represents the result for a compound that
differs in each well, the "Target GPCR" being a Gs.alpha. Fusion
Protein construct of an endogenous, constitutively active
Gs-coupled GPCR unrelated to GPR84. The results presented in FIG. 1
also provide standard deviations based upon the mean results of
each plate ("m") and the mean plus two arbitrary preference for
selection of inverse agonists as "leads" from the primary screen
involves selection of candidate compounds that that reduce the
percent response by at least the mean plate response, minus two
standard deviations. Conversely, an arbitrary preference for
selection of agonists as "leads" from the primary screen involves
selection of candidate compounds that increase the percent response
by at least the mean plate response, plus the two standard
deviations. Based upon these selection processes, the candidate
compounds in the following wells were directly identified as
putative inverse agonist (Compound A) and agonist (Compound B) to
said endogenous GPCR in wells A2 and G9, respectively. See, FIG. 1.
It is noted for clarity: these compounds have been directly
identified without any knowledge of the endogenous ligand for this
GPCR. By focusing on assay techniques that are based upon receptor
function, and not compound binding affinity, it is possible to
ascertain compounds that are able to reduce the functional activity
of this receptor (Compound A) as well as increase the functional
activity of the receptor (Compound B).
Example 7
Fluorometric Imaging Plate Reader (FLIPR) Assay for the Measurement
of Intracellular Calcium Concentration
[0809] Target Receptor (experimental) and pCMV (negative control)
stably transfected cells from respective clonal lines are seeded
into poly-D-lysine pretreated 96-well plates (Becton-Dickinson,
#356640) at 5.5.times.10.sup.4 cells/well with complete culture
medium (DMEM with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate)
for assay the next day. To prepare Fluo4-AM (Molecular Probe,
#F14202) incubation buffer stock, 1 mg Fluo4-AM is dissolved in 467
.mu.l DMSO and 467 .mu.l Pluoronic acid (Molecular Probe, #P3000)
to give a 1 mM stock solution that can be stored at -20.degree. C.
for a month. Fluo4-AM is a fluorescent calcium indicator dye.
[0810] Candidate compounds are prepared in wash buffer
(1.times.HBSS/2.5 mM Probenicid/20 mM HEPES at pH 7.4).
[0811] At the time of assay, culture medium is removed from the
wells and the cells are loaded with 100 .mu.l of 4 .mu.M
Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20 mM HEPES/complete
medium at pH 7.4. Incubation at 37.degree. C./5% CO.sub.2 is
allowed to proceed for 60 min.
[0812] After the 1 hr incubation, the Fluo4-AM incubation buffer is
removed and the cells are washed 2.times. with 100 .mu.l wash
buffer. In each well is left 100 .mu.l wash buffer. The plate is
returned to the incubator at 37.degree. C./5% CO.sub.2 for 60
min.
[0813] FLIPR (Fluorometric Imaging Plate Reader; Molecular Device)
is programmed to add 50 .mu.l candidate compound on the 30th second
and to record transient changes in intracellular calcium
concentration ([Ca.sup.2+]) evoked by the candidate compound for
another 150 seconds. Total fluorescence change counts are used to
determine agonist activity using the FLIPR software. The instrument
software normalizes the fluorescent reading to give equivalent
initial readings at zero.
[0814] In some embodiments, the cells comprising Target Receptor
further comprise G.alpha.15, G.alpha.16, or Gq(del)/Gi chimeric G
protein.
[0815] Although the foregoing provides a FLIPR assay for agonist
activity using stably transfected cells, a person of ordinary skill
in the art would readily be able to modify the assay in order to
characterize antagonist activity. The person of ordinary skill in
the art would also readily appreciate that, alternatively,
transiently transfected cells could be used.
Example 8
Map Kinase Assay
[0816] MAP kinase (mitogen activated kinase) may be monitored to
evaluate receptor activation. MAP kinase can be detected by several
approaches. One approach is based on an evaluation of the
phosphorylation state, either unphosphorylated (inactive) or
phosphorylated (active). The phosphorylated protein has a slower
mobility in SDS-PAGE and can therefore be compared with the
unstimulated protein using Western blotting. Alternatively,
antibodies specific for the phosphorylated protein are available
(New England Biolabs) which can be used to detect an increase in
the phosphorylated kinase. In either method, cells are stimulated
with the test compound and then extracted with Laemmli buffer. The
soluble fraction is applied to an SDS-PAGE gel and proteins are
transferred electrophoretically to nitrocellulose or Immobilin.
Immunoreactive bands are detected by standard Western blotting
technique. Visible or chemiluminescent signals are recorded on film
and may be quantified by densitometry.
[0817] Another approach is based on evaluation of the MAP kinase
activity via a phosphorylation assay. Cells are stimulated with the
test compound and a soluble extract is prepared. The extract is
incubated at 30.degree. C. for 10 min with gamma-.sup.32P-ATP, an
ATP regenerating system, and a specific substrate for MAP kinase
such as phosphorylated heat and acid stable protein regulated by
insulin, or PHAS-I. The reaction is terminated by the addition of
H.sub.3PO.sub.4 and samples are transferred to ice. An aliquot is
spotted onto Whatman P81 chromatography paper, which retains the
phosphorylated protein. The chromatography paper is washed and
counted for .sup.32P is a liquid scintillation counter.
Alternatively, the cell extract is incubated with
gamma-.sup.32P-ATP, an ATP regenerating system, and biotinylated
myelin basic protein bound by streptavidin to a filter support. The
myelin basic protein is a substrate for activated MAP kinase. The
phosphorylation reaction is carried out for 10 min at 30.degree. C.
The extract can then be aspirated through the filter, which
retains, the phosphorylated myelin basic protein. The filter is
washed and counted for .sup.32P by liquid scintillation
counting.
Example 9
Melanophore Technology
[0818] Melanophores are skin cells found in lower vertebrates. They
contain pigmented organelles termed melanosomes. Melanophores are
able to redistribute these melanosomes along a microtubule network
upon G-protein coupled receptor (GPCR) activation. The result of
this pigment movement is an apparent lightening or darkening of the
cells. In melanophores, the decreased levels of intracellular cAMP
that result from activation of a Gi-coupled receptor cause
melanosomes to migrate to the center of the cell, resulting in a
dramatic lightening in color. If cAMP levels are then raised,
following activation of a Gs-coupled receptor, the melanosomes are
re-dispersed and the cells appear dark again. The increased levels
of diacylglycerol that result from activation of Gq-coupled
receptors can also induce this re-dispersion. In addition, the
technology is also suited to the study of certain receptor tyrosine
kinases. The response of the melanophores takes place within
minutes of receptor activation and results in a simple, robust
color change. The response can be easily detected using a
conventional absorbance microplate reader or a modest video imaging
system. Unlike other skin cells, the melanophores derive from the
neural crest and appear to express a full complement of signaling
proteins. In particular, the cells express an extremely wide range
of G-proteins and so are able to functionally express almost all
GPCRs.
[0819] Melanophores can be utilized to identify compounds,
including natural ligands, against GPCRs. This method can be
conducted by introducing test cells of a pigment cell line capable
of dispersing or aggregating their pigment in response to a
specific stimulus and expressing an exogenous clone coding for the
GCPR. A stimulant, e.g., melatonin, sets an initial state of
pigment disposition wherein the pigment is aggregated within the
test cells if activation of the GPCR induces pigment dispersion.
However, stimulating the cell with a stimulant to set an initial
state of pigment disposition wherein the pigment is dispersed if
activation of the GPCR induces pigment aggregation. The test cells
are then contacted with chemical compounds, and it is determined
whether the pigment disposition in the cells changed from the
initial state of pigment disposition. Dispersion of pigments cells
due to the candidate compound, including but not limited to a
ligand, coupling to the GPCR will appear dark on a petri dish,
while aggregation of pigments cells will appear light.
[0820] Materials and methods can be followed according to the
disclosure of U.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386.
These patent disclosures are herein incorporated by reference in
their entirety.
[0821] The cells are plated in e.g. 96-well plates (one receptor
per plate). 48 hours post-transfection, half of the cells on each
plate are treated with 10 nM melatonin. Melatonin activates an
endogenous Gi-coupled receptor in the melanophores and causes them
to aggregate their pigment. The remaining half of the cells are
transferred to serum-free medium 0.7.times.L-15 (Gibco). After one
hour, the cells in serum-free media remain in a pigment-dispersed
state while the melatonin-treated cells are in a pigment-aggregated
state. At this point, the cells are treated with a dose response of
a test/candidate compound. If the plated GPCRs bind to the
test/candidate compound, the melanophores would be expected to
undergo a color change in response to the compound. If the receptor
were either a Gs or Gq coupled receptor, then the
melatonin-aggregated melanophores would undergo pigment dispersion.
In contrast, if the receptor was a Gi-coupled receptor, then the
pigment-dispersed cells would be expected to undergo a
dose-dependent pigment aggregation.
Example 10
Monocyte/Macrophage Expression of GPR84
[0822] A. Microarray Analysis of GPR84 Expression
[0823] GPR84 expression across a panel of normal human tissues and
isolated cells was assessed by microarray analysis of total RNA and
is shown in FIG. 2A. The microarray analysis used 26 probes for
GPR84 to assess expression. The expression levels in FIG. 2A are
the average and SD of two replicates. GPR84 expression was found to
be enriched in human monocyte and macrophage cell types.
[0824] B. Expression of GPR84 in Human Macrophage
[0825] Taqman analysis of GPR84 in human macrophage was carried as
shown in FIG. 2B. TaqMan analysis was performed on cultured
monocyte-derived macrophages from two donors as well as the stable
cell line HL-60 differentiated into macrophage-like cells by TPA
treatment for 24 hrs. Cells were either vehicle treated or
stimulated with 1 ng/ml of LPS. For quantitative PCR (qPCR)
analysis, the cDNA was synthesized from total RNA using the Bio-Rad
cDNA synthesis kit following the manufacturer's protocol. The
Bio-Rad PCR machine was used for this synthesis.
[0826] The qPCR reactions were prepared from the cDNA using the
Bio-Rad qPCR kit, following the manufacturer's protocol. The qPCR
reactions were performed using the ABI Prism 7900HT machine with
the following temperatures and times:
50.degree. C., 2 minutes; 95.degree. C., 10 minutes; followed by 40
cycles of 95.degree. C., 15 seconds and 60.degree. C., 1 minute.
Primers and probe sequences used for qPCR analysis human GPR84 were
as follows:
TABLE-US-00005 (SEQ ID NO: 16) Forward Primer-
5'-TCCTTTTTGCCTCCAATTCTGT-3' (SEQ ID NO: 17) Reverse Primer-
5'-GCGTCCCAGTGCGATGAG-3' (SEQ ID NO: 18) Taqman MGB Probe-
6FAM-5'-TCCATCCTGACCCTCT-3'
GPR84 was found to-be expressed in human macrophage, and the level
of GPR84 expression in human macrophage was found to be
up-regulated by LPS (FIG. 2B).
[0827] C. Expression of GPR84 in Mouse Macrophage
[0828] Microarray analysis of mouse GPR84 expression in the
macrophage cell lines RAW and Jaws2 was carried out. Cell lines
were cultured to subconfluency and then stimulated with 10 ng/ml of
LPS for the indicated times. Total RNA was extracted from treated
cells and analyzed by microarray analysis. Expression levels are
the average and SD of two replicates.
[0829] GPR84 was found to be expressed in mouse macrophage lines,
and the level of GPR84 expression in mouse macrophage was found to
be up-regulated by LPS (FIG. 2C).
Example 11
Upregulation of GPR84 mRNA in human monocytes by proinflammatory
Cytokines
[0830] Regulation of GPR84 mRNA expression in human monocytes by
the proinflammatory cytokines tumor necrosis factor alpha
(TNF.alpha.) and interferon gamma (IFN.gamma.) was assessed by
QRT-PCR as described below.
[0831] Peripheral blood leukocytes were separated from erythrocytes
by sedimentation in a dextran containing saline solution. The
leukocytes were washed, and mononuclear cells were isolated on a
Ficoll density gradient. Peripheral blood mononuclear cells were
then washed and negatively selected using a MACS.RTM. monocyte
isolation kit II (Miltenyi Biotec, Auburn, Calif.) and LS MACS.RTM.
cell separation columns according to manufacturer's instructions.
Freshly isolated monocytes were then resuspended in culture medium
alone or culture medium supplemented with TNF.alpha. (10 ng/ml) or
IFN.gamma. (10 or 100 ng/ml) and allowed to adhere to wells
precoated with vitronectin. Cultures were harvested at the
indicated times. GPR84 TaqMan analysis was performed essentially as
described in Part A of Example 15, infra. Multiple replicates were
obtained from different individual donors. Data are expressed as
the mean of the ratio of the GPR84 signal to the HS9 (Human
ribosomal protein S9, housekeeping gene) signal .+-.SEM.
[0832] From FIG. 3A, it is apparent that overnight incubation of
primary human monocytes with TNF.alpha. under the conditions of the
assay led to a significant increase in GPR84 mRNA expression
(p=0.0252 by Student's t test, with a value of p<0.05 considered
significant). From FIG. 3B, it is apparent that overnight
incubation of primary monocytes with IFN.gamma. under the
conditions of the assay led to a dose-dependent increase in GPR84
mRNA expression.
Example 12
Analysis of GPR84 mRNA Expression in Rheumatoid Arthritis Synovium
by In Situ Hybridization
[0833] The expression of GPR84 mRNA in rheumatoid arthritis
synovial tissue was examined by in situ hybridization. Sections of
human tissue obtained from biopsy were embedded in paraffin and
subjected to in situ hybridization as described here.
[0834] Tissue sections were deparaffinized and rehydrated, followed
by acetylation to quench nonspecific binding of probe to tissue.
Single-stranded sense and anti-sense .sup.33P-labeled RNA probes
were generated by in vitro transcription from the cDNA encoding
human GPR84 (the 820 bp cDNA fragment corresponding to nucleotides
372 to 1191 of SEQ ID NO:1). Hybridization with the probes was
performed at 55.degree. C. for 16 h, after which the sections were
washed in 0.1.times.SSC buffer, followed by digestion with
ribonuclease A. Slides were then washed in 2.times.SSC, 1.times.SSC
and 0.5.times.SSC (5 min) prior to incubation in 0.1.times.SSC at
65.degree. C. for 1 hour. Following incubation, slides were washed
in 0.1.times.SSC (room temp) and dehydrated. Slides were exposed to
film for 3-5 days and images were captured with MTI CCD camera
(Compix Inc.). Tissues were stained with hematoxalin and eosin for
histology, including for the characterization of leukocyte
infiltration. CD68.sup.+ macrophages were identified by
immunohistochemistry.
[0835] GPR84 mRNA expression in rheumatoid arthritis synovial
tissue was found to be associated with areas of leukocyte
infiltration. A representative analysis is shown in FIG. 4. The
sense probe (Panel A) did not show hybridization, evidencing the
specificity of the GPR84 signal obtained using the antisense probe
(Panel B). Analysis of the boxed area in Panel B at increased
magnification showed that the area of high GPR84 mRNA expression
corresponded to an area of high infiltration by CD68.sup.+
macrophages (Panels C and D).
Example 13
Identification of Candidate Compounds as Agonists of GPR84
[0836] A. Identification of Compound 1 as an Agonist of GPR84
[0837] Melanophores were transiently transfected with plasmid DNA
encoding human GPR84 and used in aggregation assay. Cells were
cultured for 48 hours, plated into assay plates and initial
absorbance readings collected (Ai). Serial diluted compound was
added to assay plates. After a one hour incubation, a final
absorbance reading was collected (Af). Absorbance was calculated
(Af-Ai)-1 and plotted as percent of positive control versus molar
compound concentration. These results demonstrate that Compound 1
is a potent agonist at GPR84 in vitro. These results also are
consistent with GPR84 being a Gi-coupled receptor. In the
representative melanophore assay shown in FIG. 5A, Compound 1 was
found to have an EC.sub.50 at GPR84 of 4.36 nM. As Compound 1 has
an EC.sub.50 at GPR88 of greater than 100 .mu.M (not shown),
Compound 1 is a selective GPR84 agonist.
[0838] B. Identification of Compound 2 as an Agonist of GPR84
[0839] Melanophores were transiently transfected with plasmid DNA
encoding human GPR84 and used in aggregation assay. Cells were
cultured for 48 hours, plated into assay plates and initial
absorbance readings collected (Ai). Serial diluted compound was
added to assay plates. After a one hour incubation, a final
absorbance reading was collected (Af). Absorbance was calculated
(Af-Ai)-1 and plotted as percent of positive control versus molar
compound concentration. These results demonstrate that Compound 2
is a potent agonist at GPR84 in vitro. These results also are
consistent with GPR84 being a Gi-coupled receptor. In the
representative melanophore assay shown in FIG. 5B, Compound 2 was
found to have an EC.sub.50 at GPR84 of 48.2 nM. As Compound 2 has
an EC.sub.50 at GPR88 of greater than 10 .mu.M (not shown),
Compound 2 is a selective GPR84 agonist.
[0840] C. Identification of Compound 3 as an Agonist of GPR84
[0841] Melanophores were transiently transfected with plasmid DNA
encoding human GPR84 and used in aggregation assay. Cells were
cultured for 48 hours, plated into assay plates and initial
absorbance readings collected (Ai). Serial diluted compound was
added to assay plates. After a one hour incubation, a final
absorbance reading was collected (Af). Absorbance was calculated
(Af-Ai)-1 and plotted as percent of positive control versus molar
compound concentration. These results demonstrate that Compound 3
is a potent agonist at GPR84 in vitro. These results also are
consistent with GPR84 being a Gi-coupled receptor. In the
representative melanophore assay shown in FIG. 5C, Compound 3 was
found to have an EC.sub.50 at GPR84 of 30.8 nM. As Compound 3 has
an EC.sub.50 at GPR88 of greater than 10 .mu.M, Compound 3 is a
selective GPR84 agonist.
Example 14
Evidence for GPR84 as a Gi-coupled GPCR
[0842] A. Agonist to GPR84 Stimulates GTP.gamma.S Binding to
Membrane
[0843] GTP.gamma.S assay of GPR84 activity was carried out.
Membranes prepared from GPR84 transfected CHO-KI cells were
dissolved in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10
mM MgCl.sub.2) and homogenized briefly using a polytron. Membrane
protein concentration was determined by Bradford assay and the
membranes were diluted to 0.4 mg/ml in binding buffer. 50 ul of the
membranes were pre-incubated with Compound 1 in the absence or
presence of pertussis toxin (PTX) diluted in assay buffer
containing 40 mM GDP (final [GDP] was 10 mM) in Wallac Scintistrip
plates for 10 minutes before the addition of 25 .mu.l of 0.3 nM
.sup.35S-GTP.gamma.S. Binding was allowed to proceed for one hour
before centrifuging the plates at room temperature to pellet the
membrane and subsequent counting in a TopCount scintillation
counter.
[0844] In the representative GTP.gamma.S assay shown in FIG. 6A,
Compound 1 was found to have an EC.sub.50 at GPR84 of 540 nM.
Pertussis toxin was found to inhibit the stimulation of GTP.gamma.S
binding to membrane by Compound 1, consistent with GPR84 being a
Gi-coupled receptor (not shown).
[0845] B. Agonist to GPR84 Deceases Intracellular Camp
[0846] Thyroid-stimulating hormone (TSH, or thyrotropin) receptor
(TSHR) causes the accumulation of intracellular cAMP on activation
by its ligand TSH. An effective technique for measuring the
decrease in production of cAMP corresponding to activation of a
Gi-coupled receptor is to co-transfect TSHR with the Gi-coupled
receptor and to carry out the assay in the presence of TSH to raise
the level of basal cAMP, whereby TSHR acts as a "signal window
enhancer." Such an approach was used here.
[0847] Cyclase assay of GPR84 activity was carried out. 293 cells
were transfected with either vector alone or vector containing
polynucleotide encoding GPR84. 48 hrs after transfection, 50,000
cells were treated for 1 hr with or without Compound 1 as GPR84
agonist and analyzed using the cAMP Flashplate kit from Perkin
Elmer.
[0848] Compound 1 was found to decrease the level of intracellular
cAMP (FIG. 6B). The results are consistent with GPR84 being a
Gi-coupled receptor.
[0849] C. Agonist to GPR84 Increases IP.sub.3 Accumulation in the
Presence of Gq(DEL)/Gi Chimeric G Protein
[0850] IP.sub.3 Assay of GPR84 activity was carried out. 293 cells
were transfected with either vector alone or vector containing
polynucleotide encoding GPR84. The 293 cells comprised or did not
comprise Gq(del)/Gi chimeric G protein. 24 hrs after transfection,
cells were plated into 96 well plates, labeled overnight with
.sup.3H-myoinositol. The following day, cells were stimulated for 3
hrs with 10 .mu.M Compound 1 as GPR84 agonist or vehicle and IP
accumulation was measured.
[0851] Compound 1 was found to increase the level of intracellular
IP accumulation in 293 cells comprising Gq(del)/Gi chimeric G
protein, but not in 293 cells which did not comprise Gq(del)/Gi
(FIG. 6C). The results are consistent with GPR84 being a Gi-coupled
receptor.
Example 15
Agonist to GPR84 Selectively Modulates Gene Expression in
Monocytes/Macrophages
[0852] A. Agonist to GPR84 Increases Steady-State ABCA1 mRNA Level
in Human Macrophage
[0853] The effect of Compound 1 treatment on steady-state ABCA1
mRNA level in human macrophage was assessed. Macrophages were
derived from monocytes as previously indicated. Cells were treated
for 24 hours with vehicle or Compound 1. Vehicle was dimethyl
sufloxide (DMSO); the final concentration of DMSO in the culture
was 0.1% (v/v). Total RNA was extracted from the cells and analyzed
by TaqMan analysis as previously indicated to assess steady-state
ABCA1 mRNA level. Data are the average and SD of three
replicates.
[0854] The qPCR reactions were prepared from the cDNA using the
Bio-Rad qPCR kit, following the manufacturer's protocol. The qPCR
reactions were performed using the ABI Prism 7900HT machine with
the following temperatures and times: 50.degree. C.-2 min,
95.degree. C.-10 min followed by 40 cycles of 95.degree. C.-15 sec
and 60.degree. C.-1 min.
Primers and probe sequences used for qPCR analysis human ABCA1 are
as follows:
TABLE-US-00006 (SEQ ID NO: 21) Forward primer
5'-TCCAGGCCAGTACGGAATTC-3' (SEQ ID NO: 22) Reverse primer
5'-ACTTTCCTCGCCAAACCAGTAG-3' (SEQ ID NO: 23) Taqman MGB Probe
5'-CTGGTATTTTCCTTGCACCAA-3'
[0855] Compound 1 was found to increase the steady-state level of
ABCA1 mRNA in human macrophage (FIG. 7A).
[0856] B. Agonist to GPR84 Selectively Modulates Steady-State
Chemokine mRNA Level in Human Macrophage
[0857] The effect of Compound 1 treatment on steady state MCP-1 and
IL-8 mRNA level in human macrophage was assessed by microarray
analysis. Human peripheral blood monocytes were isolated from buffy
coat by dextran and ficoll purification. Monocytes were then
purified using a monocyte isolation kit from Miltenyi Biotec.
Monocytes were cultured for 7 days with 10 ng/ml GM-CSF to derive
macrophage cells. Cells were treated with vehicle or 10 .mu.M
Compound 1 for 24 hours. MCP-1 and IL-8 steady-state mRNA levels
were assessed by microarray analysis. Vehicle was dimethyl
sufloxide (DMSO); the final concentration of DMSO in the culture
was 0.1% (v/v). Expression levels are the average and SD of two
replicates.
[0858] Compound 1 was found to decrease the steady-state level of
MCP-1 mRNA and to increase the steady-state level of IL-8 mRNA in
human macrophage (FIG. 7B).
[0859] C. Agonist to GPR84 does not Increase TNF.alpha. Secretion
in Human Macrophage
[0860] The effect of Compound 1 treatment TNF.alpha. secretion by
human macrophage in the absence or presence of LPS was assessed.
Human peripheral blood monocytes were isolated from buffy coat by
dextran and ficoll purification. Monocytes were then purified using
a monocyte isolation kit from Miltenyi Biotec. Monocytes were
cultured for 7 days with 10 ng/ml GM-CSF to derive macrophage
cells. Cells were treated with vehicle or 10 .mu.M Compound 1 for
24 hrs. Vehicle was dimethyl sufloxide (DMSO); the final
concentration of DMSO in the culture was 0.1% (v/v). Cells were
then stimulated with the indicated concentrations of LPS for 24
hours. Supernatants were collected and TNF.alpha. levels were
assayed using a human specific TNF.alpha. ELISA kit from
Biosource.
[0861] Compound 1 was found to have no effect on TNF.alpha.
secretion by human macrophage (FIG. 7C) under the conditions of the
assay.
[0862] D. Agonist to GPR84 does not Increase TNF.alpha. Secretion
in Mouse Macrophage
[0863] The effect of Compound 1 treatment on TNF.alpha. secretion
by the mouse macrophage cell line RAW in the absence or presence of
LPS was assessed. RAW cells were cultured to subconfluency. Cells
were treated with vehicle or 1 .mu.M Compound 1 for 24 hours.
Vehicle was dimethyl sufloxide (DMSO); the final concentration of
DMSO in the culture was 0.1% (v/v). Cells were then stimulated with
the indicated concentrations of LPS for 24 hours. Supernatants were
collected and TNF.alpha. levels were assayed using a mouse specific
TNF.alpha. ELISA kit from Biosource.
[0864] Compound 1 was found to have no effect on TNF.alpha.
secretion by mouse macrophage cell line RAW (FIG. 7D) under the
conditions of the assay.
Example 16
In Vivo Effects of a GPR84 Agonist on Atherogenesis in Mice
[0865] Male apoE.sup.-/- mice (Jackson Laboratory, Bar Harbor, Me.)
are fed a normal chow. The apolipoprotein E-deficient
(apoE.sup.-/-) mouse is an established animal model of
atherosclerosis, developing extensive atherosclerotic lesions on a
chow diet [Zhang et al, Science (1992) 258:468-471]. At the age of
12 weeks, an agonist of GPR84 having agonist activity at mouse
GPR84 or vehicle alone is injected daily into the tail vein. A
preferred dose of the GPR84 agonist is 0.1-100 mg/kg. Other
preferred dose is selected from the group consisting of 0.1 mg/kg,
0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0866] The mice are anesthetized with an intraperitoneal injection
of pentobarbital (50 mg/kg), and the hearts, which contain the
aoritic sinus and aortic arch, are harvested 14 days after the
injection of the GPR84 agonist. Hearts are also harvested from
uninjected mice at the start of the experiment. Ten mice are used
for each of the three experimental groups.
[0867] The frozen cross-sections (10 .mu.M thick) of aortic sinus
embedded in Optimal Cutting Temperature (OCT; Sakura Finetechnical
Co., Ltd) compound after overnight fixation in 10% formalin are
mounted on slides. For the analysis of plaque size, 3 sections (100
.mu.M apart) from each mouse were stained with Oil Red O. The
lesion size and the diameter of lipid droplets in the lesions are
quantified with an image analyzing computer software, and the mean
values are determined. The mean value for the group injection with
the GPR84 agonist is compared with the mean value for the group
injected with vehicle alone.
[0868] Data are presented as means.+-.SEM and are analyzed by
Student's t test or the Mann-Whitney U test, depending on their
distribution pattern. A value of P<0.05 is considered
statistically significant. See, e.g., Okamoto et al, Circulation
(2002) 106:2767-2770.
[0869] These results can demonstrate that the GPR84 agonist is an
inhibitor of atherogenesis.
[0870] It is expressly contemplated that in other embodiment a
GPR84 agonist can be shown to be an inhibitor of atherogenesis in
the ApoE.sup.-/- mice using a non-invasive in vivo technique [see,
e.g., Fayad et al, Circulation (1998) 98:1541-1547]. It is
expressly contemplated that in other embodiment, administration of
the agonist is other than intravenous, for example that
administration of the agonist is intraperitoneal or oral. It is
expressly contemplated that in other embodiment, treatment begins
other than at 12 weeks of age, either earlier or later than at 12
weeks of age. It is expressly contemplated that in other embodiment
treatment continues for less than or more than 14 days. It is
expressly contemplated that injection may be other than daily.
Example 17
In Vivo Effects of a GPR84 Agonist on Chronic Inflammatory
Arthritis in Mice
[0871] Both male and female MRL-lpr mice (Jackson Laboratory, Bar
Harbor, Me.) are used at 13-14 weeks of age. MRL-lpr mice
spontaneously develop a chronic inflammatory arthritis with similar
characteristics to human rheumatoid arthritis including cell
infiltration, pannus formation, bone and cartilage breakdown, and
the presence of serum rheumatoid factor. The disease normally
develops towards the end of the animal's life span; however,
injection with complete Freund's adjuvant (CFA) initiates early
onset and increases the severity of arthritis [Rakay et al, J
Immunol (1993) 151:5081-5087].
[0872] On Day 0 of each experiment, all groups of mice are injected
with CFA intradermally into a thoracic and an inguinal site with
0.05 ml CFA supplemented to 10 mg/ml with heat inactivated
Mycobacterium tuberculosis H37 Ra (Difco, Detroit, Mich.).
Immediately, an agonist of GPR84 having agonist activity at mouse
GPR84 or vehicle alone is injected daily into the tail vein. A
preferred dose of the GPR84 agonist is 0.1-100 mg/kg. Other
preferred dose is selected from the group consisting of 0.1 mg/kg,
0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0873] Treatment is continued for 30 days. For quantifying
swelling, ankle widths are measured with a micrometer. The
statistical comparison of paired sets of ankle width measurements
is carried out using the Student's t test.
[0874] Histopathological Analysis
[0875] At day 30 after CFA priming, the hind paws are fixed in
buffered formalin. After decalcification in 10% formic acid for 48
hours, the tissues are processed for paraffin embedding.
[0876] Serial sections of the tarso-metatarsal joints are cut to a
thickness of 5 mm and stained with hematoxylin and eosin. Sections
are examined by an individual without knowledge of the experimental
protocol. A minimum of 10 sections/joint are assessed and scored to
provide a semiquantitative measure of subsynovial inflammation (0,
normal; 1 focal inflammatory infiltrates; 2, inflammatory
infiltrate that dominates the cellular histology), synovial
hyperplasia (0, normal; 1, a continuous, minimum three-layer thick,
synovial lining seen in one joint; 2, minimum three-layer thick,
synovial lining detetected in several joints), pannus formation and
cartilage erosion (0, normal; 1, pannus partially covered cartilage
surfaces without evident cartilage loss; 2, pannus connected to
evident cartilage loss), bone destruction (0, normal; 1, detectable
destruction of bone by the pannus or osteoclast activity; 2, the
pannus or osteoclast activity has destroyed a significant part of
the bone), and finally, overall pathology is the overall assessment
derived by the summation of the values for these criteria [see,
e.g., Gong et al, J Exp Med (1997) 186:131-137]. Statistical
analysis of the histopathology indices is done using the Student's
t test.
[0877] These results can demonstrate that the GPR84 agonist is an
inhibitor of a chronic inflammatory arthritis, for example that the
GPR84 agonist is an inhibitor of rheumatoid arthritis.
[0878] It is expressly contemplated that in other embodiment,
administration of the agonist is other than intravenous, for
example that administration of the agonist is intraperitoneal or
oral. It is expressly contemplated that in other embodiment,
treatment begins other than at 13-14 weeks of age, either earlier
or later than at 13-14 weeks of age. It is expressly contemplated
that in other embodiment treatment continues for less than or more
than 30 days. It is expressly contemplated that injection may be
other than daily.
Example 18
Yeast Reporter Assay for GPR84 Agonist Activity
[0879] The yeast cell-based reporter assays have previously been
described in the literature (e.g., see Miret et al, J Biol Chem
(2002) 277:6881-6887; Campbell et al, Bioorg Med Chem Lett (1999)
9:2413-2418; King et al, Science (1990) 250:121-123; WO 99/14344;
WO 00/12704; and U.S. Pat. No. 6,100,042). Briefly, yeast cells
have been engineered such that the endogenous yeast G-alpha (GPA1)
has been deleted and replaced with G-protein chimeras constructed
using multiple techniques. Additionally, the endogenous yeast
alpha-cell GPCR, Step 3 has been deleted to allow for a homologous
expression of a mammalian GPCR of choice. In the yeast, elements of
the pheromone signaling transduction pathway, which are conserved
in eukaryotic cells (for example, the mitogen-activated protein
kinase pathway), drive the expression of Fus1. By placing
.beta.-galactosidase (LacZ) under the control of the Fus1 promoter
(Fus1p), a system has been developed whereby receptor activation
leads to an enzymatic readout.
[0880] Yeast cells are transformed by an adaptation of the lithium
acetate method described by Agatep et al (Agatep et al, 1998,
Transformation of Saccharomyces cerevisiae by the lithium
acetate/single-stranded carrier DNA/polyethylene glycol
(LiAc/ss-DNA/PEG) protocol. Technical Tips Online, Trends Journals,
Elsevier). Briefly, yeast cells are grown overnight on yeast
tryptone plates (YT). Carrier single-stranded DNA (10 .mu.g), 2
.mu.g of each of two Fus1p-LacZ reporter plasmids (one with URA
selection marker and one with TRP), 2 .mu.g of GPR84 (e.g., human
receptor) in yeast expression vector (2 .mu.g origin of
replication) and a lithium acetate/polyethylene glycol/TE buffer is
pipetted into an Eppendorf tube. The yeast expression plasmid
containing the receptor/no receptor control has a LEU marker. Yeast
cells are inoculated into this mixture and the reaction proceeds at
30.degree. C. for 60 min. The yeast cells are then heat-shocked at
42.degree. C. for 15 min. The cells are then washed and spread on
selection plates. The selection plates are synthetic defined yeast
media minus LEU, URA and TRP (SD-LUT). After incubating at
30.degree. C. for 2-3 days, colonies that grow on the selection
plates are then tested in the LacZ assay.
[0881] In order to perform fluorimetric enzyme assays for
.beta.-galactosidase, yeast cells carrying the subject GPR84
receptor are grown overnight in liquid SD-LUT medium to an
unsaturated concentration (i.e. the cells are still dividing and
have not yet reached stationary phase). They are diluted in fresh
medium to an optimal assay concentration and 90 .mu.l of yeast
cells are added to 96-well black polystyrene plates (Costar). Test
compounds, dissolved in DMSO and diluted in a 10% DMSO solution to
10.times. concentration, are added to the plates and the plates
placed at 30.degree. C. for 4 h. After 4 h, the substrate for the
.beta.-galactosidase is added to each well. In these experiments,
Fluorescein di(.beta.-D-galactopyranoside) is used (FDG), a
substrate for the enzyme that releases fluorescein, allowing a
fluorimetric read-out. 20 .mu.l per well of 500 .mu.M FDG/2.5%
Triton X100 is added (the detergent is necessary to render the
cells permeable). After incubation of the cells with the substrate
for 60 min, 20 .mu.l per well of 1 .mu.M sodium carbonate is added
to terminate the reaction and enhance the fluorescent signal. The
plates are then read in a fluorimeter at 485/535 nm.
[0882] An increase in fluorescent signal in GPR84-transformed yeast
cells over that in yeast cells transformed with empty vector is
indicative of a test compound being a compound that stimulates
GPR84 receptor functionality. In certain embodiments, compounds of
the invention give an increase in fluorescent signal above that of
the background signal (the signal obtained in the presence of
vehicle alone).
Example 19
Receptor Binding Assay
[0883] A test compound can be evaluated for its ability to reduce
formation of the complex between a compound known to be a ligand of
a G protein-coupled receptor of the invention and the receptor. In
certain embodiments, the known ligand is radiolabeled. The
radiolabeled known ligand can be used in a screening assay to
identify/evaluate compounds. In general terms, a newly synthesized
or identified compound (i.e., test compound) can be evaluated for
its ability to reduce binding of the radiolabeled known ligand to
the receptor, by its ability to reduce formation of the complex
between the radiolabeled known ligand and the receptor.
[0884] In other aspect, a test compound can be radiolabeled and
shown to be a ligand of a subject GPCR of the invention by
evaluating its ability to bind to a cell comprising the subject
GPCR or to membrane comprising the subject GPCR.
[0885] A level of specific binding of the radiolabled known ligand
in the presence of the test compound less than a level of specific
binding of the radiolabeled known ligand in the absence of the test
compound is indicative of less of the complex between said
radiolabeled known ligand and said receptor being formed in the
presence of the test compound than in the absence of the test
compound.
Assay Protocol for Detecting the Complex Between a Compound Known
to be a Ligand of a G Protein-Coupled Receptor of the Invention and
the Receptor
[0886] A. Preparation of the Receptor
[0887] 293 cells are transiently transfected with 10 ug expression
vector comprising a polynucleotide encoding a G protein-coupled
receptor of the invention using 60 ul Lipofectamine (per 15-cm
dish). The transiently transfected cells are grown in the dish for
24 hours (75% confluency) with a media change and removed with 10
ml/dish of Hepes-EDTA buffer (20 mM Hepes+10 mM EDTA, pH 7.4). The
cells are then centrifuged in a Beckman Coulter centrifuge for 20
minutes, 17,000 rpm (JA-25.50 rotor). Subsequently, the pellet is
resuspended in 20 mM Hepes+1 mM EDTA, pH 7.4 and homogenized with a
50-ml Dounce homogenizer and again centrifuged. After removing the
supernatant, the pellets are stored at -80.degree. C., until used
in binding assay. When used in the assay, membranes are thawed on
ice for 20 minutes and then 10 mL of incubation buffer (20 mM
Hepes, 1 mM MgCl.sub.2, 100 mM NaCl, pH 7.4) added. The membranes
are then vortexed to resuspend the crude membrane pellet and
homogenized with a Brinkmann PT-3100 Polytron homogenizer for 15
seconds at setting 6. The concentration of membrane protein is
determined using the BRL Bradford protein assay.
[0888] B. Binding Assay
[0889] For total binding, a total volume of 50 ul of appropriately
diluted membranes (diluted in assay buffer containing 50 mM Tris
HCl (pH 7.4), 10 mM MgCl.sub.2, and 1 mM EDTA; 5-50 ug protein) is
added to 96-well polyproylene microtiter plates followed by
addition of 100 ul of assay buffer and 50 ul of a radiolabeled
known ligand. For nonspecific binding, 50 ul of assay buffer is
added instead of 100 ul and an additional 50 ul of 10 uM said known
ligand which is not radiolabeled is added before 50 ul of said
radiolabeled known ligand is added. Plates are then incubated at
room temperature for 60-120 minutes. The binding reaction is
terminated by filtering assay plates through a Microplate Devices
GF/C Unifilter filtration plate with a Brandell 96-well plate
harvestor followed by washing with cold 50 mM Tris HCl, pH 7.4
containing 0.9% NaCl. Then, the bottom of the filtration plate are
sealed, 50 ul of Optiphase Supermix is added to each well, the top
of the plates are sealed, and plates are counted in a Trilux
MicroBeta scintillation counter. For determining whether less of
the complex between said radiolabeled known ligand and said
receptor is formed in the presence of a test compound, instead of
adding 100 ul of assay buffer, 100 ul of appropriately diluted said
test compound is added to appropriate wells followed by addition of
50 ul of said radiolabled known ligand.
Sequence CWU 1
1
2311191DNAHomo Sapiens 1atgtggaaca gctctgacgc caacttctcc tgctaccatg
agtctgtgct gggctatcgt 60tatgttgcag ttagctgggg ggtggtggtg gctgtgacag
gcaccgtggg caatgtgctc 120accctactgg ccttggccat ccagcccaag
ctccgtaccc gattcaacct gctcatagcc 180aacctcacac tggctgatct
cctctactgc acgctccttc agcccttctc tgtggacacc 240tacctccacc
tgcactggcg caccggtgcc accttctgca gggtatttgg gctcctcctt
300tttgcctcca attctgtctc catcctgacc ctctgcctca tcgcactggg
acgctacctc 360ctcattgccc accctaagct ttttccccaa gttttcagtg
ccaaggggat agtgctggca 420ctggtgagca cctgggttgt gggcgtggcc
agctttgctc ccctctggcc tatttatatc 480ctggtacctg tagtctgcac
ctgcagcttt gaccgcatcc gaggccggcc ttacaccacc 540atcctcatgg
gcatctactt tgtgcttggg ctcagcagtg ttggcatctt ctattgcctc
600atccaccgcc aggtcaaacg agcagcacag gcactggacc aatacaagtt
gcgacaggca 660agcatccact ccaaccatgt ggccaggact gatgaggcca
tgcctggtcg tttccaggag 720ctggacagca ggttagcatc aggaggaccc
agtgagggga tttcatctga gccagtcagt 780gctgccacca cccagaccct
ggaaggggac tcatcagaag tgggagacca gatcaacagc 840aagagagcta
agcagatggc agagaaaagc cctccagaag catctgccaa agcccagcca
900attaaaggag ccagaagagc tccggattct tcatcggaat ttgggaaggt
gactcgaatg 960tgttttgctg tgttcctctg ctttgccctg agctacatcc
ccttcttgct gctcaacatt 1020ctggatgcca gagtccaggc tccccgggtg
gtccacatgc ttgctgccaa cctcacctgg 1080ctcaatggtt gcatcaaccc
tgtgctctat gcagccatga accgccaatt ccgccaagca 1140tatggctcca
ttttaaaaag agggccccgg agtttccata ggctccatta g 11912396PRTHomo
Sapiens 2Met Trp Asn Ser Ser Asp Ala Asn Phe Ser Cys Tyr His Glu
Ser Val 1 5 10 15 Leu Gly Tyr Arg Tyr Val Ala Val Ser Trp Gly Val
Val Val Ala Val 20 25 30 Thr Gly Thr Val Gly Asn Val Leu Thr Leu
Leu Ala Leu Ala Ile Gln 35 40 45 Pro Lys Leu Arg Thr Arg Phe Asn
Leu Leu Ile Ala Asn Leu Thr Leu 50 55 60 Ala Asp Leu Leu Tyr Cys
Thr Leu Leu Gln Pro Phe Ser Val Asp Thr 65 70 75 80 Tyr Leu His Leu
His Trp Arg Thr Gly Ala Thr Phe Cys Arg Val Phe 85 90 95 Gly Leu
Leu Leu Phe Ala Ser Asn Ser Val Ser Ile Leu Thr Leu Cys 100 105 110
Leu Ile Ala Leu Gly Arg Tyr Leu Leu Ile Ala His Pro Lys Leu Phe 115
120 125 Pro Gln Val Phe Ser Ala Lys Gly Ile Val Leu Ala Leu Val Ser
Thr 130 135 140 Trp Val Val Gly Val Ala Ser Phe Ala Pro Leu Trp Pro
Ile Tyr Ile 145 150 155 160 Leu Val Pro Val Val Cys Thr Cys Ser Phe
Asp Arg Ile Arg Gly Arg 165 170 175 Pro Tyr Thr Thr Ile Leu Met Gly
Ile Tyr Phe Val Leu Gly Leu Ser 180 185 190 Ser Val Gly Ile Phe Tyr
Cys Leu Ile His Arg Gln Val Lys Arg Ala 195 200 205 Ala Gln Ala Leu
Asp Gln Tyr Lys Leu Arg Gln Ala Ser Ile His Ser 210 215 220 Asn His
Val Ala Arg Thr Asp Glu Ala Met Pro Gly Arg Phe Gln Glu 225 230 235
240 Leu Asp Ser Arg Leu Ala Ser Gly Gly Pro Ser Glu Gly Ile Ser Ser
245 250 255 Glu Pro Val Ser Ala Ala Thr Thr Gln Thr Leu Glu Gly Asp
Ser Ser 260 265 270 Glu Val Gly Asp Gln Ile Asn Ser Lys Arg Ala Lys
Gln Met Ala Glu 275 280 285 Lys Ser Pro Pro Glu Ala Ser Ala Lys Ala
Gln Pro Ile Lys Gly Ala 290 295 300 Arg Arg Ala Pro Asp Ser Ser Ser
Glu Phe Gly Lys Val Thr Arg Met 305 310 315 320 Cys Phe Ala Val Phe
Leu Cys Phe Ala Leu Ser Tyr Ile Pro Phe Leu 325 330 335 Leu Leu Asn
Ile Leu Asp Ala Arg Val Gln Ala Pro Arg Val Val His 340 345 350 Met
Leu Ala Ala Asn Leu Thr Trp Leu Asn Gly Cys Ile Asn Pro Val 355 360
365 Leu Tyr Ala Ala Met Asn Arg Gln Phe Arg Gln Ala Tyr Gly Ser Ile
370 375 380 Leu Lys Arg Gly Pro Arg Ser Phe His Arg Leu His 385 390
395 31191DNAMus Musculus 3atgtggaaca gctcagatgc caacttctcc
tgctaccatg agtctgtgtt gggctatcga 60tactttgcaa ttatctgggg cgtggcagtg
gctgtgacag gcacggtggg caatgtgctc 120actctgctgg ccttggccat
tcgtcccaag ctccgaaccc gcttcaacct gctcattgcc 180aacctcaccc
tggctgatct actctactgc acgctcctgc agcctttctc cgtggacaca
240tacctccacc tccattggcg taccggcgcg gtcttctgta gaatatttgg
actcctcctc 300tttacttcca attctgtctc catcctcacc ctctgtctca
ttgctctagg acgctacctc 360ctcattgccc accctaagct ctttccccag
gttttcagtg ccaaggggat cgtgctggca 420ctggtgggca gctgggttgt
gggggtgacc agctttgccc ccctctggaa tgtttttgtc 480ttggtgccag
ttgtctgcac ctgcagcttt gaccgcatgc gaggccggcc ttacaccacc
540atcctcatgg gcatctactt tgtgcttggg ctcagcagcg tgggcgtctt
ctactgcctc 600atccaccggc aagtgaagcg tgcggctcga gcactggacc
aatacgggct gcatcaggcc 660agcatccgct ctcatcaggt ggctgggaca
caagaagcca tgcctggcca cttccaggag 720ctagacagcg gggttgcctc
aagagggccc agcgagggga tttcatctga gccagtcagt 780gctgcgacca
cgcagaccct ggaaggtgat tcgtcagaag ctgggggcca gggcattaga
840aaggcagctc aacagatcgc agagagaagc cttccagaag tgcatcgcaa
gccccgggaa 900actgcaggag ctcgcagagc cacagatgcc ccatcagagt
tcgggaaggt gacccgtatg 960tgcttcgcag tgttcctctg cttcgccctc
agctacatcc ccttcctgtt gctcaacatt 1020ctggacgcca ggggccgtgc
tccacgagta gtgcacatgg tggctgccaa cctcacctgg 1080ctcaacagct
gcatcaaccc tgtgctctat gcagccatga accgccagtt tcgccacgcg
1140tatggctcca tcctgaaacg cgggccacag agtttccgcc ggttccatta a
11914396PRTMus Musculus 4Met Trp Asn Ser Ser Asp Ala Asn Phe Ser
Cys Tyr His Glu Ser Val 1 5 10 15 Leu Gly Tyr Arg Tyr Phe Ala Ile
Ile Trp Gly Val Ala Val Ala Val 20 25 30 Thr Gly Thr Val Gly Asn
Val Leu Thr Leu Leu Ala Leu Ala Ile Arg 35 40 45 Pro Lys Leu Arg
Thr Arg Phe Asn Leu Leu Ile Ala Asn Leu Thr Leu 50 55 60 Ala Asp
Leu Leu Tyr Cys Thr Leu Leu Gln Pro Phe Ser Val Asp Thr 65 70 75 80
Tyr Leu His Leu His Trp Arg Thr Gly Ala Val Phe Cys Arg Ile Phe 85
90 95 Gly Leu Leu Leu Phe Thr Ser Asn Ser Val Ser Ile Leu Thr Leu
Cys 100 105 110 Leu Ile Ala Leu Gly Arg Tyr Leu Leu Ile Ala His Pro
Lys Leu Phe 115 120 125 Pro Gln Val Phe Ser Ala Lys Gly Ile Val Leu
Ala Leu Val Gly Ser 130 135 140 Trp Val Val Gly Val Thr Ser Phe Ala
Pro Leu Trp Asn Val Phe Val 145 150 155 160 Leu Val Pro Val Val Cys
Thr Cys Ser Phe Asp Arg Met Arg Gly Arg 165 170 175 Pro Tyr Thr Thr
Ile Leu Met Gly Ile Tyr Phe Val Leu Gly Leu Ser 180 185 190 Ser Val
Gly Val Phe Tyr Cys Leu Ile His Arg Gln Val Lys Arg Ala 195 200 205
Ala Arg Ala Leu Asp Gln Tyr Gly Leu His Gln Ala Ser Ile Arg Ser 210
215 220 His Gln Val Ala Gly Thr Gln Glu Ala Met Pro Gly His Phe Gln
Glu 225 230 235 240 Leu Asp Ser Gly Val Ala Ser Arg Gly Pro Ser Glu
Gly Ile Ser Ser 245 250 255 Glu Pro Val Ser Ala Ala Thr Thr Gln Thr
Leu Glu Gly Asp Ser Ser 260 265 270 Glu Ala Gly Gly Gln Gly Ile Arg
Lys Ala Ala Gln Gln Ile Ala Glu 275 280 285 Arg Ser Leu Pro Glu Val
His Arg Lys Pro Arg Glu Thr Ala Gly Ala 290 295 300 Arg Arg Ala Thr
Asp Ala Pro Ser Glu Phe Gly Lys Val Thr Arg Met 305 310 315 320 Cys
Phe Ala Val Phe Leu Cys Phe Ala Leu Ser Tyr Ile Pro Phe Leu 325 330
335 Leu Leu Asn Ile Leu Asp Ala Arg Gly Arg Ala Pro Arg Val Val His
340 345 350 Met Val Ala Ala Asn Leu Thr Trp Leu Asn Ser Cys Ile Asn
Pro Val 355 360 365 Leu Tyr Ala Ala Met Asn Arg Gln Phe Arg His Ala
Tyr Gly Ser Ile 370 375 380 Leu Lys Arg Gly Pro Gln Ser Phe Arg Arg
Phe His 385 390 395 51191DNARattus Norvegicus 5atgtggaaca
gctcagatga caacttctcc tgctaccatg agtctgtatt gggctatcga 60tactttgcag
ttatctgggg catggtagtg gctgcaacag gcaccgtggg caatgtgctc
120accctgttgg ccttggccat ccgtcccaaa ctccgaaccc gtttcaacct
gctcattgcc 180aacctcaccc tggctgatct actctactgc acgctcctgc
agcctttctc cgtggacaca 240tacctccacc tccattggcg caccggcgcc
atcttctgta gaatattcgg actcctcctc 300tttacttcca attctgtctc
cattcttacc ctctgtctca ttgctctagg acgctacctt 360ctcattgccc
accctaagct ctttccccag gttttcagtg ccaaggggat cgtgctggca
420ctagtgggca gctgggttgt gggggtgacc agctttgccc ccctctggaa
tgtttatgtc 480ttggtgccag ttgtctgcac ctgcagcttt gaccgcgtgc
gaggccggcc ttacaccacc 540atcctcatgg gcatcttctt tgtggttggg
ctcagcagcg tgggcgtctt ctactgcctc 600atccaccgcc aagtgaagcg
tgcggctcga gcgctggaca aatatgggct gcaggaggcc 660agcatgcgct
cccatcaggt gtctgggaca catgaagctg tgccaggcca cttccaggag
720ctagacagcg ggcttgcatc aagaggtccc agcgaaggga tttcatctga
gccagtcagt 780gctgcgacga cacagaccct ggaaggtgat tcgtcagaag
cgggggacca gggcatgaga 840aaggcagctc agcagatctc agagagaagc
cttccagaag tgcatcgcaa gactggagga 900gctgcaggag cacgcagagc
cacggatgca ccatcggagt tcgggaaggt gacccgtatg 960tgctttgcag
tgttcctttg cttcgtcctc agctacatcc ctttcctgct gctcaacatt
1020ctggacgcca ggggccgcgc tccacgagta gtgcatatgg ttgctgccaa
cctcacctgg 1080ctcaacagct gcatcaaccc tgtgctctat gcagccatga
accgccagtt tcgccaggct 1140tatggctcca tcctgaaacg cgggccacag
agtttccgac ggttccatta g 11916396PRTRattus Norvegicus 6Met Trp Asn
Ser Ser Asp Asp Asn Phe Ser Cys Tyr His Glu Ser Val 1 5 10 15 Leu
Gly Tyr Arg Tyr Phe Ala Val Ile Trp Gly Met Val Val Ala Ala 20 25
30 Thr Gly Thr Val Gly Asn Val Leu Thr Leu Leu Ala Leu Ala Ile Arg
35 40 45 Pro Lys Leu Arg Thr Arg Phe Asn Leu Leu Ile Ala Asn Leu
Thr Leu 50 55 60 Ala Asp Leu Leu Tyr Cys Thr Leu Leu Gln Pro Phe
Ser Val Asp Thr 65 70 75 80 Tyr Leu His Leu His Trp Arg Thr Gly Ala
Ile Phe Cys Arg Ile Phe 85 90 95 Gly Leu Leu Leu Phe Thr Ser Asn
Ser Val Ser Ile Leu Thr Leu Cys 100 105 110 Leu Ile Ala Leu Gly Arg
Tyr Leu Leu Ile Ala His Pro Lys Leu Phe 115 120 125 Pro Gln Val Phe
Ser Ala Lys Gly Ile Val Leu Ala Leu Val Gly Ser 130 135 140 Trp Val
Val Gly Val Thr Ser Phe Ala Pro Leu Trp Asn Val Tyr Val 145 150 155
160 Leu Val Pro Val Val Cys Thr Cys Ser Phe Asp Arg Val Arg Gly Arg
165 170 175 Pro Tyr Thr Thr Ile Leu Met Gly Ile Phe Phe Val Val Gly
Leu Ser 180 185 190 Ser Val Gly Val Phe Tyr Cys Leu Ile His Arg Gln
Val Lys Arg Ala 195 200 205 Ala Arg Ala Leu Asp Lys Tyr Gly Leu Gln
Glu Ala Ser Met Arg Ser 210 215 220 His Gln Val Ser Gly Thr His Glu
Ala Val Pro Gly His Phe Gln Glu 225 230 235 240 Leu Asp Ser Gly Leu
Ala Ser Arg Gly Pro Ser Glu Gly Ile Ser Ser 245 250 255 Glu Pro Val
Ser Ala Ala Thr Thr Gln Thr Leu Glu Gly Asp Ser Ser 260 265 270 Glu
Ala Gly Asp Gln Gly Met Arg Lys Ala Ala Gln Gln Ile Ser Glu 275 280
285 Arg Ser Leu Pro Glu Val His Arg Lys Thr Gly Gly Ala Ala Gly Ala
290 295 300 Arg Arg Ala Thr Asp Ala Pro Ser Glu Phe Gly Lys Val Thr
Arg Met 305 310 315 320 Cys Phe Ala Val Phe Leu Cys Phe Val Leu Ser
Tyr Ile Pro Phe Leu 325 330 335 Leu Leu Asn Ile Leu Asp Ala Arg Gly
Arg Ala Pro Arg Val Val His 340 345 350 Met Val Ala Ala Asn Leu Thr
Trp Leu Asn Ser Cys Ile Asn Pro Val 355 360 365 Leu Tyr Ala Ala Met
Asn Arg Gln Phe Arg Gln Ala Tyr Gly Ser Ile 370 375 380 Leu Lys Arg
Gly Pro Gln Ser Phe Arg Arg Phe His 385 390 395
726DNAArtificialprimer 7actaagcttc tatcatgtgg aacagc
26818DNAArtificialprimer 8aggagacagt cctgaatt
1896PRTArtificialSynthetic peptide N-terminal of Gaq 9Thr Leu Glu
Ser Ile Met 1 5 105PRTArtificialSynthetic peptide C-terminal of Gaq
10Glu Tyr Asn Leu Val 1 5 115PRTArtificialSynthetic peptide
C-terminal of Gai 11Asp Cys Gly Leu Phe 1 5 1236DNAArtificialprimer
12gatcaagctt ccatggcgtg ctgcctgagc gaggag 361353DNAArtificialprimer
13gatcggatcc ttagaacagg ccgcagtcct tcaggttcag ctgcaggatg gtg
53141062DNAArtificialchimeric polynucleotide 14atggcgtgct
gcctgagcga ggaggccaag gaagcccgga ggatcaacga cgagatcgag 60cggcagctgc
gcagggacaa gcgcgacgcc cgccgggagc tcaagctgct gctgctgggg
120acaggggaga gtggcaagtc gaccttcatc aagcagatga ggatcatcca
cgggtcgggc 180tactctgacg aagacaagcg cggcttcacc aagctggtgt
atcagaacat cttcacggcc 240atgcaggcca tgatcagagc gatggacaca
ctcaagatcc catacaagta tgaacacaat 300aaggctcatg cacaattggt
tcgagaggtt gatgtggaga aggtgtctgc ttttgacgtc 360cccgactacg
cggcaataaa gagcttgtgg aatgatcctg gaatccagga gtgctacgac
420agacgacggg aatatcagtt atctgactct accaaatact atctgaatga
cttggaccgt 480gtagccgacc cttcctatct gcctacacaa caagacgtgc
ttagagttcg agtccccact 540acagggatca tcgaataccc ctttgactta
caaagtgtca ttttcagaat ggtcgatgta 600gggggccaaa ggtcagagag
aagaaaatgg atccactgct ttgaaaatgt cacctccatc 660atgtttctag
tagcgcttag cgaatatgat caagttcttg tggagtcaga caatgagaac
720cgcatggagg agagcaaagc actctttaga acaattatca cctacccctg
gttccagaac 780tcctctgtga ttctgttctt aaacaagaaa gatcttctag
aggagaaaat catgtattcc 840cacctagtcg actacttccc agaatatgat
ggaccccaga gagatgccca ggcagctcga 900gaattcatcc tgaaaatgtt
cgtggacctg aaccccgaca gtgacaaaat catctactcc 960cacttcacgt
gcgccacaga taccgagaac atccgcttcg tctttgcagc cgtcaaggac
1020accatcctgc agctgaacct gaaggactgc ggcctgttct aa
106215353PRTArtificialchimeric polypeptide 15Met Ala Cys Cys Leu
Ser Glu Glu Ala Lys Glu Ala Arg Arg Ile Asn 1 5 10 15 Asp Glu Ile
Glu Arg Gln Leu Arg Arg Asp Lys Arg Asp Ala Arg Arg 20 25 30 Glu
Leu Lys Leu Leu Leu Leu Gly Thr Gly Glu Ser Gly Lys Ser Thr 35 40
45 Phe Ile Lys Gln Met Arg Ile Ile His Gly Ser Gly Tyr Ser Asp Glu
50 55 60 Asp Lys Arg Gly Phe Thr Lys Leu Val Tyr Gln Asn Ile Phe
Thr Ala 65 70 75 80 Met Gln Ala Met Ile Arg Ala Met Asp Thr Leu Lys
Ile Pro Tyr Lys 85 90 95 Tyr Glu His Asn Lys Ala His Ala Gln Leu
Val Arg Glu Val Asp Val 100 105 110 Glu Lys Val Ser Ala Phe Asp Val
Pro Asp Tyr Ala Ala Ile Lys Ser 115 120 125 Leu Trp Asn Asp Pro Gly
Ile Gln Glu Cys Tyr Asp Arg Arg Arg Glu 130 135 140 Tyr Gln Leu Ser
Asp Ser Thr Lys Tyr Tyr Leu Asn Asp Leu Asp Arg 145 150 155 160 Val
Ala Asp Pro Ser Tyr Leu Pro Thr Gln Gln Asp Val Leu Arg Val 165 170
175 Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr Pro Phe Asp Leu Gln Ser
180 185 190 Val Ile Phe Arg Met Val Asp Val Gly Gly Gln Arg Ser Glu
Arg Arg 195 200 205 Lys Trp Ile His Cys Phe Glu Asn Val Thr Ser Ile
Met Phe Leu Val 210 215 220 Ala Leu Ser Glu Tyr Asp Gln Val Leu Val
Glu Ser Asp Asn Glu Asn 225 230 235 240 Arg Met Glu Glu Ser Lys Ala
Leu Phe Arg Thr Ile Ile Thr Tyr Pro 245 250 255 Trp Phe Gln Asn Ser
Ser Val Ile Leu Phe Leu Asn Lys Lys Asp Leu 260 265 270 Leu Glu Glu
Lys Ile Met Tyr Ser His Leu Val Asp Tyr Phe
Pro Glu 275 280 285 Tyr Asp Gly Pro Gln Arg Asp Ala Gln Ala Ala Arg
Glu Phe Ile Leu 290 295 300 Lys Met Phe Val Asp Leu Asn Pro Asp Ser
Asp Lys Ile Ile Tyr Ser 305 310 315 320 His Phe Thr Cys Ala Thr Asp
Thr Glu Asn Ile Arg Phe Val Phe Ala 325 330 335 Ala Val Lys Asp Thr
Ile Leu Gln Leu Asn Leu Lys Asp Cys Gly Leu 340 345 350 Phe
1622DNAArtificialprimer 16tcctttttgc ctccaattct gt
221718DNAArtificialprimer 17gcgtcccagt gcgatgag
181816DNAArtificialprimer 18tccatcctga ccctct
16191227DNAArtificialchimeric polynucleotide 19atgtacccat
acgacgtccc agactacgct ggaagcttgt ggaacagctc tgacgccaac 60ttctcctgct
accatgagtc tgtgctgggc tatcgttatg ttgcagttag ctggggggtg
120gtggtggctg tgacaggcac cgtgggcaat gtgctcaccc tactggcctt
ggccatccag 180cccaagctcc gtacccgatt caacctgctc atagccaacc
tcacactggc tgatctcctc 240tactgcacgc tccttcagcc cttctctgtg
gacacctacc tccacctgca ctggcgcacc 300ggtgccacct tctgcagggt
atttgggctc ctcctttttg cctccaattc tgtctccatc 360ctgaccctct
gcctcatcgc actgggacgc tacctcctca ttgcccaccc taagcttttt
420ccccaagttt tcagtgccaa ggggatagtg ctggcactgg tgagcacctg
ggttgtgggc 480gtggccagct ttgctcccct ctggcctatt tatatcctgg
tacctgtagt ctgcacctgc 540agctttgacc gcatccgagg ccggccttac
accaccatcc tcatgggcat ctactttgtg 600cttgggctca gcagtgttgg
catcttctat tgcctcatcc accgccaggt caaacgagca 660gcacaggcac
tggaccaata caagttgcga caggcaagca tccactccaa ccatgtggcc
720aggactgatg aggccatgcc tggtcgtttc caggagctgg acagcaggtt
agcatcagga 780ggacccagtg aggggatttc atctgagcca gtcagtgctg
ccaccaccca gaccctggaa 840ggggactcat cagaagtggg agaccagatc
aacagcaaga gagctaagca gatggcagag 900aaaagccctc cagaagcatc
tgccaaagcc cagccaatta aaggagccag aagagctccg 960gattcttcat
cggaatttgg gaaggtgact cgaatgtgtt ttgctgtgtt cctctgcttt
1020gccctgagct acatcccctt cttgctgctc aacattctgg atgccagagt
ccaggctccc 1080cgggtggtcc acatgcttgc tgccaacctc acctggctca
atggttgcat caaccctgtg 1140ctctatgcag ccatgaaccg ccaattccgc
caagcatatg gctccatttt aaaaagaggg 1200ccccggagtt tccataggct ccattag
122720408PRTArtificialchimeric polypeptide 20Met Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala Gly Ser Leu Trp Asn Ser 1 5 10 15 Ser Asp Ala
Asn Phe Ser Cys Tyr His Glu Ser Val Leu Gly Tyr Arg 20 25 30 Tyr
Val Ala Val Ser Trp Gly Val Val Val Ala Val Thr Gly Thr Val 35 40
45 Gly Asn Val Leu Thr Leu Leu Ala Leu Ala Ile Gln Pro Lys Leu Arg
50 55 60 Thr Arg Phe Asn Leu Leu Ile Ala Asn Leu Thr Leu Ala Asp
Leu Leu 65 70 75 80 Tyr Cys Thr Leu Leu Gln Pro Phe Ser Val Asp Thr
Tyr Leu His Leu 85 90 95 His Trp Arg Thr Gly Ala Thr Phe Cys Arg
Val Phe Gly Leu Leu Leu 100 105 110 Phe Ala Ser Asn Ser Val Ser Ile
Leu Thr Leu Cys Leu Ile Ala Leu 115 120 125 Gly Arg Tyr Leu Leu Ile
Ala His Pro Lys Leu Phe Pro Gln Val Phe 130 135 140 Ser Ala Lys Gly
Ile Val Leu Ala Leu Val Ser Thr Trp Val Val Gly 145 150 155 160 Val
Ala Ser Phe Ala Pro Leu Trp Pro Ile Tyr Ile Leu Val Pro Val 165 170
175 Val Cys Thr Cys Ser Phe Asp Arg Ile Arg Gly Arg Pro Tyr Thr Thr
180 185 190 Ile Leu Met Gly Ile Tyr Phe Val Leu Gly Leu Ser Ser Val
Gly Ile 195 200 205 Phe Tyr Cys Leu Ile His Arg Gln Val Lys Arg Ala
Ala Gln Ala Leu 210 215 220 Asp Gln Tyr Lys Leu Arg Gln Ala Ser Ile
His Ser Asn His Val Ala 225 230 235 240 Arg Thr Asp Glu Ala Met Pro
Gly Arg Phe Gln Glu Leu Asp Ser Arg 245 250 255 Leu Ala Ser Gly Gly
Pro Ser Glu Gly Ile Ser Ser Glu Pro Val Ser 260 265 270 Ala Ala Thr
Thr Gln Thr Leu Glu Gly Asp Ser Ser Glu Val Gly Asp 275 280 285 Gln
Ile Asn Ser Lys Arg Ala Lys Gln Met Ala Glu Lys Ser Pro Pro 290 295
300 Glu Ala Ser Ala Lys Ala Gln Pro Ile Lys Gly Ala Arg Arg Ala Pro
305 310 315 320 Asp Ser Ser Ser Glu Phe Gly Lys Val Thr Arg Met Cys
Phe Ala Val 325 330 335 Phe Leu Cys Phe Ala Leu Ser Tyr Ile Pro Phe
Leu Leu Leu Asn Ile 340 345 350 Leu Asp Ala Arg Val Gln Ala Pro Arg
Val Val His Met Leu Ala Ala 355 360 365 Asn Leu Thr Trp Leu Asn Gly
Cys Ile Asn Pro Val Leu Tyr Ala Ala 370 375 380 Met Asn Arg Gln Phe
Arg Gln Ala Tyr Gly Ser Ile Leu Lys Arg Gly 385 390 395 400 Pro Arg
Ser Phe His Arg Leu His 405 2120DNAArtificialprimer 21tccaggccag
tacggaattc 202222DNAArtificialprimer 22actttcctcg ccaaaccagt ag
222321DNAArtificialprobe 23ctggtatttt ccttgcacca a 21
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