U.S. patent application number 11/986225 was filed with the patent office on 2008-11-20 for ligand for g-protein coupled receptor gpr43 and uses thereof.
This patent application is currently assigned to Euroscreen s.a.. Invention is credited to Stephane Brezillon, Michel Detheux, Vincent Lannoy, Emmanuel LePoul, Marc Parmentier.
Application Number | 20080286806 11/986225 |
Document ID | / |
Family ID | 23359173 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080286806 |
Kind Code |
A1 |
LePoul; Emmanuel ; et
al. |
November 20, 2008 |
Ligand for G-protein coupled receptor GPR43 and uses thereof
Abstract
The present invention is related to the G-protein coupled orphan
receptor GPR43 and the identification of short chain fatty acids as
natural ligands of the receptor. The invention further relates to
assays for the identification of agents that modulate GPR43 ligand
binding and signalling activity, as well as compositions consisting
essentially of an isolated GPR43 polypeptide and an isolated short
chain fatty acid. The invention also relates to diagnostic methods
and kits that take advantage of the novel interaction of GPR43 with
short chain fatty acids.
Inventors: |
LePoul; Emmanuel; (Cessy,
FR) ; Detheux; Michel; (Marche-Lez-Ecausines, BE)
; Brezillon; Stephane; (Cortiontreue, FR) ;
Lannoy; Vincent; (Liernu, BE) ; Parmentier; Marc;
(Beersel, BE) |
Correspondence
Address: |
Edwards Angell Palmer & Dodge LLP
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
Euroscreen s.a.
|
Family ID: |
23359173 |
Appl. No.: |
11/986225 |
Filed: |
November 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10337992 |
Jan 7, 2003 |
7303889 |
|
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11986225 |
|
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60346396 |
Jan 7, 2002 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 37/02 20180101; A61P 7/06 20180101; A61P 25/32 20180101; A61P
39/02 20180101; A61K 31/00 20130101; A61P 1/04 20180101; A61K 31/19
20130101; G01N 2333/726 20130101; A61P 1/02 20180101; A61P 31/00
20180101; A61P 43/00 20180101; C07K 14/70567 20130101; A61P 29/00
20180101; C07K 14/705 20130101; A61P 1/16 20180101 |
Class at
Publication: |
435/7.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of detecting the presence of a disease characterized by
the dysregulation of GPR43, comprising: a) contacting a GPR43
polypeptide present in the membrane of a PMN cell with a short
chain fatty acid; b) measuring the binding of said GPR43
polypeptide to said short chain fatty acid; and c) comparing the
binding detected in step (b) with a standard, wherein a difference
in binding relative to said standard is indicative of the presence
of a disease characterized by the dysregulation of GPR43.
2. A method of detecting the presence of a disease characterized by
the dysregulation of GPR43, comprising: a) contacting a GPR43
polypeptide present in the membrane of a PMN cell with a short
chain fatty acid; b) measuring a signalling activity of said GPR43
polypeptide; and c) comparing the signalling activity detected in
step (b) with a standard, wherein a difference in binding relative
to said standard is indicative of the presence of a disease
characterized by the dysregulation of GPR43.
3. A method of diagnosing a disease or disorder characterized by
dysregulation of GPR43 signalling, said method comprising: a)
contacting a tissue sample comprising a GPR43 polypeptide with a
short chain fatty acid b) detecting binding of said short chain
fatty acid to said GPR43; and c) comparing the binding detected in
step (b) with a standard, wherein a difference in binding relative
to said standard is indicative of the presence of a disease
characterized by the dysregulation of GPR43.
4. A method of diagnosing a disease or disorder characterized by
dysregulation of GPR43 signaling, said method comprising: a)
contacting a tissue sample comprising a GPR43 polypeptide with a
short chain fatty acid b) detecting a signaling activity of said
GPR43 polypeptide in said tissue sample; and c) comparing the
signaling activity detected in step (b) with a standard, wherein a
difference in said signaling activity relative to said standard is
indicative of the presence of a disease characterized by the
dysregulation of GPR43.
5. The method of claim 1 or 3, wherein said measuring is performed
using a method selected from the group consisting of label
displacement, surface plasmon resonance, fluorescence resonance
energy transfer, fluorescence quenching, and fluorescence
polarization.
6. The method of claim 2 or 4, wherein said step of measuring a
signalling activity of said GPR43 polypeptide comprises detecting a
change in the level of a second messenger.
7. The method of claim 2 or 4, wherein the step of measuring a
signalling activity comprises measurement of guanine nucleotide
binding or exchange, adenylate cyclase activity, cAMP, Protein
Kinase C activity, phosphatidylinosotol breakdown, diacylglycerol,
inositol triphosphate, intracellular calcium, arachinoid acid, MAP
kinase activity, tyrosine kinase activity, or reporter gene
expression.
8. The method of claim 6, wherein said measuring a signaling
activity comprises using an aequorin-based assay.
Description
PRIORITY
[0001] This application is a Divisional of U.S. Ser. No.
10/337,992, filed Jan. 7, 2003, which claims priority to U.S.
Provisional Application No. 60/346,396, filed Jan. 7, 2002.
FIELD OF THE INVENTION
[0002] The present invention is related to the natural ligand for
an orphan G protein coupled receptor and methods of use.
BACKGROUND OF THE INVENTION AND STATE OF THE ART
[0003] G-protein coupled receptors (GPCRs) are proteins responsible
for transducing a signal within a cell. GPCRs have usually seven
transmembrane domains. Upon binding of a ligand to an
extra-cellular portion or fragment of a GPCR, a signal is
transduced within the cell that results in a change in a biological
or physiological property or behaviour of the cell. GPCRs, along
with G-proteins and effectors (intracellular enzymes and channels
modulated by G-proteins), are the components of a modular
signalling system that connects the state of intra-cellular second
messengers to extra-cellular inputs.
[0004] GPCR genes and gene products can modulate various
physiological processes and are potential causative agents of
disease. The GPCRs seem to be of critical importance to both the
central nervous system and peripheral physiological processes.
[0005] The GPCR protein superfamily is represented in five
families: Family I, receptors typified by rhodopsin and the
beta2-adrenergic receptor and currently represented by over 200
unique members; Family II, the parathyroid
hormone/calcitonin/secretin receptor family; Family III, the
metabotropic glutamate receptor family, Family IV, the CAMP
receptor family, important in the chemotaxis and development of D.
discoideum; and Family V, the fungal mating pheromone receptor such
as STE2.
[0006] G proteins represent a family of heterotrimeric proteins
composed of .alpha., .beta. and .gamma. subunits, that bind guanine
nucleotides. These proteins are usually linked to cell surface
receptors (receptors containing seven transmembrane domains) for
signal transduction. Indeed, following ligand binding to the GPCR,
a conformational change is transmitted to the G protein, which
causes the .alpha.-subunit to exchange a bound GDP molecule for a
GTP molecule and to dissociate from the .beta..gamma.-subunits.
[0007] The GTP-bound form of the .alpha., .beta. and
.gamma.-subunits typically functions as an effector-modulating
moiety, leading to the production of second messengers, such as
cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or
inositol phosphates.
[0008] Greater than 20 different types of .alpha.-subunits are
known in humans. These subunits associate with a small pool of
.beta. and .gamma. subunits. Examples of mammalian G proteins
include Gi, Go, Gq, Gs and Gt. G proteins are described extensively
in Lodish et al., Molecular Cell Biology (Scientific American Books
Inc., New York, N.Y., 1995; and also by Downes and Gautam, 1999,
The G-Protein Subunit Gene Families. Genomics 62:544-552), the
contents of both of which are incorporated herein by reference.
[0009] Known and uncharacterized GPCRs currently constitute major
targets for drug action and development. There are ongoing efforts
to identify new G protein coupled receptors which can be used to
screen for new agonists and antagonists having potential
prophylactic and therapeutic properties.
[0010] More than 300 GPCRs have been cloned to date, excluding the
family of olfactory receptors. Mechanistically, approximately
50-60% of all clinically relevant drugs act by modulating the
functions of various GPCRs (Cudermann et al., J. Mol. Med.,
73:51-63, 1995).
[0011] GPR43 is a member of the rhodopsin like receptors family,
cloned in 1997. It shows a homology of 38% with another orphan
GPCR, GPR41 and 27% with transmembrane domains of mouse PAR1
receptor. The gene encoding GPR43 coding gene is located on human
chromosome 19q31 (Sawzdargo et al., 1997). GPR43 has been described
as a gene induced by IL-9 in mouse cytokine dependent T helper cell
lines and bone marrow derived primary mast cells. In addition,
GPR43 mRNA transcription is stimulated in the lung, intestine and
stomach of transgenic mice overexpressing IL-9. GPR43 mRNA is also
induced in splenoytes by mitogens, such as concanavalin A, and this
induction is blocked by aminosterol compounds (see WO99/15656).
GPR43 polynucleotide and amino acid sequences are disclosed in U.S.
Pat. Nos. 5,910,430 and 6,180,365B1 and in WO00/28083, WO98/40483,
WO99/15656 and WO00/22129, each of which is incorporated herein by
reference.
[0012] Short chain fatty acids (SCFA) include but are not limited
to acetate, propionate, butyrate and valerate. SCFA are produced by
microbial fermentation in the hindgut in considerable amounts. Most
of the anions in hindgut contents are SCFA, mainly acetate,
propionate and butyrate. SCFA are rapidly absorbed, and the total
SCFA concentration in peripheral blood reaches 79 .mu.M (Cummings,
1987). Among the different SCFAs, acetate is the principal anion
and can also be produced in different tissues by biochemical
synthesis (Bergman, 1990). Acetate is present in the plasma at a
concentration of 59 to 85 .mu.M and its concentration can be
increased by 20 fold after ethanol administration (Lundquist et
al., 1960). It is believed that most plasma acetate is derived from
the splanchnic bed and is used by other tissues where it can
account for almost 7% of basal energy expenditure. Butyrate is
produced by bacterial fermentation of dietary fibers in the colon
lumen, and dramatically affects the proliferation of colon cancer
cells in in vitro experiments. Various periodontal and root canal
pathogens, such as the Bacteroides species, can produce significant
amounts of short chain fatty acids. (SCFA). Short-chain fatty acids
are also physiological regulators of growth and differentiation in
the gastrointestinal tract and can act as antibacterial agents.
There is some evidence that SCFA metabolism is involved in the
development of colitis ulcerosa, diverticulosis and colorectal
cancer. The differences between the effects of SCFA on cell
proliferation, differentiation and apoptosis of colonocytes in vivo
and in vitro indicate that in addition to direct effects of SCFA,
systemic effects such as neural and humoral factors are also of
crucial importance. The opposing effects of SCFA on proliferation
and apoptosis in normal colonocytes and in colon cancer cells
demonstrate possibilities for prevention and/or therapy of colonic
diseases.
SUMMARY OF THE INVENTION
[0013] The invention is based on the discovery that short chain
fatty acids (SCFAs) are natural ligands of the orphan receptor
GPR43. This invention thus relates to the SCFA ligand/receptor
(identified hereafter as SEQ ID NO. 2) pair, and to functional
homologs of the receptor which also bind SCFAs and cells
transformed by a vector comprising the nucleotide sequence encoding
the receptor (SEQ ID NO: 1) in combination with the SCFA ligand.
The invention also relates to a composition consisting essentially
of an isolated GPR43 polypeptide and an isolated SCFA, as well as
to methods of identifying agents that modulate the activities of
GPR43 polypeptides. The methods are useful for the identification
of agonist, inverse agonist or antagonist compounds useful for the
development of new drugs. The interaction of GPR43 with SCFAs is
also useful, for the development of diagnostics for diseases
related to GPR43 activity.
[0014] The invention encompasses a method of identifying an agent
that modulates the function of GPR43, the method comprising: a)
contacting a GPR43 polypeptide with a short chain fatty acid in the
presence and absence of a candidate modulator under conditions
permitting the binding of the short chain fatty acid to the GPR43
polypeptide; and b) measuring binding of the GPR43 polypeptide to
the short chain fatty acid wherein a decrease in binding in the
presence of the candidate modulator, relative to binding in the
absence of the candidate modulator, identifies the candidate
modulator as an agent that modulates the function of GPR43.
[0015] The invention further encompasses a method of detecting, in
a sample, the presence of an agent that modulates the function of
GPR43, the method comprising: a) contacting a GPR43 polypeptide
with a short chain fatty acid in the presence and absence of the
sample under conditions permitting the binding of the short chain
fatty acid to the GPR43 polypeptide; and b) measuring binding of
the GPR43 polypeptide to the short chain fatty acid wherein a
decrease in binding in the presence of the sample, relative to
binding in the absence of the sample, indicates the presence, in
the sample of an agent that modulates the function of GPR43.
[0016] In one embodiment of either of the preceding methods, the
measuring is performed using a method selected from label
displacement, surface plasmon resonance, fluorescence resonance
energy transfer, fluorescence quenching, and fluorescence
polarization.
[0017] The invention further encompasses a method of identifying an
agent that modulates the function of GPR43, the method comprising:
a) contacting a GPR43 polypeptide with a short chain fatty acid in
the presence and absence of a candidate modulator; and b) measuring
a signalling activity of the GPR43 polypeptide, wherein a change in
the activity in the presence of the candidate modulator relative to
the activity in the absence of the candidate modulator identifies
the candidate modulator as an agent that modulates the function of
GPR43.
[0018] The invention further encompasses a method of identifying an
agent that modulates the function of GPR43, the method comprising:
a) contacting a GPR43 polypeptide with a candidate modulator; b)
measuring a signalling activity of the GPR43 polypeptide in the
presence of the candidate modulator; and c) comparing the activity
measured in the presence of the candidate modulator to the activity
measured in a sample in which the GPR43 polypeptide is contacted
with a short chain fatty acid at its EC.sub.50, wherein the
candidate modulator is identified as an agent that modulates the
function of GPR43 when the amount of the activity measured in the
presence of the candidate modulator is at least 20% of the amount
induced by the short chain fatty acid present at its EC.sub.50.
[0019] The invention further encompasses a method of detecting the
presence, in a sample, of an agent that modulates the function of
GPR43, the method comprising: a) contacting a GPR43 polypeptide
with short chain fatty acid in the presence and absence of the
sample; b) measuring a signalling activity of the GPR43
polypeptide; and c) comparing the amount of the activity measured
in a reaction containing GPR43 and short chain fatty acid without
the sample to the amount of the activity measured in a reaction
containing GPR43, short chain fatty acid and the sample, wherein a
change in the activity in the presence of the sample relative to
the activity in the absence of the sample indicates the presence,
in the sample, of an agent that modulates the function of
GPR43.
[0020] The invention further encompasses a method of detecting the
presence, in a sample, of an agent that modulates the function of
GPR43, the method comprising: a) contacting a GPR43 polypeptide
with the sample; b) measuring a signalling activity of the GPR43
polypeptide in the presence of the sample; and c) comparing the
activity measured in the presence of the sample to the activity
measured in a reaction in which the GPR43 polypeptide is contacted
with a short chain fatty acid present at its EC.sub.50, wherein an
agent that modulates the function of GPR43 is detected if the
amount of the activity measured in the presence of the sample is at
least 20% of the amount induced by the short chain fatty acid
present at its EC.sub.50.
[0021] In one embodiment of each of the preceding methods, the
short chain fatty acid is detectably labeled. In a preferred
embodiment, the short chain fatty acid is detectably labeled with a
moiety selected from the group consisting of a radioisotope, a
fluorophore, a quencher of fluorescence, an enzyme, and an affinity
tag.
[0022] In an embodiment of each of the preceding methods, the
contacting is performed in or on a cell expressing the GPR43
polypeptide.
[0023] In an embodiment of each of the preceding methods the
contacting is performed in or on synthetic liposomes.
[0024] In an embodiment of each of the preceding methods the
contacting is performed in or on virus-induced budding membranes
containing a GPR43 polypeptide.
[0025] In an embodiment of each of the preceding methods the
contacting is performed using a membrane fraction from cells
expressing the GPR43 polypeptide.
[0026] In an embodiment of each of the preceding methods the agent
is selected from the group consisting of a natural or synthetic
peptide or polypeptide, an antibody or antigen-binding fragment
thereof, a lipid, a carbohydrate, a nucleic acid, and a small
organic molecule.
[0027] In one embodiment of the methods wherein a signalling
activity is measured, the step of measuring a signalling activity
of the GPR43 polypeptide comprises detecting a change in the level
of a second messenger.
[0028] In another embodiment of the methods wherein a signalling
activity is measured, the step of measuring a signalling activity
comprises measurement of guanine nucleotide binding or exchange,
adenylate cyclase activity, cAMP, Protein Kinase C activity,
phosphatidylinositol breakdown, diacylglycerol, inositol
triphosphate, intracellular calcium, arachinoid acid, MAP kinase
activity, tyrosine kinase activity, or reporter gene
expression.
[0029] In one embodiment, the step of measuring a signalling
activity comprises using an aequorin-based assay.
[0030] The invention further comprises a method of modulating the
activity of a GPR43 polypeptide in a cell, the method comprising
the step of delivering to the cell an agent that modulates the
activity of a GPR43 polypeptide, such that the activity of GPR43 is
modulated.
[0031] The invention further encompasses a method of diagnosing a
disease or disorder characterized by dysregulation of GPR43
signalling, the method comprising: a) contacting a tissue sample
with an antibody specific for a GPR43 polypeptide; b) detecting
binding of the antibody to the tissue sample; and c) comparing the
binding detected in step (b) with a standard, wherein a difference
in binding relative to the standard is diagnostic of a disease or
disorder characterized by dysregulation of GPR43.
[0032] The invention further encompasses a method of diagnosing a
disease or disorder characterized by dysregulation of GPR43
signalling, the method comprising: a) isolating nucleic acid from a
tissue sample; b) amplifying a GPR43 polynucleotide, using the
nucleic acid as a template; and c) comparing the amount of
amplified GPR43 polynucleotide produced in step (b) with a
standard, wherein a difference in the amount of amplified GPR43
polynucleotide relative to the standard is diagnostic of a disease
or disorder characterized by dysregulation of GPR43.
[0033] The invention further encompasses a method of diagnosing a
disease or disorder characterized by dysregulation of GPR43
signalling, the method comprising: a) isolating nucleic acid from a
tissue sample; b) amplifying a GPR43 polynucleotide, using the
nucleic acid as a template; and c) comparing the sequence of the
amplified GPR43 polynucleotide produced in step (b) with a
standard, wherein a difference in the sequence, relative to the
standard is diagnostic of a disease or disorder characterized by
dysregulation of GPR43. In one embodiment, the step of amplifying
comprises RT/PCR. In another embodiment, the standard is SEQ ID NO:
1. In another embodiment, the step of comparing the sequence
comprises minisequencing. In another embodiment, the step of
comparing the amount is performed using a microarray.
[0034] The invention further encompasses a composition consisting
essentially of an isolated GPR43 polypeptide and an isolated short
chain fatty acid. An isolated GPR43 polypeptide and an isolated
short chain fatty acid together can form a complex that is useful
for the identification of agents that modulate their interaction,
the identification of agents that modulate the activity of GPR43
polypeptides, and the identification of individuals suffering from
a disease or disorder mediated by or involving GPR43. Complexed or
uncomplexed (i.e., bound or unbound) isolated GPR43 polypeptide and
isolated short chain fatty acid is thus the essential element or
basis of the assays and methods of the invention. The composition
"consisting essentially of" an isolated GPR43 polypeptide and an
isolated short chain fatty acid can comprise additional components,
however, such additional components are not essential to the novel
interaction upon which the invention is based. The composition
"consisting essentially of" an isolated GPR43 polypeptide and an
isolated short chain fatty acid is distinct from and excludes
naturally occurring complexes between GPR43 polypeptides and short
chain fatty acids, present e.g., in cells, tissues or in cell or
tissue extracts. The composition of the invention is also distinct
from and excludes complexes between GPR43 polypeptides expressed
from recombinant constructs and naturally-occurring short chain
fatty acids.
[0035] The invention further encompasses a kit comprising an
isolated GPR43 polypeptide and an isolated short chain fatty acid
or salt thereof. In one embodiment, the short chain fatty acid or
salt thereof is linear. In another embodiment, the short chain
fatty acid or salt thereof is branched. In another embodiment, one
or more non-carbonyl carbons in the short chain fatty acid or salt
thereof is substituted with a non-carbon-containing substituent. In
another embodiment, the short chain fatty acid salt is selected
from the group consisting of sodium butyrate, sodium propionate,
sodium acetate, sodium valerate and sodium formate.
[0036] The invention further encompasses a kit comprising an
isolated polynucleotide encoding a GPR43 polypeptide and an
isolated short chain fatty acid salt. In one embodiment, the short
chain fatty acid or salt thereof is linear. In another embodiment,
the short chain fatty acid or salt thereof is branched. In another
embodiment, one or more non-carbonyl carbons in the short chain
fatty acid or salt thereof is substituted with a
non-carbon-containing substituent. In another embodiment, the short
chain fatty acid salt is selected from the group consisting of
sodium butyrate, sodium propionate, sodium acetate, sodium valerate
and sodium formate.
[0037] The invention further encompasses a kit comprising a cell
transformed with a polynucleotide encoding a GPR43 polypeptide and
an isolated short chain fatty acid or salt thereof. In one
embodiment, the short chain fatty acid or salt thereof is linear.
In another embodiment, the short chain fatty acid or salt thereof
is branched. In another embodiment, one or more non-carbonyl
carbons in the short chain fatty acid or salt thereof is
substituted with a non-carbon-containing substituent. In another
embodiment, the short chain fatty acid salt is selected from the
group consisting of sodium butyrate, sodium propionate, sodium
acetate, sodium valerate and sodium formate.
[0038] The invention further encompasses a kit comprising a
cellular membrane fraction comprising a GPR43 polypeptide, and
packaging materials therefor. In one embodiment, the kit further
comprises an isolated short chain fatty acid or salt thereof. In
one embodiment, the short chain fatty acid or salt thereof is
linear. In another embodiment, the short chain fatty acid or salt
thereof is branched. In another embodiment, one or more
non-carbonyl carbons in the short chain fatty acid or salt thereof
is substituted with a non-carbon-containing substituent. In another
embodiment, the short chain fatty acid salt is selected from the
group consisting of sodium butyrate, sodium propionate, sodium
acetate, sodium valerate and sodium formate.
[0039] Kits according to the invention are useful, for example, for
screening for agents that modulate the activity of GPR43,
identifying the presence of an agent that modulates GPR43 in a
sample, or for diagnosis of a disease or disorder characterized by
dysregulation of GPR43. Kits according to the invention will
additionally comprise packaging materials necessary for such kits.
Kits according to the invention can additionally comprise a
standard. In one embodiment, the standard is a sample from an
individual not affected by a disease or disorder characterized by
dysregulation of GPR43.
[0040] As used herein, the term "GPR43 polypeptide" refers to a
polypeptide having two essential properties: 1) a GPR43 polypeptide
has at least 80% amino acid identity, preferably 85%, 90%, 95%, or
higher, up to and including 100% identity, with SEQ ID NO. 2; and
2) a GPR43 polypeptide has GPR43 activity including either or both
of GPR43 ligand binding activity (wherein SCFA ligands bind with
affinity at least equivalent to acetate or propionate binding) or
GPR43 signalling activity as defined herein.
[0041] As used herein, "GPR43 activity" refers to SCFA binding to
or signalling by a GPR43 polypeptide as defined herein. A
polypeptide that has "GPR43 activity" will bind to acetate and
propionate with an affinity that is at least 100-fold greater than
that of formate.
[0042] A homologous sequence (which may exist in other mammal
species or specific groups of human populations), where homology
indicates sequence identity, means a sequence which presents a high
sequence identity (more than 80%, 85%, 90%, 95% or 98% sequence
identity) with the complete human nucleotide or amino acid sequence
of SEQ ID NO: 2. A functional homolog is characterized by the
ability to bind a short chain fatty acid ligand as defined herein
or by the ability to initiate or propagate a signal in response to
ligand binding, or both. A functional homolog will bind natural
ligands of wt GPR43 with affinity as follows:
propionate=acetate>butyrate>formate, where propionate and
acetate bind with at least 100.times. greater affinity than
formate.
[0043] Homologous sequences of a sequence according to the
invention may include an amino acid or nucleotide sequence encoding
a similar receptor which exists in other animal species (rat,
mouse, cat, dog, etc.) or in specific human population groups, but
which are involved in the same biochemical pathway.
[0044] Such homologous sequences may comprise additions, deletions
or substitutions of one or more amino acids or nucleotides, which
do not substantially alter the functional characteristics of the
receptor according to the invention. That is, homologs will have at
least 90% of the activity of wt full length human GPR43 and will
bind acetate and propionate with at least 100.times. greater
affinity than formate.
[0045] Such homologous sequences can also be nucleotide sequences
of more than 400, 600, 800 or 1000 nucleotides which are able to
hybridize to the complete human GPR43 sequence under stringent
hybridisation conditions (such as the ones described by SAMBROOK et
al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor
Laboratory press, New York). An example of "stringent hybridization
conditions" is as follows: hybridize in 50% formamide, 5.times.SSC,
50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,
5.times.Denhardt's solution, 50 .mu.g/ml sonicated salmon sperm
DNA, 0.1% SDS and 10% dextran sulfate at 42.degree. C.; and wash at
42.degree. C. (or higher, e.g., up to two degrees C. below the
T.sub.m of the perfect complement of the probe sequence) in
0.2.times.SSC and 0.1% SDS.
[0046] As used herein, the term "GPR43 signalling activity" refers
to the initiation or propagation of signalling by a GPR43
polypeptide. GPR43 signalling activity is monitored by measuring a
detectable step in a signalling cascade by assaying one or more of
the following: stimulation of GDP for GTP exchange on a G protein;
alteration of adenylate cyclase activity; protein kinase C
modulation; phosphatidylinositol breakdown (generating second
messengers diacylglycerol, and inositol triphosphate);
intracellular calcium flux; activation of MAP kinases; modulation
of tyrosine kinases; or modulation of gene or reporter gene
activity. A detectable step in a signalling cascade is considered
initiated or mediated if the measurable activity is altered by 10%
or more above or below a baseline established in the substantial
absence of a SCFA relative to any of the GPR43 activity assays
described herein below. The measurable activity can be measured
directly, as in, for example, measurement of cAMP or diacylglycerol
levels. Alternatively, the measurable activity can be measured
indirectly, as in, for example, a reporter gene assay.
[0047] As used herein, the terms "short chain fatty acid" and
"short fatty carobxylic acid" refer to a molecule of the general
structure C.sub.xH.sub.(2x+1)--COO wherein x is 0 to 5, or to
related molecules in which non-carbon-containing substitutents,
including, for example, OH, NH.sub.3, PO.sub.4, O, and halogens are
branched on the carbonyl chain. An SCFA according to the invention
can be linear or branched, saturated or unsaturated. An SCFA
according to the invention will bind to a GPR43 polypeptide as
defined herein with an affinity at least equivalent to acetate or
propionate and at least 100.times. stronger than formate. An SCFA
according to the invention may additionally stimulate a GPR43
signalling activity. Examples of SCFA include, but are not limited
to acetate, propionate, n-buyrate, n-pentanoate (valerate) and
formate. Examples of SCFAs according to the invention and their
relative activity on GPR43 are shown in FIG. 12.
[0048] As used herein, the term "detectable step" refers to a step
that can be measured, either directly, e.g., by measurement of a
second messenger or detection of a modified (e.g., phosphorylated)
protein, or indirectly, e.g., by monitoring a downstream effect of
that step. For example, adenylate cyclase activation results in the
generation of cAMP. The activity of adenylate cyclase can be
measured directly, e.g., by an assay that monitors the production
of cAMP in the assay, or indirectly, by measurement of actual
levels of cAMP.
[0049] Preferably, a recombinant cell according to the invention is
a recombinant cell transformed by a plasmid, cosmid or viral
vector, preferably a baculovirus, an adenovirus, or a semliki
forest virus, and the cell is preferably selected from the group
consisting of bacterial cells, yeast cells, insect cells or mammal
cells.
[0050] According to a preferred embodiment of the present
invention, the cell is selected from the group consisting of COS-7
cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293 cell,
K-562 cell or a 1321N1 astrocytoma cell. Other transfectable cell
lines are also useful, however. Preferably, the vector comprises
regulatory elements operatively linked to the polynucleotide
sequence encoding the receptor according to the invention, so as to
permit expression thereof.
[0051] Another aspect of the present invention is related to the
use of a specific active portion of the sequences. As used herein,
an "active portion" refers to a portion of a sequence that is of
sufficient size to exhibit normal or near normal pharmacology
(e.g., receptor activity (as defined herein), the response to an
activator or inhibitor, or ligand binding are at least 90% of the
level of activity, response, or binding exhibited by a wild type
receptor). "A portion" as it refers to a sequence encoding a
receptor, refers to less than 100% of the sequence (i.e., 99, 90,
80, 70, 60, 50% etc. . . . ). The active portion could be a
receptor which comprises a partial deletion of the complete
nucleotide or amino acid sequence and which still maintains the
active site(s) and protein domain(s) necessary for the binding of
and interaction with a specific ligand, preferably acetate and
propionate.
[0052] In another embodiment of any of the preceding methods, the
contacting is performed in or on synthetic liposomes (Mirzabekov et
al., 2000) or virus-induced budding membranes containing a GPR43
polypeptide. (see Patent application WO0102551, Virus-like
particles, their Preparation and their Use preferably in
Pharmaceutical Screening and Functional Genomics (2001)
incorporated herein by reference).
[0053] As used herein, "ligand" refers to a moiety that is capable
of associating or binding to a receptor. According to the method of
the invention, a ligand and a receptor have a binding constant that
is sufficiently strong to allow detection of binding by an assay
method that is appropriate for detection of a ligand binding to a
receptor (e.g. a second messenger assay to detect an increase or
decrease in the production of a second messenger in response to
ligand binding to the receptor, a binding assay to measure
protein-ligand binding or an immunoassay to measure
antibody-antigen interactions). A ligand according to the invention
includes the actual molecule that binds a receptor (e.g. propionate
is the ligand for GPR43) or a ligand may be any nucleotide,
antibody, antigen, enzyme, peptide, polypeptide or nucleic acid
capable of binding to the receptor. A ligand is preferably a short
chain carboxylic acid but can also include a polypeptide, a peptide
or a nucleic acid sequence. According to the method of the
invention, a ligand and receptor specifically bind to each other
(e.g. via covalent or hydrogen bonding or via an interaction
between, for example, a protein and a ligand, an antibody and an
antigen or protein subunits).
[0054] Another aspect of the present invention is related to a
method for the screening, detection and recovery of candidate
modulators of a receptor of the invention comprising the steps of:
contacting a cell expressing GPR43 with an SCFA under conditions
which permit binding of acetate or propionate to GPR43, in the
presence of the candidate modulator, performing a second messenger
assay, and comparing the results of the second messenger assay
obtained in the presence and absence of the candidate
modulator.
[0055] Another aspect of the present invention is related to a
method for the screening, detection and possible recovery of
candidate modulators of a receptor of the invention comprising the
steps of: contacting a cell membrane expressing GPR43 with an SCFA
under conditions which permit binding of acetate or propionate to
GPR43, performing a second messenger assay, and comparing the
results of the second messenger assay obtained in the presence and
absence of the candidate modulator.
[0056] In another embodiment, the step of measuring a signalling
activity of the GPR43 polypeptide comprises detecting a change in
the level of a second messenger.
[0057] A further aspect of the present invention is related to the
unknown agonist and/or antagonist compounds identified and/or
recovered by the method of the invention, as well as to a
diagnostic kit comprising the (unknown) compounds or a
pharmaceutical composition (including a vaccine) comprising an
adequate pharmaceutical carrier and a sufficient amount of the
(unknown) compound.
[0058] An antagonist compound according to the invention means a
molecule or a group of molecules able to bind to the receptor
according to the invention and block the binding of natural
compounds (propionate or acetate or related short chain carboxylic
acids).
[0059] The invention further encompasses a method of diagnosing a
disease or disorder characterized by dysregulation of GPR43
signalling, the method comprising: a) contacting a tissue sample
with an antibody specific for a GPR43 polypeptide and an antibody
specific for a GPR43 ligand; b) detecting binding of the antibodies
to the tissue sample; and c) comparing the binding detected in step
(b) with a standard, wherein a difference in binding of either
antibody or both, relative to the standard, is diagnostic of a
disease or disorder characterized by dysregulaltion of GPR43
[0060] The invention further encompasses a method of diagnosing a
disease or disorder characterized by dysregulation of GPR43
signalling, the method comprising: a) isolating a tissue sample; b)
measuring the concentration of SCFA; and c) comparing the amount of
SCFA measured in step (b) with a standard, wherein a difference in
the amount of SCFA relative to the standard is diagnostic of a
disease or disorder characterized by dysregulation of GPR43.
[0061] A further aspect of the present invention is related to a
non-human mammal comprising a homozygous null mutation (homozygous
"knock-out") of the polynucleotide sequence encoding the GPR43
receptor according to the invention, or a transgenic non-human
mammal that over expresses a GPR43 polypeptide above the natural
level of expression. As used herein. "above the natural level of
expression" refers to a level that is at least 2-fold, preferably
5-fold, more preferably 10-fold and most preferably 100-fold or
more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold,
10,000-fold etc.) as compared to the level of expression of the
endogenous receptor in its normal native context. A transgenic
non-human mammal according to the invention will express the
transgene in at least one tissue or cell type but can express the
GPR43 transgene in all tissues and cells. A transgenic non-human
mammal can be obtained by a method well known by a person skilled
in the art, for instance, as described in document WO 98/20112
using the classical technique based upon the transfection of
embryonic stem cells, preferably according to the method described
by Carmeliet et al. (Nature, Vol. 380, p. 435-439, 1996).
[0062] "Gene targeting" is a type of homologous recombination that
occurs when a fragment of genomic DNA is introduced into a
mammalian cell and that fragment locates and recombines with
endogenous homologous sequences as exemplified in U.S. Pat. No.
5,464,764, and U.S. Pat. No. 5,777,195, the contents of which are
hereby incorporated by reference herein in their entireties. As
used herein the term "transgenic animal" refers to a non-human
animal in which one or more, and preferably essentially all, of the
cells of the animal contain a transgene introduced by way of human
intervention, such as by transgenic techniques known in the art.
The transgene can be introduced into the cell, directly or
indirectly by introduction into a precursor of the cell, by way of
deliberate genetic manipulation, such as by microinjection or by
infection with a recombinant virus.
[0063] Preferably, the transgenic non-human mammal overexpressing
the polynucleotide encoding the GPR43 receptor according to the
invention comprises the polynucleotide incorporated in a DNA
construct with an inducible promoter allowing the overexpression of
the receptor and possibly also tissue and cell-specific regulatory
elements.
[0064] In one embodiment, the kits according to the invention
comprise reagents for measuring the binding of a short chain fatty
acid to a GPR43 polypeptide. In another embodiment, the kit
comprises reagents for measuring a signalling activity of a GPR43
polypeptide.
[0065] In one embodiment, a screening or diagnostic kit according
to the invention includes a GPR43 receptor polypeptide or a
cellular membrane preparation comprising a GPR43 polypeptide and
one or more SCFAs in separate containers. Such kits can
additionally comprise all the necessary means and media for
performing a detection of specific binding (for example of
propionate) to the GPR43 receptor according to the invention.
Binding or signalling activity can be correlated with a method of
monitoring one or more of the symptoms of the diseases described
hereafter.
[0066] The diagnostic kits can thus further comprise elements
necessary for a specific diagnostic measurement, or, for example,
the measurements of bound compounds using high throughput screening
techniques known to the person skilled in the art, e.g., the
techniques described in WO 00/02045. Such kits can be used, e.g. to
monitor dosage and effectiveness of GPR43 modulating agents used
for treatment. The high throughput screening diagnostic dosage and
monitoring can be performed by using various solid supports, such
as microtiter plates or biochips selected by the person skilled in
the art.
[0067] In a pharmaceutical composition according to the invention,
the adequate pharmaceutical carrier is a carrier of solid, liquid
or gaseous form, which can be selected by the person skilled in the
art according to the type of administration and the possible side
effects of the compound administered to modulate GPR43 activity.
The pharmaceutical carrier useful according to the invention does
not include tissue culture medium or other media comprising serum.
The ratio between the pharmaceutical carrier and the specific
compound can be selected by the person skilled in the art according
to the patient treated, the administration and the possible side
effects of the compound, as well as the type of disease of disorder
treated or sought to be prevented.
[0068] The pharmaceutical composition finds advantageous
applications in the field of treatment and/or prevention of various
diseases or disorders, preferably selected from the group
consisting of ostatic hypertrophy, migraine, vomiting, psychotic
and neurological disorders, including anxiety, schizophrenia, manic
depression, depression, delirium, dementia and severe mental
retardation, degenerative diseases, neurodegenerative diseases such
as Alzheimer's disease or Parkinson's disease, and dyskinasias,
such as Huntington's disease or Gilles de la Tourett's syndrome and
other related diseases including thrombosis and other
cardiovascular diseases, autoimmune and inflammatory diseases.
[0069] Among the mentioned diseases the preferred applications are
related to therapeutic agents targeting 7TM receptors that can play
a function in preventing, improving or correcting dysfunctions or
diseases, including, but not limited to fertility, fetal
development, infections such as bacterial, fungal, protozoan and
viral infections, particularly infections caused by HIV1 and HIV2,
pain, cancer, anorexia, bulimia, asthma, Parkinson's disease, acute
heart failure, hypertension, urinary retention, osteoporosis,
angina pectoris, myocardial infarction, ulcers, asthma, allergies,
benign prostatic hypertrophy, psychotic and neurological disorders
including anxiety, depression, migraine, vomiting, stroke,
schizophrenia, manic depression, delirium, dementia, severe mental
retardation and dyskinesias, such as Huntington's disease or Gilles
de la Tourette's syndrome including thrombosis and other
cardiovascular diseases, autoimmune and inflammatory diseases.
[0070] The present invention also provides a method of modulating
PMN chemotaxis in a mammal comprising contacting PMN cells bearing
the cell surface receptor GPR43 with a modulator of GPR43
signalling activity, sufficient to modulate said PMN
chemotaxis.
[0071] In one embodiment, the present invention provides a method
of modulating PMN chemotaxis in a patient in need thereof,
comprising administering to the patient, an inhibitor of GPR43
signalling activity.
[0072] In one embodiment, PMN chemotaxis is decreased by contacting
said PMN cell with an inhibitor of GPR43 signalling activity.
[0073] In one embodiment, PMN chemotaxis is decreased by contacting
said PMN cell with an antagonist of GPR43 signalling activity.
[0074] In a further embodiment, PMN chemotaxis is increased by
contacting said PMN cell with an agonist of GPR43 signalling
activity.
[0075] The present invention also includes a method for identifying
an agent which modulates PMN chemotaxis, comprising contacting a
GPR43 polypeptide with a short chain fatty acid in the presence and
absence of a candidate agent under conditions permitting binding of
the short chain fatty acid to said GPR43 polypeptide; and measuring
a signalling activity of the GPR43 polypeptide wherein an increase
or decrease in signalling activity of the GPR43 in the presence of
said candidate agent, relative to the signalling activity in the
absence of the candidate agent, identifies said candidate agent as
an agent which modulates PMN chemotaxis.
[0076] The present invention also provides a method for identifying
an agent for the treatment of a PMN chemotaxis related disease
comprising contacting a GPR43 polypeptide with a short chain fatty
acid in the presence and absence of a candidate agent under
conditions permitting binding of the short chain fatty acid to said
GPR43 polypeptide; and measuring a signalling activity of the GPR43
polypeptide wherein an increase or decrease in signalling activity
of the GPR43 in the presence of said candidate agent, relative to
the signalling activity in the absence of the candidate agent,
identifies said candidate agent as an agent for the treatment of a
PMN chemotaxis-related disease.
[0077] The present invention provides a method for identifying an
agent which modulates PMN chemotaxis, comprising contacting a GPR43
polypeptide with a short chain fatty acid in the presence and
absence of a candidate agent under conditions permitting binding of
the short chain fatty acid to the GPR43 polypeptide; and measuring
binding of the GPR43 polypeptide to the short chain fatty acid,
wherein a decrease in binding in the presence of the candidate
agent, relative to binding in the absence of the candidate agent,
identifies said candidate agent as an agent which modulates PMN
chemotaxis.
[0078] The present invention still further provides a method for
identifying an agent for the treatement of a PMN chemotaxis-related
disease comprising contacting a GPR43 polypeptide with a short
chain fatty acid in the presence and absence of a candidate agent
under conditions permitting binding of the short chain fatty acid
to the GPR43 polypeptide; and measuring binding of the GPR43
polypeptide to the short chain fatty acid, wherein a decrease in
binding in the presence of the candidate agent, relative to binding
in the absence of the candidate agent, identifies said candidate
agent as an agent for the treatment of a PMN chemotaxis-related
disease.
[0079] In one embodiment, the GPR43 receptor is present in the cell
membrane of a PMN cell.
[0080] In one embodiment, the short chain fatty acid is detectably
labeled.
[0081] In a further embodiment, the short chain fatty acid is
detectably labeled with a moiety selected from the group consisting
of a radioisotope, a fluorophore, a quencher of fluorescence, an
enzyme, and an affinity tag.
[0082] As used herein, the term "polymorphonuclear cell" or "PMN"
refers to a leukocyte of granulocytic lineage of between 10-14
.mu.m in diameter. A "PMN" according to the invention has a nucleus
with coarse, clumped, deeply staining chromatin with two or more
lobes or segments (although immature PMN cells may have unlobated,
band nuclei). A "PMN" according to the invention also has a
granular cytoplasm containing small, weakly staining, or large
strongly staining basophilic granules, or large (0.5-1 .mu.m)
eosinophilic granules. The morphology of a PMN cell according to
the invention is well known to those of skill in the art.
[0083] As used herein, "PMN chemotaxis" refers to the to the
directed movement of a PMN cell in response to, and either towards
or away from a chemotactic factor. Chemotactic factors include, but
are not limited to bacterial factors (N-formylated peptides such as
FMLP, which are unique to the initiation of bacterial protein),
plasma proteins (e.g., C5a, one of the activated products of either
the classical or alternative pathways of complement activation, and
leukotrines), and cells (e.g., TGF-beta and other cytokines,
polypeptides released from lymphocytes, mast cells, and basophils,
G.sub.c-globulin, opsonins). PMN chemotaxis may be measured,
according to the invention, by procedures originally developed by
S. Boyden in 1962. (See, S. Boyden, The Chemotactic Effect of
Mixtures of Antibody and Antigen on Polymorphonuclear Leucocytes,
J. Exp. Med. 115: pp. 453-466, 1962). Briefly, the procedure
involves placing a suspension of PMN cells and a chemical agent in
two separate chambers, which chambers are separated by a
polycarbonate filter. The PMN may, for example, be prepared from
the peripheral blood of a mammal. After a predetermined period of
time, the filter is removed and cells on the filter surface closest
to the chamber containing the cell suspension are carefully
removed. The remaining cells on the filter are then fixed and
stained. Using a high power microscope, the filter is examined and
the number of cells appearing on the underside of the filter (i.e.,
the side of the filter closest to the chamber containing the
chemical agent) are counted manually. A positive chemotactic
response is indicated by the cells having migrated or "crawled"
through the filter to the side closest to the chamber containing
the chemical agent. Because of the time required to do so,
typically the entire filter is not examined. Rather, representative
sample areas are examined and counted. According to the invention,
"PMN chemotaxis" is said to have occurred where there are at least
10% more PMN cells on the filter surface aposed to the chamber
containing the chemotactic factor when the chemotactic factor is
present in the chamber, than when the chemotactic factor is not
present.
[0084] As used herein, an "antagonist" is a ligand which
competitively binds to a receptor at the same site as an agonist,
but does not activate an intracellular response initiated by an
active form of the receptor. An antagonist thereby inhibits the
intracellular response induced by an agonist, for example
propionate, by at least 10%, preferably 15-25%, more preferably
25-50% and most preferably, 50-100%, as compared to the
intracellular response in the presence of an agonist and in the
absence of an antagonist.
[0085] As used herein, an "agonist" refers to a ligand that
activates an intracellular response when it binds to a receptor at
concentrations equal to or lower than propionate concentrations
which induce an intracellular response. An agonist according to the
invention can increase the intracellular response mediated by a
receptor by at least 2-fold, preferably 5-fold, more preferably
10-fold and most preferably 100-fold or more (i.e., 150-fold,
200-fold, 250-fold, 500-fold, 1000-fold, 10.000-fold etc. . . . ),
as compared to the intracellular response in the absence of
agonist. An agonist according to the invention may decrease
internalization of a cell surface receptor such that the cell
surface expression of a receptor is increased by at least 2-fold,
preferably 5-fold, more preferably 10-fold and most preferably,
100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold,
1000-fold, 10.000-fold etc. . . . ), as compared to the number of
cell surface receptors present on the surface of a cell in the
absence of an agonist. In another embodiment of the invention, an
agonist stablizes a cell surface receptor and increases the cell
surface expression of a receptor by at least 2-fold, preferably
5-fold, more preferably 10-fold and most preferably, 100-fold or
more (i.e., 200-fold, 250-fold, 500-fold, 1000-fold, 10.000-fold
etc. . . . ), as compared to the number of cell surface receptors
present on the surface of a cell in the absence of agonist.
[0086] As used herein, an "inverse agonist" refers to a ligand
which decreases a constitutive activity of a cell surface receptor
when it binds to a receptor. An inverse agonist according to the
invention can decrease the constitutive intracellular response
mediated by a receptor by at least 2-fold, preferably 5-fold, more
preferably 10-fold and most preferably 100-fold or more (i.e.,
150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10.000-fold etc.
. . . ), as compared to the intracellular response in the absence
of inverse agonist.
[0087] An "inhibitor" compound according to the invention is a
molecule directed against the receptor or against the natural
ligand for the receptor that decreases the binding of the ligand to
the receptor by at least 10%, preferably 15-25%, more preferably
25-50% and most preferably, 50-100%, in the presence of acetate or
propionate, as compared to the binding in the presence of acetate
or propionate and in the absence of inhibitor. An "inhibitor"
compound of the invention can decrease the intracellular response
induced by an agonist, for example acetate or propionate, by at
least 10%, preferably 15-25%, more preferably 25-50% and most
preferably, 50-100%. An "inhibitor" also refers to a nucleotide
sequence encoding an inhibitor compound of the invention. An
inhibitor, useful according to the present invention, includes, but
is not limited to an antibody which specifically binds to at least
a portion of GPR43 which is required for signal transduction
through GPR43 (such as the ligand binding site), or chemical
compounds which are capable of blocking or reducing (e.g., by at
least 10%) the signal transduction pathway which is coupled to the
GPR43 receptor. Such inhibitors include, but are not limited to
sub-lethal doses of pertussis toxin, N-ethylmaleimide (NEM; Sigma),
dibutyryl cAMP (Boehringer Mannheim, Corp.), and H-89
(N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide-HCL;
Calbiochem).
[0088] As used herein, "natural ligand" refers to a naturally
occurring ligand, found in nature, which binds to a receptor in a
manner that is at least equivalent to acetate or propionate (i.e.,
with an affinity for the receptor that is greater than the affinity
of formate (acetate=propionate>formate)). A "natural ligand"
does not refer to an engineered ligand that is not found in nature
and that is engineered to bind to a receptor, where it did not
formerly do so in a manner different, either in degree or kind,
from that which it was engineered to do. Such an engineered ligand
is no longer naturally-occurring but is "non-natural" and is
derived from a naturally occurring molecule.
[0089] As used herein, a "modulator" refers to a compound that
increases or decreases the cell surface expression of a receptor of
the invention, increases or decreases the binding of a ligand to a
receptor of the invention, or any compound that increases or
decreases the intracellular response initiated by an active form of
the receptor of the invention, either in the presence or absence or
an agonist, and in the presence of a ligand for the receptor, for
example acetate or propionate. A modulator includes an agonist,
antagonist, inhibitor or inverse agonist, as defined herein. A
modulator can be for example, a polypeptide, a peptide, an antibody
or antigen-binding fragment thereof, a lipid, a carbohydrate, a
nucleic acid, and a small organic molecule. Candidate modulators
can be natural or synthetic compounds, including, for example,
synthetic small molecules, compounds contained in extracts of
animal, plant, bacterial or fungal cells, as well as conditioned
medium from such cells.
[0090] As used herein, "increase" and "decrease" refer to a change
in ligand binding to the GPR43 receptor and/or cell signalling
through GPR43 of at least 10%. An "increase" or "decrease" in
binding or signalling is preferably measured in response to
contacting GPR43 with a ligand in the presence of a candidate
modulator, wherein the change in binding or signalling is relative
to the binding or signalling in the absence of the candidate
modulator.
[0091] As used herein, the term "small molecule" refers to a
compound having molecular mass of less than 3000 Daltons,
preferably less than 2000 or 1500, still more preferably less than
1000, and most preferably less than 600 Daltons. A "small organic
molecule" is a small molecule that comprises carbon.
[0092] As used herein, the terms "change", "difference",
"decrease", or "increase" as applied to e.g., binding or signalling
activity or amount of a substance refer to an at least 10% increase
or decrease in binding, signalling activity, or for example, level
of mRNA, polypeptide or ligand relative to a standard in a given
assay.
[0093] As used herein, the term "dysregulation" refers to the
signalling activity of GPR43 in a sample wherein:
[0094] a) a 10% or greater increase or decrease in the amount of
one or more of GPR43 polypeptide, ligand or mRNA level is measured
relative to a standard, as defined herein, in a given assay or;
[0095] b) at least a single base pair change in the GPR43 coding
sequence is detected relative to SEQ ID NO: 1, and results in an
alteration of GPR43 ligand binding or signalling activity as
defined in paragraphs a), c) or d) or;
[0096] c) a 10% or greater increase or decrease in the amount of
GPR43 ligand binding activity is measured relative to a standard,
as defined herein, in a given assay or;
[0097] d) a 10% or greater increase or decrease in a second
messenger, as defined herein, is measured relative to the standard,
as defined herein, in a given assay.
[0098] As used herein, the term "conditions permitting the binding
of SFCA to a GPR43 polypeptide" refers to conditions of, for
example, temperature, salt concentration, pH and protein
concentration under which SCFA, (e.g., acetate or propionate) binds
GPR43. Exact binding conditions will vary depending upon the nature
of the assay, for example, whether the assay uses viable cells or
only a membrane fraction of cells. However, because GPR43 is a cell
surface protein favored conditions will generally include
physiological salt (90 mM) and pH (about 7.0 to 8.0). Temperatures
for binding can vary from 15.degree. C. to 37.degree. C., but will
preferably be between room temperature and about 30.degree. C. The
concentration of SCFA in a binding reaction will also vary, but
will preferably be about 1 .mu.M (e.g., in a reaction with
radiolabelled tracer SCFA, e.g., propionate, where the
concentration is generally below the K.sub.d) to 10 mM (e.g.,
propionate as competitor).
[0099] As used herein, the term "sample" refers to the source of
molecules being tested for the presence of an agent or modulator
compound that modulates binding to or signalling activity of a
GPR43 polypeptide. A sample can be an environmental sample, a
natural extract of animal, plant yeast or bacterial cells or
tissues, a clinical sample, a synthetic sample, or a conditioned
medium from recombinant cells or a fermentation process. The term
"tissue sample" refers to a tissue that is tested for the presence,
abundance, quality or an activity of a GPR43 polypeptide, a nucleic
acid encoding a GPR43 polypeptide, a GPR43 ligand or an agent or
compound that modifies the ligand binding or activity of a GPR43
polypeptide.
[0100] As used herein, a "tissue" is an aggregate of cells that
perform a particular function in am organism. The term "tissue" as
used herein refers to cellular material from a particular
physiological region. The cells in a particular tissue can comprise
several different cell types. A non-limiting example of this would
be brain tissue that further comprises neurons and glial cells, as
well as capillary endothelial cells and blood cells, all contained
in a given tissue section or sample. In addition to solid tissues,
the term "tissue" is also intended to encompass non-solid tissues,
such as blood.
[0101] As used herein, the term "membrane fraction" refers to a
preparation of cellular lipid membranes comprising a GPR43
polypeptide. As the term is used herein, a "membrane fraction" is
distinct from a cellular homogenate, in that at least a portion
(i.e., at least 10%, and preferably more) of
non-membrane-associated cellular constituents has been removed. The
term "membrane associated" refers to those cellular constituents
that are either integrated into a lipid membrane or are physically
associated with a component that is integrated into a lipid
membrane.
[0102] As used herein, the "second messenger assay" preferably
comprises the measurement of guanine nucleotide binding or
exchange, adenylate cyclase, intra-cellular cAMP, intracellular
inositol phosphate, intra-cellular diacylglycerol concentration,
arachidonic acid concentration, MAP kinase(s) or tyrosine
kinase(s), protein kinase C activity, or reporter gene expression
or an aequorin-based assay according to methods known in the art
and defined herein.
[0103] As used herein, the term "second messenger" refers to a
molecule, generated or caused to vary in concentration by the
activation of a G-Protein Coupled Receptor, that participates in
the transduction of a signal from that GPCR. Non-limiting examples
of second messengers include cAMP, diacylglycerol, inositol
triphosphate, arachidonic acid release, inositol triphosphates and
intracellular calcium. The term "change in the level of a second
messenger" refers to an increase or decrease of at least 10% in the
detected level of a given second messenger relative to the amount
detected in an assay performed in the absence of a candidate
modulator.
[0104] As used herein, the term "aequorin-based assay" refers to an
assay for GPCR activity that measures intracellular calcium flux
induced by activated GPCRs, wherein intracellular calcium flux is
measured by the luminescence of aequorin expressed in the cell.
[0105] As used herein, the term "binding" refers to the physical
association of a ligand (e.g., am SCFA such as propionate, or an
antibody) with a receptor (e.g., GPR43). As the term is used
herein, binding is "specific" if it occurs with an EC.sub.50 or a
K.sub.d of 1 mM less, generally in the range of 1 mM to 10 nM For
example, binding is specific if the EC.sub.50 or K.sub.d is 1 mM,
500 .mu.M, 100 .mu.M, 10 .mu.M, 9.5 .mu.M, 9 .mu.M, 8.5 .mu.M, 8
.mu.M, 7.5 .mu.M, 7 .mu.M, 6.5 .mu.M, 6 .mu.M, 5.5 .mu.M, 5 .mu.M,
4.5 .mu.M, 4 .mu.M, 3.5 .mu.M, 3 .mu.M, 2.5 .mu.M, 2 .mu.M, 1.5
.mu.M, 1 .mu.M, 750 nM, 500 nM, 250 nM or 100 nM or less.
[0106] As used herein, the term "EC.sub.50," refers to that
concentration of a compound at which a given activity, including
binding of propionate or other ligand and a functional activity of
a GPR43 polypeptide, is 50% of the maximum for that GPR43 activity
measurable using the same assay in the absence of compound. Stated
differently, the "EC.sub.50" is the concentration of compound that
gives 50% activation, when 100% activation is set at the amount of
activity that does not increase with the addition of more agonist.
It should be noted that the "EC.sub.50" of an analog, of, for
example, propionate, will vary according to the identity of the
analogue used in the assay; for example, propionate analogues can
have EC.sub.50 values higher than, lower than or the same as
propionate. Therefore, where a propionate analogue differs from
propionate, one of skill in the art can determine the EC.sub.50 for
that analogue according to conventional methods. The EC.sub.50 of a
given SCFA is measured by performing an assay for the activity of a
fixed amount of GPR43 polypeptide in the presence of doses of SCFA
that increase at least until the GPR43 response is saturated or
maximal, and then plotting the measured GPR43 activity versus the
concentration of SCFA.
[0107] As used herein, the term "saturation" refers to the
concentration of propionate or other ligand at which further
increases in ligand concentration fail to increase the binding of
ligand or GPR43-specific signalling activity.
[0108] As used herein, the term "IC.sub.50" is the concentration of
an antagonist or inverse agonist that reduces the maximal
activation of a GPR43 receptor by 50%.
[0109] As used herein, the term "LD50" refers to the dose of a
particular agent necessary to kill 50% of the subjects to which it
is administered.
[0110] As used herein, the term "decrease in binding" refers to a
decrease of at least 10% in the amount of ligand binding detected
in a given assay with a known or suspected modulator of GPR43
relative to binding detected in an assay lacking that known or
suspected modulator.
[0111] As used herein, the term "delivering," when used in
reference to a drug or agent, means the addition of the drug or
agent to an assay mixture, or to a cell in culture. The term also
refers to the administration of the drug or agent to an animal.
Such administration can be, for example, by injection (in a
suitable carrier, e.g., sterile saline or water) or by inhalation,
or by an oral, transdermal, rectal, vaginal, or other common route
of drug administration.
[0112] As used herein, the term "standard" refers to a sample taken
from an individual who is not affected by a disease or disorder
characterized by dysregulation of GPR43 activity. The "standard" is
used as a reference for the comparison of GPR43 mRNA or polypeptide
levels and quality (i.e., mutant vs. wild type), as well as for the
comparison of GPR43 activities. A "standard" also encompasses a
reference sequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, with which
sequences of nucleic acids or their encoded polypeptides are
compared.
[0113] As used herein, the term "amplifying," when applied to a
nucleic acid sequence, refers to a process whereby one or more
copies of a nucleic acid sequence is generated from a template
nucleic acid. A preferred method of "amplifying" is PCR or
RT/PCR.
[0114] As used herein, the term "G-Protein coupled receptor," or
"GPCR" refers to a membrane-associated polypeptide with 7 alpha
helical transmembrane domains. Functional GPCR's associate with a
ligand or agonist and also associate with and activate G-proteins.
GPR43 is a. GPCR.
[0115] As used herein, the term "antibody" is the conventional
immunoglobulin molecule, as well as fragments thereof which are
also specifically reactive with one of the subject polypeptides.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described
herein below for whole antibodies. For example, F(ab).sub.2
fragments can be generated by treating antibody with pepsin. The
resulting F(ab).sub.2 fragment cam be treated to reduce disulfide
bridges to produce Fab fragments. The antibody of the present
invention is further intended to include bispecific, single-chain,
and chimeric and humanised molecules having affinity for a
polypeptide conferred by at least one CDR region of the antibody.
In preferred embodiments, the antibody further comprises a label
attached thereto and able to be detected, (e.g., the label can be a
radioisotope, fluorescent compound, chemiluminescent compound,
enzyme, or enzyme co-factor). The antibodies, monoclonal or
polyclonal and its hypervariable portion thereof (FAB, FAB'', etc.)
as well as the hybridoma cell producing the antibodies are a
further aspect of the present invention which find a specific
industrial application in the field of diagnostics and monitoring
of specific diseases, preferably the ones hereafter described.
[0116] Inhibitors according to the invention include but are not
limited to labeled monoclonal or polyclonal antibodies or
hypervariable portions of the antibodies.
[0117] As used herein, the term "transgenic animal" refers to any
animal, preferably a non-human mammal, bird, fish or an amphibian,
in which one or more of the cells of the animal contain
heterologous nucleic acid introduced by way of human intervention,
such as by transgenic techniques well known in the art. The nucleic
acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell, by way of deliberate
genetic manipulation, such as by microinjection or by infection
with a recombinant virus. The term genetic manipulation does not
include classical cross-breeding, or in vitro fertilization, but
rather is directed to the introduction of a recombinant DNA
molecule. This molecule may be integrated within a chromosome, or
it may be extra-chromosomally replicating DNA. In the typical
transgenic animals described herein, the transgene causes cells to
express a recombinant form of one of the subject polypeptide, e.g.
either agonistic or antagonistic forms. However, transgenic animals
in which the recombinant gene is silent are also contemplated, as
for example, the FLP or CRE recombinase dependent constructs
described below. Moreover, "transgenic animal" also includes those
recombinant animals in which gene disruption of one or more genes
is caused by human intervention, including both recombination and
antisense techniques.
BRIEF DESCRIPTION OF FIGURES
[0118] FIG. 1 represents nucleotide (SEQ ID NO. 1) and deduced
amino acid (SEQ ID NO. 2) sequence of the human GPR43 receptor.
[0119] FIG. 2 is a dendrogram showing the structural relatedness of
the GPR43 receptor with related receptors. Alignment of the amino
acid sequence of GPR43 with PAR1 and other PAR related sequences
were performed using ClustalX algorithm. Then, the dendrogram was
constructed using TreeView algorithm. Proteinase activated receptor
(PAR)-1, -2, -3, -4; platelet-activating factor receptor (PAF);
G-protein coupled receptor 43 (GPR43); G-protein coupled receptor
42 (GRP42). The latter one is always a orphan receptor
[0120] FIG. 3 shows tissue distribution of the human GPR43
receptor.
[0121] FIG. 4 illustrates the inhibitory activity of SCFA on
forskolin-stimulated adenylate cyclase activity in CHO-K1 cells
stably expressing the human GPR43.
[0122] FIG. 5 illustrates the PTX-sensitivity of acetate inhibition
of forskolin-stimulated adenylate cyclase activity in CHO-K1 cells
stably expressing the human GPR43.
[0123] FIG. 6 illustrates the activity of acetate on the
accumulation of GTP.gamma.[.sup.35S] bound to i membrane
preparation from CHO-K1 cells stably expressing the human
GPR43.
[0124] FIG. 7 illustrates the equipotent activity of different
salts of acetate on the accumulation of GTP.gamma.[.sup.35S] bound
to a membrane preparation from CHO-K1 cells stably expressing the
human GPR43.
[0125] FIG. 8 illustrates the activity of SCFA and related
molecules on the accumulation of GTP.gamma.[.sup.35S] bound to a
membrane preparation from CHO-K1 cells stably expressing the human
GPR43.
[0126] FIG. 9 illustrates the activity of acetate on the
accumulation of total inositol phosphates in CHO-K1 cells stably
expressing hGPR43.
[0127] FIG. 10 illustrates the non PTX sensitive-activity of C2, C3
and C4-linear carboxylic acid on the accumulation of total inositol
phosphate metabolites on CHO-K1 cells stably expressing the human
GPR43.
[0128] FIG. 11 illustrates the activity of acetate on the
accumulation of total inositol phosphate metabolites in COS-7 cells
transiently expressing the human GPR43 and/or a chimeric G.alpha.
protein.
[0129] FIG. 12 illustrates the names and formulae of SCFAs and
related compounds tested, and their respective effects on human
GPR43 activity.
[0130] FIG. 13 shows the tissue distribution of human GPR43
transcripts using semi-quantitative RT-PCR (TaqMan) methodology
over a range of 12 selected human tissues. Data are presented as
the ratio Z of the mean mRNA copies for each tissue from 2.5 ng of
polyA+ RNA or from 25 ng of total RNA. Panel A shows the mean
(+/-S.D.) mRNA copies of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene's mRNA detected/2.5 ng of polyA+ RNA or from 25 ng of
total RNA (Y axis). Panel B shows the mean (+/-S.D.) mRNA copies of
GPR43. Y axis=copies of gene's mRNA detected/2.5 ng of polyA+ RNA
or from 25 ng of total RNA. Panel C shows the ratio (Z) of the
GPCR/GAPDH means mRNA copies for each tissue (Y axis).
[0131] FIG. 14 shows a kinetic plot of the increase of
intracellular calcium in PMN for varying concentration of Na
propionate.
[0132] FIG. 15 shows a dose response curve for the stimulation of
increased intracellular calcium levels in PMN cells induced by
increasing concentrations of Na proprionate.
[0133] FIG. 16 shows a dose response curve for the stimulation of
increased intracellular calcium levels in PMN cells induced by
increasing concentrations of Na acetate.
[0134] FIG. 17 shows neutrophil migration in response to increasing
concentrations of SCFA (acetate and propionate) reported as a
migration index. The chemotaxis data represents the mean and SEM of
5 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0135] The invention is based on the discovery that short chain
fatty acids are natural ligands for the orphan G protein coupled
receptor GPR43 and on methods of using the binding of this ligand
to the receptor in drug screening methods. The known ligand and its
interaction with the receptor GPR43 also provides for the diagnosis
of conditions involving dysregulated receptor activity. The
invention also relates to a kit comprising GPR43 and homologous
sequences, its corresponding polynucleotide and/or recombinant
cells expressing the polynucleotide, to identify agonist,
antagonist and inverse agonist compounds of the receptor
polypeptide and/or its corresponding polynucleotide. Such kits are
useful for the diagnosis, prevention and/or a treatment of diseases
and disorders related to GPR43 activity.
[0136] The invention also relates to novel agonist, antagonist and
inverse agonist compounds of the receptor polypeptide and its
corresponding polynucleotide, identified according to the method of
the invention.
[0137] All references referred to below and above are incorporated
herein by reference in their entirety.
Sequences
[0138] The invention relates to the nucleotide (SEQ ID NO: 1) and
amino acid (SEQ ID NO: 2) sequences encoding GPR43 (presented in
FIG. 1). The invention also relates to sequences that are
homologous to the nucleotide and amino acid sequences encoding
GPR43.
GPR43 Tissue Distribution
[0139] GPR43 is mainly expressed on neutrophils, and to a lower
extent on monocytes, macrophages, lymphocytes T as well as in
spleen and bone marrow. Its mRNA is also weakly detected in
eosinophils and mast cells. Its expression is enhanced by cytokine
and LPS stimulation, suggesting a possible role in leukocyte
differentiation and activation (Senga et al., 2002).
Calculation of Sequence Homology
[0140] Sequence identity with respect to any of the sequences
presented herein can be determined by a simple "eyeball" comparison
(i.e. a strict comparison) of any one or more of the sequences with
another sequence to see if that other sequence has, for example, at
least 80% sequence identity to the sequence(s).
[0141] Relative sequence identity can also be determined by
commercially available computer programs that can calculate %
identity between two or more sequences using any suitable algorithm
for determining identity, using for example default parameters. A
typical example of such a computer program is CLUSTAL. Other
computer program methods to determine identity and similarity
between two sequences include but are not limited to the GCG
program package (Devereux et al 1984 Nucleic Acids Research 12:
387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).
[0142] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0143] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0144] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example, when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension.
[0145] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research
12:387). Examples of other software that can perform sequence
comparisons include, but are not limited to, the BLAST package
(Ausubel et al., 1995, Short Protocols in Molecular Biology, 3rd
Edition, John Wiley & Sons), FASTA (Atschul et al., 1990, J.
Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools.
Both BLAST and FASTA are available for offline and online searching
(Ausubel et al., 1999 supra, pages 7-58 to 7-60).
[0146] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied. It is preferred to
use the public default values for the GCG package, or in the case
of other software, the default matrix, such as BLOSUM62.
[0147] Advantageously, the BLAST algorithm is employed, with
parameters set to default values. The BLAST algorithm is described
in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which
is incorporated herein by reference. The search parameters are
defined as follows, and can be advantageously set to the defined
default parameters.
[0148] Advantageously, "substantial identity" when assessed by
BLAST equates to sequences which match with an EXPECT value of at
least about 7, preferably at least about 9 and most preferably 10
or more. The default threshold for EXPECT in BLAST searching is
usually 10.
[0149] BLAST (Basic Local Alignment Search Tool) is the heuristic
search algorithm employed by the programs blastp, blastn, blastx,
tblastn, and tblastx; these programs ascribe significance to their
findings using the statistical methods of Karlin and Altschul
(Karlin and Altschul 1990, Proc. Natl. Acad. Sci. USA 87:2264-68;
Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-7;
see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few
enhancements. The BLAST programs are tailored for sequence
similarity searching, for example to identify homologues to a query
sequence. For a discussion of basic issues in similarity searching
of sequence databases, see Altschul et al (1994) Nature Genetics
6:119-129.
[0150] The five BLAST programs available at
http://www.ncbi.nlm.nih.gov perform the following tasks:
blastp--compares an amino acid query sequence against a protein
sequence database; blastn--compares a nucleotide query sequence
against a nucleotide sequence database; blastx--compares the
six-frame conceptual translation products of a nucleotide query
sequence (both strands) against a protein sequence database;
tblastn--compares a protein query sequence against a nucleotide
sequence database dynamically translated in all six reading frames
(both strands); tblastx--compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0151] BLAST uses the following search parameters:
[0152] HISTOGRAM--Display a histogram of scores for each search;
default is yes. (See parameter H in the BLAST Manual).
[0153] DESCRIPTIONS--Restricts the number of short descriptions of
matching sequences reported to the number specified; default limit
is 100 descriptions. (See parameter V in the manual page).
[0154] EXPECT--The statistical significance threshold for reporting
matches against database sequences; the default value is 10, such
that 10 matches are expected to be found merely by chance,
according to the stochastic model of Karlin and Altschul (1990). If
the statistical significance ascribed to a match is greater than
the EXPECT threshold, the match will not be reported. Lower EXPECT
thresholds are more stringent, leading to fewer chance matches
being reported. Fractional values are acceptable. (See parameter E
in the BLAST Manual).
[0155] CUTOFF--Cutoff score for reporting high-scoring segment
pairs. The default value is calculated from the EXPECT value (see
above). HSPs are reported for a database sequence only if the
statistical significance ascribed to them is at least as high as
would be ascribed to a lone HSP having a score equal to the CUTOFF
value. Higher CUTOFF values are more stringent, leading to fewer
chance matches being reported. (See parameter S in the BLAST
Manual). Typically, significance thresholds can be more intuitively
managed using EXPECT.
[0156] ALIGNMENTS--Restricts database sequences to the number
specified for which high-scoring segment pairs (HSPs) are reported;
the default limit is 50. If more database sequences than this
happen to satisfy the statistical significance threshold for
reporting (see EXPECT and CUTOFF below), only the matches ascribed
the greatest statistical significance are reported. (See parameter
B in the BLAST Manual).
[0157] MATRIX--Specify an alternate scoring matrix for BLASTP,
BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62
(Henikoff & Henikoff, 1992). The valid alternative choices
include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring
matrices are available for BLASTN; specifying the MATRIX directive
in BLASTN requests returns an error response.
[0158] STRAND--Restrict a TBLASTN search to just the top or bottom
strand of the database sequences; or restrict a BLASTN, BLASTX or
TBLASTX search to just reading frames on the top or bottom strand
of the query sequence.
[0159] FILTER--Mask off segments of the query sequence that have
low compositional complexity, as determined by the SEG program of
Wootton & Federhen (1993) Computers and Chemistry 17:149-163,
or segments consisting of short-periodicity internal repeats, as
determined by the XNU program of Clayerie & States (1993)
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST
program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov).
Filtering can eliminate statistically significant but biologically
uninteresting reports from the blast output (e.g., hits against
common acidic-, basic- or proline-rich regions), leaving the more
biologically interesting regions of the query sequence available
for specific matching against database sequences.
[0160] Low complexity sequence found by a filter program is
substituted using the letter "N" in nucleotide sequence (e.g.,
"NNNNNNNNNNNNN") and the letter "X" in protein sequences (e.g.,
"XXXXXXXXX").
[0161] Filtering is only applied to the query sequence (or its
translation products), not to database sequences. Default filtering
is DUST for BLASTN, SEG for other programs.
[0162] It is not unusual for nothing at all to be masked by SEG,
XNU, or both, when applied to sequences in SWISS-PROT, so filtering
should not be expected to always yield an effect. Furthermore, in
some cases, sequences are masked in their entirety, indicating that
the statistical significance of any matches reported against the
unfiltered query sequence should be suspect.
[0163] NCBI-gi--Causes NCBI gi identifiers to be shown in the
output, in addition to the accession and/or locus name.
[0164] Most preferably, sequence comparisons are conducted using
the simple BLAST search algorithm provided at
http://www.ncbi.nlm.nih.gov/BLAST. In some embodiments of the
present invention, no gap penalties are used when determining
sequence identity.
Cells
[0165] A cell that is useful according to the invention is
preferably selected from the group consisting of bacterial cells,
yeast cells, insect cells or mammalian cells.
[0166] A cell that is useful according to the invention can be any
cell into which a nucleic acid sequence encoding a receptor
according to the invention can be introduced such that the receptor
is expressed at natural levels or above natural levels, as defined
herein. Preferably a receptor of the invention that is expressed in
a cell exhibits normal or near normal pharmacology, as defined
herein. Most preferably a receptor of the invention that is
expressed in a cell comprises the nucleotide or amino acid sequence
presented in FIG. 1 or a nucleotide or amino acid sequence that is
at least 70% identical to the amino acid sequence presented in FIG.
1. Preferably, a receptor of the invention that is expressed in a
cell will bind propionate with an affinity that is at least
100-fold, preferably 500-fold and most preferably 1000-fold greater
than the affinity for IDP and UDP.
[0167] According to a preferred embodiment of the present
invention, a cell is selected from the group consisting of
COS7-cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293
cell, K-562 cell or a 1321N1 astrocytoma cell but also other
transfectable cell lines.
Assays
[0168] I. Assays For The Identification Of Agents That Modulate The
Activity Of GPR43
[0169] Agents that modulate the activity of GPR43 can be identified
in a number of ways that take advantage of the newly discovered
interaction of the receptor with SCFAs, such as acetete and
propionate. For example, the ability to reconstitute
GPR43/propionate binding either in vitro, on cultured cells or in
vivo provides a target for the identification of agents that
disrupt that binding. Assays based on disruption of binding can
identify agents, such as small organic molecules, from libraries or
collections of such molecules. Alternatively, such assays can
identify agents in samples or extracts from natural sources, e.g.,
plant, fungal or bacterial extracts or even in human tissue samples
(e.g., tumor tissue). In one aspect, the extracts can be made from
cells expressing a library of variant nucleic acids, peptides or
polypeptides. Modulators of GPR43/SCFA binding can then be screened
using a binding assay or a functional assay that measures
downstream signalling through the receptor.
[0170] Another approach that uses the GPR43/SCFA interaction more
directly to identify agents that modulate GPR43 function measures
changes in GPR43 downstream signalling induced by candidate agents
or candidate modulators. These functional assays can be performed
in isolated cell membrane fractions or on cells expressing the
receptor on their surfaces.
[0171] The discovery that SCFAs, such as acetate and propionate are
ligands of the GPR43 receptor permits screening assays to identify
agonists, antagonists and inverse agonists of receptor activity.
The screening assays have two general approaches, detailed below.
For the purposes of this section propionate is used as an exemplary
SCFA. It should be understood, however, that any SCFA as defined
herein can be used in the assays described.
[0172] 1) Ligand binding assays, in which cells expressing GPR43,
membrane extracts from such cells, or immobilized lipid membranes
comprising GPR43 are exposed to labelled and candidate compound.
Following incubation, the reaction mixture is measured for specific
binding of the labelled to the GPR43 receptor. Compounds that
interfere with binding or displace labelled can be agonists,
antagonists or inverse agonists of GPR43 activity. Subsequent
functional analysis can then be performed on positive compounds to
determine in which of these categories they belong.
[0173] 2) Functional assays, in which a signalling activity of
GPR43 is measured.
[0174] a) For agonist screening, cells expressing GPR43 or
membranes prepared from them are incubated with a candidate
compound, and a signalling activity of GPR43 is measured. The
activity induced by compounds that modulate receptor activity is
compared to that induced by the natural ligands, acetate or
propionate. An agonist or partial agonist will have a maximal
biological activity corresponding to at least 10% of the maximal
activity of propionate when the agonist or partial agonist is
present at 1 mM or less, and preferably will have a potency which
is at least as potent as acetate or propionate.
[0175] b) For antagonist or inverse agonist screening, cells
expressing GPR43 or membranes isolated from them are assayed for
signalling activity in the presence of propionate with or without a
candidate compound. Antagonists will reduce the level of
propionate-stimulated receptor activity by at least 10%, relative
to reactions lacking the antagonist in the presence of propionate.
Inverse agonists will reduce the constitutive activity of the
receptor by at least 10%, relative to reactions lacking the inverse
agonist.
[0176] c) For inverse agonist screening, cells expressing
constitutive GPR43 activity or membranes isolated from them are
used in a functional assay that measures an activity of the
receptor in the presence of a candidate compound. Inverse agonists
are those compounds that reduce the constitutive activity of the
receptor by at least 10%. Overexpression of GPR43 may lead to
constitutive activation. GPR43 can be overexpressed by placing it
under the control of a strong constitutive promoter, e.g., the CMV
early promoter. Alternatively, certain mutations of conserved GPCR
amino acids or amino acid domains tend to lead to constitutive
activity. See for example: Kjelsberg et al., 1992, J. Biol. Chem.
267:1430; McWhinney et al., 2000. J. Biol. Chem. 275:2087; Ren et
al., 1993, J. Biol. Chem. 268:16483; Samama et al., 1993, J. Biol.
Chem. 268:4625; Parma et al., 1993, Nature 365:649; Parma et al.,
1998, J. Pharmacol. Exp. Ther. 286:85; and Parent et al., 1996, J.
Biol. Chem. 271:7949.
[0177] Ligand Binding and Displacement Assays:
[0178] As noted in (1) above, one can use GPR43 polypeptides
expressed on a cell, or isolated membranes containing receptor
polypeptides, along with propionate in order to screen for
compounds that inhibit the binding of propionate to GPR43. For the
purposes of this section, propionate is used as an exemplary SCFA.
It should be understood however that any SCFA as defined herein can
be used in the assays described.
[0179] For displacement experiments, cells expressing a GPR43
polypeptide (generally 25,000 cells per assay or 1 to 100 .mu.g of
membrane extracts) are incubated in binding buffer with labelled
propionate in the presence or absence of increasing concentrations
of a candidate modulator. To validate and calibrate the assay,
control competition reactions using increasing concentrations of
unlabeled propionate can be performed. After incubation, cells are
washed extensively, and bound, labelled propionate is measured as
appropriate for the given label (e.g., scintillation counting,
fluorescence, etc.). A decrease of at least 10% in the amount of
labelled propionate bound in the presence of candidate modulator
indicates displacement of binding by the candidate modulator.
Candidate modulators are considered to bind specifically in this or
other assays described herein if they displace 50% of labelled
propionate (sub-saturating propionate dose) at a concentration of 1
mM or less.
[0180] Alternatively, binding or displacement of binding can be
monitored by surface plasmon resonance (SPR). Surface plasmon
resonance assays can be used as a quantitative method to measure
binding between two molecules by the change in mass near an
immobilized sensor caused by the binding or loss of binding of
propionate from the aqueous phase to a GPR43 polypeptide
immobilized in a membrane on the sensor. This change in mass is
measured as resonance units versus time after injection or removal
of the propionate or candidate modulator and is measured using a
Biacore Biosensor (Biacore AB). GPR43 can be immobilized on a
sensor chip (for example, research grade CM5 chip; Biacore AB) in a
thin film lipid membrane according to methods described by Salamon
et al. (Salamon et al., 1996, Biophys J. 71: 283-294; Salamon et
al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends
Biochem. Sci. 24: 213-219, each of which is incorporated herein by
reference.). Sarrio et al. demonstrated that SPR can be used to
detect ligand binding to the GPCR A(1) adenosine receptor
immobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol.
Cell. Biol. 20: 5164-5174, incorporated herein by reference).
Conditions for propionate binding to GPR43 in an SPR assay can be
fine-tuned by one of skill in the art using the conditions reported
by Sarrio et al. as a starting point.
[0181] SPR can assay for modulators of binding in at least two
ways. First, propionate can be pre-bound to immobilized GPR43
polypeptide, followed by injection of candidate modulator at a
concentration ranging from 0.1 nM to 1 .mu.M. Displacement of the
bound propionate can be quantitated, permitting detection of
modulator binding. Alternatively, the membrane-bound GPR43
polypeptide can be pre-incubated with candidate modulator and
challenged with propionate. A difference in propionate binding to
the GPR43 exposed to modulator relative to that on a chip not
pre-exposed to modulator will demonstrate binding or displacement
of propionate in the presence of modulator. In either assay, a
decrease of 10% or more in the amount of propionate bound is in the
presence of candidate modulator, relative to the amount of a
propionate bound in the absence of candidate modulator indicates
that the candidate modulator inhibits the interaction of GPR43 and
propionate.
[0182] Another method of detecting inhibition of binding of
propionate to GPR43 uses fluorescence resonance energy transfer
(FRET). FRET is a quantum mechanical phenomenon that occurs between
a fluorescence donor (D) and a fluorescence acceptor (A) in close
proximity to each other (usually <100 A of separation) if the
emission spectrum of D overlaps with the excitation spectrum of A.
The molecules to be tested, e.g. propionate and a GPR43
polypeptide, are labelled with a complementary pair of donor and
acceptor fluorophores. While bound closely together by the GPR43:
propionate interaction, the fluorescence emitted upon excitation of
the donor fluorophore will have a different wavelength than that
emitted in response to that excitation wavelength when the
propionate and GPR43 polypeptide are not bound, providing for
quantitation of bound versus unbound molecules by measurement of
emission intensity at each wavelength. Donor fluorophores with
which to label the GPR43 polypeptide are well known in the art. Of
particular interest are variants of the A. victoria GFP known as
Cyan FP (CFP, Donor (D)) and Yellow FP (YFP, Acceptor(A)). As an
example, the YFP variant can be made as a fusion protein with
GPR43. Vectors for the expression of GFP variants as fusions
(Clontech) as well as fluorophore-labeled propionate compounds
(Molecular Probes) are known in the art. The addition of a
candidate modulator to the mixture of labelled propionate and
YFP-GPR43 protein will result in an inhibition of energy transfer
evidenced by, for example, a decrease in YFP fluorescence relative
to a sample without the candidate modulator. In an assay using FRET
for the detection of GPR43: propionate interaction, a 10% or
greater decrease in the intensity of fluorescent emission at the
acceptor wavelength in samples containing a candidate modulator,
relative to samples without the candidate modulator, indicates that
the candidate modulator inhibits the GPR43: propionate
interaction.
[0183] A variation on FRET uses fluorescence quenching to monitor
molecular interactions. One molecule in the interacting pair can be
labelled with a fluorophore, and the other with a molecule that
quenches the fluorescence of the fluorophore when brought into
close apposition with it. A change in fluorescence upon excitation
is indicative of a change in the association of the molecules
tagged with the fluorophore:quencher pair. Generally, an increase
in fluorescence of the labelled GPR43 polypeptide is indicative
that the propionate molecule bearing the quencher has been
displaced. For quenching assays, a 10% or greater increase in the
intensity of fluorescent emission in samples containing a candidate
modulator, relative to samples without the candidate modulator,
indicates that the candidate modulator inhibits GPR43: propionate
interaction.
[0184] In addition to the surface plasmon resonance and FRET
methods, fluorescence polarization measurement is useful to
quantitate binding. The fluorescence polarization value for a
fluorescently-tagged molecule depends on the rotational correlation
time or tumbling rate. Complexes, such as those formed by GPR43
associating with a fluorescently labelled propionate, have higher
polarization values than uncomplexed, labelled propionate. The
inclusion of a candidate inhibitor of the GPR43: propionate
interaction results in a decrease in fluorescence polarization,
relative to a mixture without the candidate inhibitor, if the
candidate inhibitor disrupts or inhibits the interaction of GPR43
with propionate. Fluorescence polarization is well suited for the
identification of small molecules that disrupt the formation of
receptor:ligand complexes. A decrease of 10% or more in
fluorescence polarization in samples containing a candidate
modulator, relative to fluorescence polarization in a sample
lacking the candidate modulator, indicates that the candidate
modulator inhibits GPR43: propionate interaction.
[0185] Another alternative for monitoring GPR43: propionate
interactions uses a biosensor assay. ICS biosensors have been
described in the art (Australian Membrane Biotechnology Research
Institute; www.ambri.com.au/; Cornell B, Braach-Maksvytis V, King
L, Osman P, Raguse B, Wieczorek L, and Pace R. "A biosensor that
uses ion-channel switches" Nature 1997, 387, 580). In this
technology, the association of GPR43 and its ligand is coupled to
the closing of gramacidin-facilitated ion channels in suspended
membrane bilayers and thus to a measurable change in the admittance
(similar to impedence) of the biosensor. This approach is linear
over six orders of magnitude of admittance change and is ideally
suited for large scale, high throughput screening of small molecule
combinatorial libraries. A 10% or greater change (increase or
decrease) in admittance in a sample containing a candidate
modulator, relative to the admittance of a sample lacking the
candidate modulator, indicates that the candidate modulator
inhibits the interaction of GPR43 and propionate. It is important
to note that in assays testing the interaction of GPR43 with
propionate, it is possible that a modulator of the interaction need
not necessarily interact directly with the domain(s) of the
proteins that physically interact with propionate. It is also
possible that a modulator will interact at a location removed from
the site of interaction and cause, for example, a conformational
change in the GPR43 polypeptide. Modulators (inhibitors or
agonists) that act in this manner are nonetheless of interest as
agents to modulate the activity of GPR43.
[0186] It should be understood that any of the binding assays
described herein can be performed with a non-propionate ligand (for
example, agonist, antagonist, etc.) of GPR43, e.g., a small
molecule identified as described herein or propionate analogues
including but not limited to any of the propionate analogues, a
natural or synthetic peptide, a polypeptide, an antibody or
antigen-binding fragment thereof, a lipid, a carbohydrate, and a
small organic molecule.
[0187] Any of the binding assays described can be used to determine
the presence of an agent in a sample, e.g., a tissue sample, that
binds to the GPR43 receptor molecule, or that affects the binding
of propionate to the receptor. To do so, GPR43 polypeptide is
reacted with propionate or another ligand in the presence or
absence of the sample, and propionate or ligand binding is measured
as appropriate for the binding assay being used. A decrease of 10%
or more in the binding of propionate or other ligand indicates that
the sample contains an agent that modulates propionate or ligand
binding to the receptor polypeptide.
Functional Assays of Receptor Activity
[0188] i. GTPase/GTP Binding Assays:
[0189] For GPCRs such as GPR43, a measure of receptor activity is
the binding of GTP by cell membranes containing receptors. In the
method described by Traynor and Nahorski, 1995, Mol.
[0190] Pharmacol. 47: 848-854, incorporated herein by reference,
one essentially measures G-protein coupling to membranes by
detecting the binding of labelled GTP. For GTP binding assays,
membranes isolated from cells expressing the receptor are incubated
in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, and 10 mM
MgCl.sub.2, 80 pM .sup.35S-GTP.gamma.S and 3 .mu.M GDP. The assay
mixture is incubated for 60 minutes at 30.degree. C., after which
unbound labelled GTP is removed by filtration onto GF/B filters.
Bound, labelled GTP is measured by liquid scintillation counting.
In order to assay for modulation of propionate-induced GPR43
activity, membranes prepared from cells expressing a GPR43
polypeptide are mixed with propionate, and the GTP binding assay is
performed in the presence and absence of a candidate modulator of
GPR43 activity. An increase of 10% or more in labelled GTP binding
as measured by scintillation counting in an assay of this kind
containing a candidate modulator, relative to an assay without the
modulator, indicates that the candidate modulator inhibits GPR43
activity. A similar GTP-binding assay can be performed without
propionate to identify compounds that act as agonists. In this
case, propionate-stimulated GTP binding is used as a standard. A
compound is considered an agonist if it induces at least 50% of the
level of GTP binding induced by propionate when the compound is
present at 1 .mu.M or less, and preferably will induce a level the
same as or higher than that induced by propionate. GTPase activity
is measured by incubating the membranes containing a GPR43
polypeptide with .gamma..sup.=P-GTP. Active GTPase will release the
label as inorganic phosphate, which is detected by separation of
free inorganic phosphate in a 5% suspension of activated charcoal
in 20 mM H.sub.3PO.sub.4, followed by scintillation counting.
Controls include assays using membranes isolated from cells not
expressing GPR43 (mock-transfected), in order to exclude possible
non-specific effects of the candidate compound.
[0191] In order to assay for the effect of a candidate modulator on
GPR43-regulated GTPase activity, membrane samples are incubated
with propionate, with and without the modulator, followed by the
GTPase assay. A change (increase or decrease) of 10% or more in the
level of GTP binding or GTPase activity relative to samples without
modulator is indicative of GPR43 modulation by a candidate
modulator.
[0192] ii. Downstream Pathway Activation Assays:
[0193] a. Calcium flux--The Aeguorin-Based Assay.
[0194] The aequorin assay takes advantage of the responsiveness of
mitochondrial apoaequorin to intracellular calcium release induced
by the activation of GPCRs (Stables et al., 1997, Anal. Biochem.
252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508;
both of which are incorporated herein by reference). Briefly,
GPR43-expressing clones are transfected to coexpress mitochondrial
apoaequorin and G.alpha.16. Cells are incubated with 5 .mu.M
Coelenterazine H (Molecular Probes) for 4 hours at room
temperature, washed in DMEM-F12 culture medium and resuspended at a
concentration of 0.5.times.10.sup.6 cells/ml. Cells are then mixed
with test agonist molecules and light emission by the aequorin is
recorded with a luminometer for 30 sec. Results are expressed as
Relative Light Units (RLU). Controls include assays using membranes
isolated from cells not expressing GPR43 (mock transfected), in
order to exclude possible non-specific;
[0195] effects of the candidate compound.
[0196] Aequorin activity or intracellular calcium levels are
"changed" if light intensity increases or decreases by 10% or more
in a sample of cells, expressing a GPR43 polypeptide and treated
with a candidate modulator, relative to a sample of cells
expressing the GPR43 polypeptide but not treated with the candidate
modulator or relative to a sample of cells not expressing the GPR43
polypeptide (mock-transfected cells) but treated with the candidate
modulator.
[0197] When performed in the absence of propionate, the assay can
be used to identify an agonist of GPR43 activity. When the assay is
performed in the presence of propionate, it can be used to assay
for an antagonist.
[0198] b. Adenylate Cyclase Assay:
[0199] Assays for adenylate cyclase activity are described by
Kenimer & Nirenberg, 1981, Mol. Pharmacol. 20: 585-591,
incorporated herein by reference. That assay is a modification of
the assay taught by Solomon et al., 1974, Anal. Biochem. 58:
541-548, also incorporated herein by reference. Briefly, 100 .mu.l
reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mM MgCl.sub.2, 20 mM
creatine phosphate (disodium salt), 10 units (71 .mu.g of protein)
of creatine phosphokinase, 1 mM .alpha.-.sup.32P-ATP (tetrasodium
salt, 2 .mu.Ci), 0.5 mM cyclic AMP, G-.sup.-3H-labeled cyclic AMP
(approximately 10,000 cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and
50-200 .mu.g of protein homogenate to be tested (i.e., homogenate
from cells expressing or not expressing a GPR43 polypeptide,
treated or not treated with propionate with or without a candidate
modulator). Reaction mixtures are generally incubated at 37.degree.
C. for 60 minutes. Following incubation, reaction mixtures are
deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic
acid. Tubes are centrifuged at 1800.times.g for 20 minutes and each
supernatant solution is added to a Dowex AG50W-X4 column. The cAMP
fraction from the column is eluted with 4 ml of 0.1 mM
imidazole-HCl (pH 7.5) into a counting vial. Assays should be
performed in triplicate. Control reactions should also be performed
using protein homogenate from cells that do not express a GPR43
polypeptide.
[0200] According to the invention, adenylate cyclase activity is
"changed" if it increases or decreases by 10% or more in a sample
taken from cells treated with a candidate modulator of GPR43
activity, relative to a similar sample of cells not treated with
the candidate modulator or relative to a sample of cells not
expressing the GPR43 polypeptide (mock-transfected cells) but
treated with the candidate modulator.
[0201] c. cAMP Assay:
[0202] Intracellular or extracellular cAMP is measured using a cAMP
radioimmunoassay (RIA) or cAMP binding protein according to methods
widely known in the art. For example, Horton & Baxendale, 1995,
Methods Mol. Biol. 41: 91-105, which is incorporated herein by
reference, describes an RIA for cAMP.
[0203] A number of kits for the measurement of cAMP are
commercially available, such as the High Efficiency Fluorescence
Polarization-based homogeneous assay marketed by LJL Biosystems and
NEN Life Science Products. Control reactions should be performed
using extracts of mock-transfected cells to exclude possible
non-specific effects of some candidate modulators.
[0204] The level of cAMP is "changed" if the level of cAMP detected
in cells, expressing a GPR43 polypeptide and treated with a
candidate modulator of GPR43 activity (or in extracts of such
cells), using the RIA-based assay of Horton & Baxendale, 1995,
supra, increases or decreases by at least 10% relative to the cAMP
level in similar cells not treated with the candidate
modulator.
[0205] d. Phospholipid Breakdown. DAG Production and Inositol
Triphosphate Levels:
[0206] Receptors that activate the breakdown of phospholipids can
be monitored for changes due to the activity of known or suspected
modulators of GPR43 by monitoring phospholipid breakdown, and the
resulting production of second messengers DAG and/or inositol
triphosphate (IP3). Methods of detecting each of these are
described in Phospholipid Signalling Protocols, edited by Ian M.
Bird. Totowa, N.J., Humana Press, 1998, which is incorporated
herein by reference. See also Rudolph et al., 1999, J. Biol. Chem.
274: 11824-11831, incorporated herein by reference, which also
describes an assay for phosphatidylinositol breakdown. Assays
should be performed using cells or extracts of cells expressing
GPR43, treated or not treated with propionate with or without a
candidate modulator. Control reactions should be performed using
mock-transfected cells, or extracts from them in order to exclude
possible non-specific effects of some candidate modulators.
[0207] According to the invention, phosphatidylinositol breakdown,
and diacylglycerol and/or inositol triphosphate levels are
"changed" if they increase or decrease by at least 10% in a sample
from cells expressing a GPR43 polypeptide and treated with a
candidate modulator, relative to the level observed in a sample
from cells expressing a GPR43 polypeptide that is not treated with
the candidate modulator.
[0208] e. PKC Activation Assays:
[0209] Growth factor receptor tyrosine kinases can signal via a
pathway involving activation of Protein Kinase C (PKC), which is a
family of phospholipid- and calcium-activated protein kinases. PKC
activation ultimately results in the transcription of an array of
proto-oncogene transcription factor-encoding genes, including
c-fos, c-myc and c-jun, proteases, protease inhibitors, including
collagenase type I and plasminogen activator inhibitor, and
adhesion molecules, including intracellular adhesion molecule I
(ICAM I). Assays designed to detect increases in gene products
induced by PKC can be used to monitor PKC activation and thereby
receptor activity. In addition, the activity of receptors that
signal via PKC can be monitored through the use of reporter gene
constructs driven by the control sequences of genes activated by
PKC activation. This type of reporter gene-based assay is discussed
in more detail below.
[0210] For a more direct measure of PKC activity, the method of
Kikkawa et al., 1982, J. Biol. Chem. 257: 13341, incorporated
herein by reference, can be used. This assay measures
phosphorylation of a PKC substrate peptide, which is subsequently
separated by binding to phosphocellulose paper. This PKC assay
system can be used to measure activity of purified kinase, or the
activity in crude cellular extracts. Protein kinase C sample can be
diluted in 20 mM HEPES/2 mM DTT immediately prior to assay.
[0211] The substrate for the assay is the peptide
Ac-FKKSFKL-NH.sub.2 (SEQ ID NO: 3), derived from the myristoylated
alanine-rich protein kinase C substrate protein (MARCKS). The
K.sub.m of the enzyme for this peptide is approximately 50 .mu.M.
Other basic, protein kinase C-selective peptides known in the art
can also be used, at a concentration of at least 2-3 times their
K.sub.m. Cofactors required for the assay include calcium,
magnesium, ATP, phosphatidylserine and diacylglycerol. Depending
upon the intent of the user, the assay can be performed to
determine the amount of PKC present (activating conditions) or the
amount of active PKC present (non-activating conditions). For most
purposes according to the invention, non-activating conditions will
be used, such that the PKC, that is active in the sample when it is
isolated, is measured, rather than measuring the PKC that can be
activated. For non-activating conditions, calcium is omitted from
the assay in favor of EGTA.
[0212] The assay is performed in a mixture containing 20 mM HEPES,
pH 7.4, 1-2 mM DTT, 5 mM MgCl.sub.2, 100 .mu.M ATP, .about.1 .mu.Ci
.gamma.-.sup.32P-ATP, 100 .mu.g/ml peptide substrate (.about.100
.mu.M), 140 .mu.M/3.8 .mu.M phosphatidylserine/diacylglycerol
membranes, and 100 .mu.M calcium (or 500 .mu.M EGTA). 48 .mu.l of
sample, diluted in 20 mM HEPES, pH 7.4, 2 mM DTT is used in a final
reaction volume of 80 .mu.l. Reactions are performed at 30.degree.
C. for 5-10 minutes, followed by addition of 25 .mu.l of 100 mM
ATP, 100 mM EDTA, pH 8.0, which stops the reactions.
[0213] After the reaction is stopped, a portion (85 .mu.l) of each
reaction is spotted onto a Whatman P81 cellulose phosphate filter,
followed by washes: four times 500 ml in 0.4% phosphoric acid,
(5-10 min per wash); and a final wash in 500 ml 95% EtOH, for 2-5
min. Bound radioactivity is measured by scintillation counting.
Specific activity (cpm/nmol) of the labelled ATP is determined by
spotting a sample of the reaction onto P81 paper and counting
without washing. Units of PKC activity, defined as nmol phosphate
transferred per min, are calculated as follows:
[0214] The activity, in UNITS (nmol/min) is:
= ( cpm on paper ) .times. ( 105 l total / 85 l spotted ) ( assay
time , min ) ( specific activity of ATP cpm / nmol ) .
##EQU00001##
[0215] An alternative assay can be performed using a Protein Kinase
C Assay Kit sold by PanVera (Cat. # P2747).
[0216] Assays are performed on extracts from cells expressing a
GPR43 polypeptide, treated or not treated with propionate with or
without a candidate modulator. Control reactions should be
performed using mock-transfected cells, or extracts from them in
order to exclude possible non-specific effects of some candidate
modulators.
[0217] According to the invention, PKC activity is "changed" by a
candidate modulator when the units of PKC measured by either assay
described above increase or decrease by at least 10%, in extracts
from cells expressing GPR43 and treated with a candidate modulator,
relative to a reaction performed on a similar sample from cells not
treated with a candidate modulator.
[0218] f. Kinase Assays:
[0219] MAP kinase activity can be assayed using any of several kits
available commercially, for example, the p38 MAP Kinase assay kit
sold by New England Biolabs (Cat # 9820) or the FlashPlate.TM. MAP
Kinase assays sold by Perkin-Elmer Life Sciences.
[0220] MAP Kinase activity is "changed" if the level of activity is
increased or decreased by 10% or more in a sample from cells,
expressing a GPR43 polypeptide, treated with a candidate modulator
relative to MAP kinase activity in a sample from similar cells not
treated with the candidate modulator.
[0221] Direct assays for tyrosine kinase activity using known
synthetic or natural tyrosine kinase substrates and labelled
phosphate are well known, as are similar assays for other types of
kinases (e.g., Ser/Thr kinases). Kinase assays can be performed
with both purified kinases and crude extracts prepared from cells
expressing a GPR43 polypeptide, treated with or without propionate,
with or without a candidate modulator. Control reactions should be
performed using mock-transfected cells, or extracts from them in
order to exclude possible non-specific effects of some candidate
modulators. Substrates can be either full-length protein or
synthetic peptides representing the substrate. Pinna & Ruzzene
(1996, Biochem. Biophys. Acta 1314: 191-225, incorporated herein by
reference) list a number of phosphorylation substrate sites useful
for detecting kinase activities. A number of kinase substrate
peptides are commercially available. One that is particularly
useful is the "Src-related peptide," RRLIEDAEYAARG (SEQ ID NO: 4;
available from Sigma # A7433), which is a substrate for many
receptor and nonreceptor tyrosine kinases. Because the assay
described below requires binding of peptide substrates to filters,
the peptide substrates should have a net positive charge to
facilitate binding. Generally, peptide substrates should have at
least 2 basic residues and a free amino terminus. Reactions
generally use a peptide concentration of 0.7-1.5 mM.
[0222] Assays are generally carried out in a 25 .mu.l volume
comprising 5 .mu.l of 5.times. kinase buffer (5 mg/mL BSA, 150 mM
Tris-Cl (pH 7.5), 100 mM MgCl.sub.2; depending upon the exact
kinase assayed for, MnCl.sub.2 can be used in place of or in
addition to the MgCl.sub.2), 5 .mu.l of 1.0 mM ATP (0.2 mM final
concentration), .gamma.-.sup.32P-ATP (100-500 cpm/.mu.mol), 3 .mu.l
of 10 mM peptide substrate (1.2 mM final concentration), cell
extract containing kinase to be tested (cell extracts used for
kinase assays should contain a phosphatase inhibitor (e.g. 0.1-1 mM
sodium orthovanadate)), and H.sub.2O to 25 .mu.l. Reactions are
performed at 30.degree. C., and are initiated by the addition of
the cell extract.
[0223] Kinase reactions are performed for 30 seconds to about 30
minutes, followed by the addition of 45 .mu.l of ice-cold 10%
trichloroacetic acid (TCA). Samples are spun for 2 minutes in a
microcentrifuge, and 35 .mu.l of the supernatant is spotted onto
Whatman P81 cellulose phosphate filter circles. The filters are
washed three times with 500 ml cold 0.5% phosphoric acid, followed
by one wash with 200 ml of acetone at room temperature for 5
minutes. Filters are dried and incorporated .sup.32P is measured by
scintillation counting. The specific activity of ATP in the kinase
reaction (e.g., in cpm/.mu.mol) is determined by spotting a small
sample (2-5 .mu.l) of the reaction onto a P81 filter circle and
counting directly, without washing. Counts per minute obtained in
the kinase reaction (minus blank) are then divided by the specific
activity to determine the moles of phosphate transferred in the
reaction.
[0224] Tyrosine kinase activity is "changed" if the level of kinase
activity is increased or decreased by 10% or more in a sample from
cells, expressing a GPR43 polypeptide, treated with a candidate
modulator relative to kinase activity in a sample from similar
cells not treated with the candidate modulator.
[0225] g. Transcriptional Reporters for Downstream Pathway
Activation:
[0226] The intracellular signal initiated by binding of an agonist
to a receptor, e.g., GPR43, sets in motion a cascade of
intracellular events, the ultimate consequence of which is a rapid
and detectable change in the transcription or translation of one or
more genes. The activity of the receptor can therefore be monitored
by detecting the expression of a reporter gene driven by control
sequences responsive to GPR43 activation.
[0227] As used herein "promoter" refers to the transcriptional
control elements necessary for receptor-mediated regulation of gene
expression, including not only the basal promoter, but also any
enhancers or transcription-factor binding sites necessary for
receptor-regulated expression.
[0228] By selecting promoters that are responsive to the
intracellular signals resulting from agonist binding, and
operatively linking the selected promoters to reporter genes whose
transcription, translation or ultimate activity is readily
detectable and measurable, the transcription based reporter assay
provides a rapid indication of whether a given receptor is
activated.
[0229] Reporter genes such as luciferase, CAT, GFP,
.beta.-lactamase or .beta.-galactosidase are well known in the art,
as are assays for the detection of their products.
[0230] Genes particularly well suited for monitoring receptor
activity are the "immediate early" genes, which are rapidly
induced, generally within minutes of contact between the receptor
and the effector protein or ligand. The induction of immediate
early gene transcription does not require the synthesis of new
regulatory proteins. In addition to rapid responsiveness to ligand
binding, characteristics of preferred genes useful for making
reporter constructs include: low or undetectable expression in
quiescent cells; induction that is transient and independent of new
protein synthesis; subsequent shut-off of transcription requires
new protein synthesis; and mRNAs transcribed from these genes have
a short half-life. It is preferred, but not necessary that a
transcriptional control element have all of these properties for it
to be useful.
[0231] An example of a gene that is responsive to a number of
different stimuli is the c-fos proto-oncogene. The c-fos gene is
activated in a protein-synthesis-independent manner by growth
factors, hormones, differentiation-specific agents, stress, and
other known inducers of cell surface proteins. The induction of
c-fos expression is extremely rapid, often occurring within minutes
of receptor stimulation. This characteristic makes the c-fos
regulatory regions particularly attractive for use as a reporter of
receptor activation.
[0232] The c-fos regulatory elements include (see, Verma et al.,
1987, Cell 51: 513-514): a TATA box that is required for
transcription initiation; two upstream elements for basal
transcription, and an enhancer, which includes an element with dyad
symmetry and which is required for induction by TPA, serum, EGF,
and PMA.
[0233] The 20 bp c-fos transcriptional enhancer element located
between -317 and -298 bp upstream from the c-fos mRNA cap site, is
essential for serum induction in serum starved NIH 3T3 cells. One
of the two upstream elements is located at -63 to -57 and it
resembles the consensus sequence for cAMP regulation.
[0234] The transcription factor CREB (cyclic AMP responsive element
binding protein) is, as the name implies, responsive to levels of
intracellular cAMP. Therefore, the activation of a receptor that
signals via modulation of cAMP levels can be monitored by detecting
either the binding of the transcription factor, or the expression
of a reporter gene linked to a CREB-binding element (termed the
CRE, or cAMP response element). The DNA sequence of the CRE is
TGACGTCA. Reporter constructs responsive to CREB binding activity
are described in U.S. Pat. No. 5,919,649.
[0235] Other promoters and transcriptional control elements, in
addition to the c-fos elements and CREB-responsive constructs,
include the vasoactive intestinal peptide (VIP) gene promoter (cAMP
responsive; Fink et al., 1988, Proc. Natl. Acad. Sci.
85:6662-6666); the somatostatin gene promoter (cAMP responsive;
Montminy et al., 1986, Proc. Natl. Acad. Sci. 8.3:6682-6686); the
proenkephalin promoter (responsive to cAMP, nicotinic agonists, and
phorbol esters; Comb et al., 1986, Nature 323:353-356); the
phosphoenolpyruvate carboxy-kinase (PEPCK) gene promoter (cAMP
responsive; Short et al., 1986, J. Biol. Chem. 261:9721-9726).
[0236] Additional examples of transcriptional control elements that
are responsive to changes in GPCR activity include, but are not
limited to those responsive to the AP-1 transcription factor and
those responsive to NF-.kappa.B activity. The consensus AP-1
binding site is the palindrome TGA(C/G)TCA (Lee et al., 1987,
Nature 325: 368-372; Lee et al., 1987, Cell 49: 741-752). The AP-1
site is also responsible for mediating induction by tumor promoters
such as the phorbol ester 12-O-tetradecanoylphorbol-O-acetate
(TPA), and are therefore sometimes also referred to as a TRE, for
TPA-response element. AP-1 activates numerous genes that are
involved in the early response of cells to growth stimuli. Examples
of AP-1-responsive genes include, but are not limited to the genes
for Fos and Jun (which proteins themselves make up AP-1 activity),
Fos-related antigens (Fra) 1 and 2, I.kappa.B.alpha., ornithine
decarboxylase, and annexins I and II.
[0237] The NF-.kappa.B binding element has the consensus sequence
GGGGACTTTCC (SEQ ID NO: 5). A large number of genes have been
identified as NF-.kappa.B responsive, and their control elements
can be linked to a reporter gene to monitor GPCR activity. A small
sample of the genes responsive to NF-.kappa.B includes those
encoding IL-1.beta. (Hiscott et al., 1993, Mol. Cell. Biol. 13:
6231-6240), TNF-.alpha. (Shakhov et al., 1990, J. Exp. Med. 171:
35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14:
1509-1519), P-selection (Pan & McEver, 1995, J. Biol. Chem.
270: 23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol.
161: 3469-3473), GM-CSF (Schreck & Baeuerle, 1990, Mol. Cell.
Biol. 10: 1281-1286) and I.kappa.B.alpha. (Haskill et al., 1991,
Cell 65: 1281-1289). Each of these references is incorporated
herein by reference. Vectors encoding NF-.kappa.B-responsive
reporters are also known in the art or can be readily made by one
of skill in the art using, for example, synthetic NF-.kappa.B
elements and a minimal promoter, or using the
NF-.kappa.B-responsive sequences of a gene known to be subject to
NF-.kappa.B regulation. Further, NF-.kappa.B responsive reporter
constructs are commercially available from, for example,
CLONTECH.
[0238] A given promoter construct should be tested by exposing
GPR43-expressing cells, transfected with the construct, to
propionate. An increase of at least two-fold in the expression of
reporter in response to propionate indicates that the reporter is
an indicator of GPR43 activity.
[0239] In order to assay GPR43 activity with a transcriptional
reporter construct, cells that stably express a GPR43 polypeptide
are stably transfected with the reporter construct. To screen for
agonists, the cells are left untreated, exposed to candidate
modulators, or exposed to propionate, and expression of the
reporter is measured. The propionate-treated cultures serve as a
standard for the level of transcription induced by a known agonist.
An increase of at least 50% in reporter expression in the presence
of a candidate modulator indicates that the candidate is a
modulator of GPR43 activity. An agonist will induce at least as
much, and preferably the same amount or greater reporter expression
than propionate alone. This approach can also be used to screen for
inverse agonists where cells express a GPR43 polypeptide at levels
such that there is an elevated basal activity of the reporter in
the absence of propionate or another agonist. A decrease in
reporter activity of 10% or more in the presence of a candidate
modulator, relative to its absence, indicates that the compound is
an inverse agonist.
[0240] To screen for antagonists, the cells expressing GPR43 and
carrying the reporter construct are exposed to propionate (or
another agonist) in the presence and absence of candidate
modulator. A decrease of 10% or more in reporter expression in the
presence of candidate modulator, relative to the absence of the
candidate modulator, indicates that the candidate is a modulator of
GPR43 activity.
[0241] Controls for transcription assays include cells not
expressing GPR43 but carrying the reporter construct, as well as
cells with a promoterless reporter construct. Compounds that are
identified as modulators of GPR43-regulated transcription should
also be analyzed to determine whether they affect transcription
driven by other regulatory sequences and by other receptors, in
order to determine the specificity and spectrum of their
activity.
[0242] The transcriptional reporter assay, and most cell-based
assays, are well suited for screening expression libraries for
proteins for those that modulate GPR43 activity. The libraries can
be, for example, cDNA libraries from natural sources, e.g., plants,
animals, bacteria, etc., or they can be libraries expressing
randomly or systematically mutated variants of one or more
polypeptides. Genomic libraries in viral vectors can also be used
to express the mRNA content of one cell or tissue in the different
libraries used for screening of GPR43.
[0243] h) Inositol Phosphates (IP) Measurement
[0244] Cells of the invention, for example, CHO-K1 cells, are
labelled for 24 hours with 10 .mu.Ci/ml [.sup.3H] inositol in
inositol free DMEM containing 5% FCS, antibiotics, amphotericin,
sodium pyruvate and 400 .mu.g/ml G418. Cells are incubated for 2 h
in Krebs-Ringer Hepes (KRH) buffer of the following composition
(124 mM NaCl, 5 mM KCl, 1.25 mM MgSO.sub.4, 1.45 mM CaCl.sub.2,
1.25 mM KH.sub.2PO.sub.4, 25 mM Hepes (pH:7.4) and 8 mM glucose).
The cells are then challenged with various SCFA for 30 min. The
incubation is stopped by the addition of an ice cold 3% perchloric
acid solution. IP are extracted and separated on Dowex columns as
previously described (25).
[0245] GPR43 Assay
[0246] The invention provides for an assay for detecting the
activity of a receptor of the invention in a sample. For example,
GPR43 activity can be measured in a sample comprising a cell or a
cell membrane that expresses GPR43. As above, propionate is used as
an example in this section. It should be understood that any SCFA
as defined herein can be used in these assays. The assay is
performed by incubating the sample in the presence or absence of
SCFA and carrying out a second messenger assay, as described above.
The results of the second messenger assay performed in the presence
or absence of SCFA are compared to determine if the GPR43 receptor
is active. An increase of 10% or more in the detected level of a
given second messenger, as defined herein, in the presence of SCFA
relative to the amount detected in an assay performed in the
absence of SCFA is indicative of GPR43 activity.
[0247] Any of the assays of receptor activity, including but not
limited to the GTP-binding, GTPase, adenylate cyclase, cAMP,
phospholipid-breakdown, diacylglycerol, inositol triphosphate,
arachidonic acid release (see below), PKC, kinase and
transcriptional reporter assays, can be used to determine the
presence of an agent in a sample, e.g., a tissue sample, that
affects the activity of the GPR43 receptor molecule. To do so,
GPR43 polypeptide is assayed for activity in the presence and
absence of the sample or an extract of the sample. An increase in
GPR43 activity in the presence of the sample or extract relative to
the absence of the sample indicates that the sample contains an
agonist of the receptor activity. A decrease in receptor activity
in the presence of propionate or another agonist and the sample,
relative to receptor activity in the presence of propionate alone,
indicates that the sample contains an antagonist of GPR43 activity.
If desired, samples can then be fractionated and further tested to
isolate or purify the agonist or antagonist. The amount of increase
or decrease in measured activity necessary for a sample to be said
to contain a modulator depends upon the type of assay used.
Generally, a 10% or greater change (increase or decrease) relative
to an assay performed in the absence of a sample indicates the
presence of a modulator in the sample. One exception is the
transcriptional reporter assay, in which at least a two-fold
increase or 10% decrease in signal is necessary for a sample to be
said to contain a modulator. It is preferred that an agonist
stimulates at least 50%, and preferably 75% or 100% or more, e.g.,
2-fold, 5-fold, 10-fold or greater receptor activation than with
propionate alone.
[0248] Other functional assays include, for example,
microphysiometer or biosensor assays (see Hafner, 2000, Biosens.
Bioelectron. 15: 149-158, incorporated herein by reference). The
intracellular level of arachidonic acid can also be determined as
described in Gijon et al., 2000, J. Biol. Chem.,
275:20146-20156.
[0249] II. Diagnostic Assays Based upon the Interaction of GPR43
and Propionate:
[0250] Signalling through GPCRs is instrumental in the pathology of
a large number of diseases and disorders. GPR43, which is expressed
in cells of the lymphocyte lineages, platelets, spleen, stomach,
lung as well as leukemic cells, can have a role in immune
processes, cancer, thrombosis and associated disorders or
diseases.
[0251] The expression pattern of GPR43 and the knowledge with
respect to disorders generally mediated by GPCRs suggests that
GPR43 can be involved in disturbances of cell migration, cancer,
development of tumours and tumour metastasis, inflammatory and
neoplastic processes, wound and bone healing and dysfunction of
regulatory growth functions, diabetes, obesity, anorexia, bulimia,
acute heart failure, hypotension, hypertension, urinary retention,
osteoporosis, angina pectoris, myocardial infarction, restenosis,
atherosclerosis, thrombosis and other cardiovascular diseases,
autoimmune and inflammatory diseases, diseases characterized by
excessive smooth muscle cell proliferation, aneurysms, diseases
characterized by loss of smooth muscle cells or reduced smooth
muscle cell proliferation, stroke, ischemia, ulcers, allergies,
benign prostatic hypertrophy, migraine, vomiting, psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, depression, delirium, dementia and severe mental
retardation, degenerative diseases, neurodegenerative diseases such
as Alzheimer's disease or Parkinson's disease, and dyskinasias,
such as Huntington's disease or Gilles de la Tourett's syndrome and
other related diseases including thrombosis and other
cardiovascular diseases, autoimmune and inflammatory diseases.
[0252] The interaction of GPR43 with propionate can be used as the
basis of assays for the diagnosis or monitoring of diseases,
disorders or processes involving GPR43 signalling. Diagnostic
assays for GPR43-related diseases or disorders can have several
different forms. First, diagnostic assays can measure the amount of
GPR43 polypeptides, mRNA or ligand in a sample of tissue. Assays
that measure the amount of mRNA encoding GPR43 polypeptide also fit
into this category. Second, assays can evaluate the qualities of
the receptor or the ligand. For example, assays that determine
whether an individual expresses a mutant or variant form of GPR43
can be used diagnostically. Third, assays that measure one or more
activities of GPR43 polypeptide can be used diagnostically.
[0253] A. Assays that Measure the Amount of GPR43 Polypeptide
[0254] GPR43 levels can be measured and compared to standards in
order to determine whether an abnormal level of the receptor or its
ligand is present in a sample, either of which indicate probable
dysregulation of GPR43 signalling. Polypeptide levels are measured,
for example, by immunohistochemistry using antibodies specific for
the polypeptide. A sample isolated from an individual suspected of
suffering from a disease or disorder characterized by GPR43
activity is contacted with an antibody for a GPR43 polypeptide, and
binding of the antibody is measured as known in the art (e.g., by
measurement of the activity of an enzyme conjugated to a secondary
antibody).
[0255] Another approach to the measurement of GPR43 levels uses
flow cytometry analysis of cells from an affected tissue. Methods
of flow cytometry, including the fluorescent labeling of antibodies
specific for GPR43, are well known in the art. Other approaches
include radioimmunoassay or ELISA. Methods for each of these are
also well known in the art.
[0256] The amount of binding detected is compared to the binding in
a sample of similar tissue from a healthy individual, or from a
site on the affected individual that is not so affected. An
increase of 10% or more relative to the standard is diagnostic for
a disease or disorder characterized by GPR43 dysregulation.
[0257] GPR43 expression can also be measured by determining the
amount of mRNA encoding the polypeptides in a sample of tissue.
Levels of mRNA can be measured by quantitative or semi-quantitative
PCR. Methods of "quantitative" amplification are well known to
those of skill in the art, and primer sequences for the
amplification of GPR43 nucleic acid are disclosed herein. A common
method of quantitative PCR involves simultaneously co-amplifying a
known quantity of a control sequence using the same primers. This
provides an internal standard that can be used to calibrate the PCR
reaction. Detailed protocols for quantitative PCR are provided in
PCR Protocols, A Guide to Methods and Applications, Innis et al.,
Academic Press, Inc. N.Y., (1990), which is incorporated herein by
reference. An increase of 10% or more in the amount of mRNA
encoding GPR43 in a sample, relative to the amount expressed in a
sample of like tissue from a healthy individual or in a sample of
tissue from an unaffected location in an affected individual is
diagnostic for a disease or disorder characterized by dysregulation
of GPR43 signalling.
[0258] B. Qualitative Assays
[0259] Assays that evaluate whether or not a GPR43 polypeptide or
the mRNA encoding it are wild-type or not can be used
diagnostically. In order to diagnose a disease or disorder
characterized by GPR43 dysregulation in this manner, RNA isolated
from a sample is used as a template for PCR amplification of GPR43.
The amplified sequences are then either directly sequenced using
standard methods, or are first cloned into a vector, followed by
sequencing. A, difference in the sequence that changes one or more
encoded amino acids relative to the sequence of wild-type GPR43 can
be diagnostic of a disease or disorder characterized by
dysregulation of GPR43 signalling. It can be useful, when a change
in coding sequence is identified in a sample, to express the
variant receptor or ligand and compare its activity to that of wild
type GPR43. Among other benefits, this approach can provide novel
mutants, including constitutively active and null mutants.
[0260] In addition to standard sequencing methods, amplified
sequences can be assayed for the presence of specific mutations
using, for example, hybridization of molecular beacons that
discriminate between wild type and variant sequences. Hybridization
assays that discriminate on the basis of changes as small as one
nucleotide are well known in the art. Alternatively, any of a
number of "minisequencing" assays can be performed, including,
those described, for example, in U.S. Pat. Nos. 5,888,819,
6,004,744 and 6,013,431 (incorporated herein by reference). These
assays and others known in the art can determine the presence, in a
given sample, of a nucleic acid with a known polymorphism.
[0261] If desired, array or microarray-based methods can be used to
analyze the expression or the presence of mutation, in GPR43
sequences. Array-based methods for minisequencing and for
quantitation of nucleic acid expression are well known in the
art.
[0262] C. Functional Assays.
[0263] Diagnosis of a disease or disorder characterized by the
dysregulation of GPR43 signalling can also be performed using
functional assays. To do so, cell membranes or cell extracts
prepared from a tissue sample are used in an assay of GPR43
activity as described herein (e.g., ligand binding assays, the
GTP-binding assay, GTPase assay, adenylate cyclase assay, cAMP
assay, arachidonic acid level, phospholipid breakdown, diacyl
glycerol or inositol triphosphate assays, PKC activation assay, or
kinase assay). The activity detected is compared to that in a
standard sample taken from a healthy individual or from an
unaffected site on the affected individual. As an alternative, a
sample or extract of a sample can be applied to cells expressing
GPR43, followed by measurement of GPR43 signalling activity
relative to a standard sample. A difference of 10% or more in the
activity measured in any of these assays, relative to the activity
of the standard, is diagnostic for a disease or disorder
characterized by dysregulation of GPR43 signalling.
Modulation of GPR43 Activity in a Cell According to the
Invention
[0264] The discovery of propionate as a ligand of GPR43 provides
methods of modulating the activity of a GPR43 polypeptide in a
cell. GPR43 activity is modulated in a cell by delivering to that
cell an agent that modulates the function of a GPR43 polypeptide.
This modulation can be performed in cultured cells as part of an
assay for the identification of additional modulating agents, or,
for example, in an animal, including a human. Agents include
propionate and other SCFAs as defined herein, as well as additional
modulators identified using the screening methods described herein
including but not limited to any of the propionate analogues.
[0265] An agent can be delivered to a cell by adding it to culture
medium. The amount to deliver will vary with the identity of the
agent and with the purpose for which it is delivered. For example,
in a culture assay to identify antagonists of GPR43 activity, one
will preferably add an amount of agent, e.g., propionate that
half-maximally activates the receptors (e.g., approximately
EC.sub.50), preferably without exceeding the dose required for
receptor saturation. This dose can be determined by titrating the
amount of propionate to determine the point at which further
addition of propionate has no additional effect on GPR43
activity.
[0266] When a modulator of GPR43 activity is administered to an
animal for the treatment of a disease or disorder, the amount
administered can be adjusted by one of skill in the art on the
basis of the desired outcome. Successful treatment is achieved when
one or more measurable aspects of the pathology (e.g., tumor cell
growth, accumulation of inflammatory cells) is changed by at least
10% relative to the value for that aspect prior to treatment.
Candidate Modulators Useful According to the Invention
[0267] The invention provides for a compound that is a modulator of
a receptor of the invention.
[0268] Preferably a candidate modulator is a Short chain fatty acid
or a carboxylic acid.
[0269] The candidate compound can be a synthetic compound, or a
mixture of compounds, or may be a natural product (e.g. a plant
extract or culture supernatant). A candidate compound according to
the invention includes but is not limited to a small molecule that
can be synthesized, a natural extract, peptides, polypeptides,
carbohydrates, lipids, an antibody or antigen-binding fragment
thereof, nucleic acids, and a small organic molecules.
[0270] Candidate modulator compounds from large libraries of
synthetic or natural compounds can be screened. Numerous means are
currently used for random and directed synthesis of saccharide,
peptide, and nucleic acid based compounds. Synthetic compound
libraries are commercially available from a number of companies
including Maybridge Chemical Co. (Trevillet, Cornwall, UK),
Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.),
and Microsource (New Milford, Conn.). A rare chemical library is
available from Aldrich (Milwaukee, Wis.). Combinatorial libraries
are available and can be prepared. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available from e.g., Pan Laboratories (Bothell,
Wash.) or MycoSearch (NC), or are readily producible by methods
well known in the art. Additionally, natural and synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical, and biochemical means.
[0271] Useful compounds may be found within numerous chemical
classes. Useful compounds may be organic compounds, or small
organic compounds. Small organic compounds have a molecular weight
of more than 50 yet less than about 2,500 daltons, preferably less
than about 750, more preferably less than about 350 daltons.
Exemplary classes include heterocycles, peptides, saccharides,
steroids, and the like. The compounds may be modified to enhance
efficacy, stability, pharmaceutical compatibility, and the like.
Structural identification of an agent may be used to identify,
generate, or screen additional agents. For example, where peptide
agents are identified, they may be modified in a variety of ways to
enhance their stability, such as using an unnatural amino acid,
such as a D-amino acid, particularly D-alanine, by functionalizing
the amino or carboxylic terminus, e.g. for the amino group,
acylation or alkylation, and for the carboxyl group, esterification
or amidification, or the like.
[0272] For primary screening, a useful concentration of a candidate
compound according to the invention is from about 10 .mu.M to about
100 .mu.M or more (i.e. 1 mM, 10 mM, 100 mM, or even 1M), but can
also be 1 nM and higher, 1 .mu.M and higher, or 1 fM and higher.
The primary screening concentration will be used as an upper limit,
along with nine additional concentrations, wherein the additional
concentrations are determined by reducing the primary screening
concentration at half-log intervals (e.g. for 9 more
concentrations) for secondary screens or for generating
concentration curves.
Antibodies Useful According to the Invention
[0273] The invention provides for antibodies to GPR43. Antibodies
can be made using standard protocols known in the art (See, for
example, Antibodies: A Laboratory Manual ed. by Harlow and Lane
(Cold Spring Harbor Press: 1988)). A mammal, such as a mouse,
hamster, or rabbit can be immunized with an immunogenic form of the
peptide (e.g., GPR43 polypeptide or an antigenic fragment which is
capable of eliciting an antibody response, or a fusion protein as
described herein above). Immunogens for raising antibodies are
prepared by mixing the polypeptides (e.g., isolated recombinant
polypeptides or synthetic peptides) with adjuvants. Alternatively,
GPR43 polypeptides or peptides are made as fusion proteins to
larger immunogenic proteins. Polypeptides can also be covalently
linked to other larger immunogenic proteins, such as keyhole limpet
hemocyanin. Alternatively, plasmid or viral vectors encoding GPR43
polypeptide, or a fragment of these proteins, can be used to
express the polypeptides and generate an immune response in an
animal as described in Costagliola et al., 2000, J. Clin. Invest.
105:803-811, which is incorporated herein by reference. In order to
raise antibodies, immunogens are typically administered
intradermally, subcutaneously, or intramuscularly to experimental
animals such as rabbits, sheep, and mice. In addition to the
antibodies discussed above, genetically engineered antibody
derivatives can be made, such as single chain antibodies.
[0274] The progress of immunization can be monitored by detection
of antibody titers in plasma or serum. Standard ELISA, flow
cytometry or other immunoassays can also be used with the immunogen
as antigen to assess the levels of antibodies. Antibody
preparations can be simply serum from an immunized animal, or if
desired, polyclonal antibodies can be isolated from the serum by,
for example, affinity chromatography using immobilized
immunogen.
[0275] To produce monoclonal antibodies, antibody-producing
splenocytes can be harvested from an immunized animal and fused by
standard somatic cell fusion procedures with immortalizing cells
such as myeloma cells to yield hybridoma cells. Such techniques are
well known in the art, and include, for example, the hybridoma
technique (originally developed by Kohler and Milstein, (1975)
Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar
et al., (1983)
[0276] Immunology Today, 4: 72), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., (1985) Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96).
Hybridoma cells can be screened immunochemically for production of
antibodies specifically reactive with GPR43 polypeptide, and
monoclonal antibodies isolated from the media of a culture
comprising such hybridoma cells.
High Throughput Screening Kit
[0277] A high throughput screening kit according to the invention
comprises all the necessary means and media for performing the
detection of a modulator compound including an agonist, antagonist,
inverse agonist or inhibitor to the receptor of the invention in
the presence of propionate, preferably at a concentration in the
range of 1 .mu.M to 1 mM. The kit comprises materials to perform
the following successive steps. Recombinant cells of the invention,
comprising and expressing the nucleotide sequence encoding the
GPR43 receptor, are grown on a solid support, such as a microtiter
plate, more preferably a 96 well microtiter plate, according to
methods well known to the person skilled in the art, especially as
described in WO 00/02045. Modulator compounds according to the
invention, at concentrations from about 1 .mu.M to 1 mM or more,
are added to the culture media of defined wells in the presence of
an appropriate concentration of propionate (preferably in the range
of 1 .mu.M to 1 .mu.M).
[0278] Kits according to the invention can also comprise materials
necessary for second messenger assays amenable to high throughput
screening analysis, including but not limited to the measurement of
intracellular levels of cAMP, intracellular inositol phosphate,
intracellular diacylglycerol concentrations, arachinoid acid
concentration or MAP kinase or tyrosine kinase activity (as
described above). For example, the GPR43 activity, as measured in a
cyclic AMP assay, is quantified by a radioimmunoassay as previously
described (26). Results are compared to the baseline level of GPR43
activity obtained from recombinant cells according to the invention
in the presence of propionate but in the absence of added modulator
compound. Wells showing at least 2 fold, preferably 5 fold, more
preferably 10 fold and most preferably a 100 fold or more increase
or decrease in GPR43 activity as compared to the level of activity
in the absence of modulator, are selected for further analysis.
Other Kits Useful According to the Invention
[0279] The invention provides for kits useful for screening for
modulators of GPR43 activity, as well as kits useful for diagnosis
of diseases or disorders characterized by dysregulation of GPR43
signalling. Kits useful according to the invention can include an
isolated GPR43 polypeptide (including a membrane- or
cell-associated GPR43 polypeptide, e.g., on isolated membranes,
cells expressing GPR43, or on an SPR chip). A kit can also comprise
an antibody specific for GPR43. Alternatively, or in addition, a
kit can contain cells transformed to express GPR43 polypeptide. In
a further embodiment, a kit according to the invention can contain
a polynucleotide encoding a GPR43 polypeptide. In a still further
embodiment, a kit according to the invention may comprise the
specific primers useful for amplification of GPR43 as described
below. All kits according to the invention will comprise the stated
items or combinations of items and packaging materials therefor.
Kits will also include instructions for use.
Transgenic Animals
[0280] Transgenic mice provide a useful tool for genetic and
developmental biology studies and for the determination of the
function of a novel sequence. According to the method of
conventional transgenesis, additional copies of normal or modified
genes are injected into the male pronucleus of the zygote and
become integrated into the genomic DNA of the recipient mouse. The
transgene is transmitted in a Mendelian manner in established
transgenic strains. Constructs useful for creating transgenic
animals comprise genes under the control of either their normal
promoters or an inducible promoter, reporter genes under the
control of promoters to be analyzed with respect to their patterns
of tissue expression and regulation, and constructs containing
dominant mutations, mutant promoters, and artificial fusion genes
to be studied with regard to their specific developmental outcome.
Typically, DNA fragments on the order of 10 kilobases or less are
used to construct a transgenic animal (Reeves, 1998, New. Anat.,
253:19). Transgenic animals can be created with a construct
comprising a candidate gene containing one or more polymorphisms
according to the invention. Alternatively, a transgenic animal
expressing a candidate gene containing a single polymorphism can be
crossed to a second transgenic animal expressing a candidate gene
containing a different polymorphism and the combined effects of the
two polymorphisms can be studied in the offspring animals.
Other Transgenic Animals
[0281] The invention provides for transgenic animals that include
but are not limited to transgenic mice, rabbits, rats, pigs, sheep,
horses, cows, goats, etc. A protocol for the production of a
transgenic pig can be found in White and Yannoutsos, Current Topics
in Complement Research: 64.sup.th Forum in Immunology, pp. 88-94;
U.S. Pat. No. 5,523,226; U.S. Pat. No. 5,573,933: PCT Application
WO93/25071; and PCT Application WO95/04744. A protocol for the
production of a transgenic mouse can be found in U.S. Pat. No.
5,530,177. A protocol for the production of a transgenic rat can be
found in Bader and Ganten, Clinical and Experimental Pharmacology
and Physiology, Supp. 3:S81-S87, 1996. A protocol for the
production of a transgenic cow can be found in Transgenic Animal
Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press,
Inc. A protocol for the production of a transgenic rabbit can be
found in Hammer et al., Nature 315:680-683, 1985 and Taylor and
Fan, Frontiers in Bioscience 2:d298-308, 1997.
Knock Out Animals
[0282] i. Standard
[0283] Knock out animals are produced by the method of creating
gene deletions with homologous recombination. This technique is
based on the development of embryonic stem (ES) cells that are
derived from embryos, are maintained in culture and have the
capacity to participate in the development of every tissue in the
mouse when introduced into a host blastocyst. A knock out animal is
produced by directing homologous recombination to a specific target
gene in the ES cells, thereby producing a null allele of the gene.
The potential phenotypic consequences of this null allele (either
in heterozygous or homozygous offspring) can be analyzed (Reeves,
supra).
[0284] ii. In vivo Tissue Specific Knock Out in Mice Using
Cre-lox.
[0285] The method of targeted homologous recombination has been
improved by the development of a system for site-specific
recombination based on the bacteriophage PI site specific
recombinase Cre. The Cre-loxP site-specific DNA recombinase from
bacteriophage PI is used in transgenic mouse assays in order to
create gene knockouts restricted to defined tissues or
developmental stages. Regionally restricted genetic deletion, as
opposed to global gene knockout, has the advantage that a phenotype
can be attributed to a particular cell/tissue (Marth, 1996, Clin.
Invest. 97: 1999). In the Cre-loxP system one transgenic mouse
strain is engineered such that loxP sites flank one or more exons
of the gene of interest. Homozygotes for this so called `floxed
gene` are crossed with a second transgenic mouse that expresses the
Cre gene under control of a cell/tissue type transcriptional
promoter. Cre protein then excises DNA between loxP recognition
sequences and effectively removes target gene function (Sauer,
1998, Methods, 14:381). There are now many in vivo examples of this
method, including the inducible inactivation of mammary tissue
specific genes (Wagner et al., 1997, Nucleic Acids Res.,
25:4323).
[0286] iii. Bac Rescue of Knock Out Phenotype
[0287] In order to verify that a particular genetic
polymorphism/mutation is responsible for altered protein function
in vivo one can "rescue" the altered protein function by
introducing a wild-type copy of the gene in question. In vivo
complementation with bacterial artificial chromosome (BAC) clones
expressed in transgenic mice can be used for these purposes. This
method has been used for the identification of the mouse circadian
Clock gene (Antoch et al., 1997, Cell 89: 655).
Materials
[0288] Trypsin was from Flow Laboratories (Bioggio, Switzerland).
Culture media, G418, fetal bovine serum (FBS), restriction enzymes,
Platinum Pfx and Taq DNA polymerases were purchased from Life
Technologies, Inc. (Merelbeke, Belgium). The radioactive product
myo-D-[2-.sup.3H]inositol (17.7 Cl/mmol) was from Amersham (Ghent,
Belgium). Dowex AG1X8 (formate form) was from Bio-Rad Laboratories
(Richmond, Calif.). ATP, propionate, acetate, formate, butyrate,
valerate, beta-hydroxybutyrate, gamma-hydroxybutyrate and other
carboxylic acids were obtained from Sigma Chemical Co. (St. Louis,
Mo.). Forskolin was purchased from Calbiochem (Bierges, Belgium).
Rolipram was a gift from the Laboratories Jacques Logeais (Trappes,
France). pEFIN5 is an expression vector developed by Euroscreen
(Brussels, Belgium). Monoclonal antibody specific for the dually
phosphorylated forms of Erk1 and Erk2 (at Thr.sup.202 and
Tyr.sup.204) was obtained from New England Biolabs (Beverly,
Mass.).
Dosage and Mode of Administration
[0289] By way of example, a patient can be treated as follows by
the administration of a modulator of GPR43 (for example, an
agonist, antagonist or inhibitor of GPR43, of the invention). A
modulator of GPR43 the invention can be administered to the
patient, preferably in a biologically compatible solution or a
pharmaceutically acceptable delivery vehicle, by ingestion,
injection, inhalation or any number of other methods. The dosages
administered will vary from patient to patient; a "therapeutically
effective dose" can be determined, for example, by the level of
enhancement of function (e.g., as determined in a second messenger
assay described herein). Monitoring propionate binding will also
enable one skilled in the art to select and adjust the dosages
administered. The dosage of a modulator of GPR43 of the invention
may be repeated daily, weekly, monthly, yearly, or as considered
appropriate by the treating physician.
[0290] In one embodiment, a patient can be treated to modulate the
signalling activity of a GPR43 receptor by administering to a
patient a sublethal dose of an agent which inhibits or promotes the
signalling activity of GPR43. A sublethal dose according to the
invention, refers to a dose of an agent for inhibiting or
stimulating a GPR43 signalling activity which is at or below the
LD50 for the particular agent. In one embodiment, the dose of an
agent which inhibits the signalling activity of GPR43 is between 1
aM and 1 M, preferably between 1 fM and 1 mM, and more preferably
between 1 nM and 1 .mu.M. In one embodiment, an agent useful for
the modulation of GPR43 signalling may be an antibody which
specifically binds to the ligand binding site of GPR43. An amount
of anti-GPR43 antibody needed to achieve a dosage useful for the
modulation of GPR43 signalling will depend upon the level of
expression of GPR43, localization of receptor expression, and
general state of the patient's own immune system, but generally
range from 0.0005 to 5.0 mg of anti-GPR43 antibody or binding
protein thereof per kilogram of body weight, with doses of 0.05 to
2.0 mg/kg/dose being more commonly used.
Pharmaceutical Compositions
[0291] The invention provides for compositions comprising a GPR43
modulator according to the invention admixed with a physiologically
compatible carrier. As used herein, "physiologically compatible
carrier" refers to a physiologically acceptable diluent such as
water, phosphate buffered saline, or saline, and further may
include an adjuvant. Adjuvants such as incomplete Freund's
adjuvant, aluminium phosphate, aluminium hydroxide, or alum are
materials well known in the art.
[0292] The invention also provides for pharmaceutical compositions.
In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carrier preparations which can be used pharmaceutically.
[0293] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for ingestion by the patient.
[0294] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores.
[0295] Suitable excipients are carbohydrate or protein fillers such
as sugars, including lactose, sucrose, mannitol, or sorbitol;
starch from corn, wheat, rice, potato, or other plants; cellulose
such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethyl cellulose; and gums including arabic and tragacanth;
and proteins such as gelatin and collagen. If desired,
disintegrating or solubilizing agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt
thereof, such as sodium alginate.
[0296] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0297] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders such as lactose or starches, 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 glycol with or without stabilizers.
[0298] Pharmaceutical formulations for parenteral administration
include aqueous solutions of active compounds. For injection, the
pharmaceutical compositions of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hank's solution, Ringer' solution, or physiologically
buffered saline. Aqueous injection suspensions may contain
substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,
suspensions of the active solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Optionally, the suspension
may also contain suitable stabilizers or agents which increase the
solubility of the compounds to allow for the preparation of highly
concentrated solutions.
[0299] For nasal administration, penetrants appropriate to the
particular barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art.
[0300] The pharmaceutical compositions of the present invention may
be manufactured in a manner known in the art, e.g. by means of
conventional mixing, dissolving, granulating, dragee-making,
levitating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0301] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. . . . Salts tend to be more soluble in aqueous or other
protonic solvents that are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH
range of 4.5 to 5.5 that is combined with buffer prior to use.
[0302] After pharmaceutical compositions comprising a compound of
the invention formulated in a acceptable carrier have been
prepared, they can be placed in an appropriate container and
labeled for treatment of an indicated condition with information
including amount, frequency and method of administration.
Modulation of Chemotaxis
[0303] The present invention provides a method for the modulation
of the chemotaxis of PMN, and related cells in vitro or in vivo by
contacting the cells with short chain fatty acid molecules of the
invention. Migration of immune cells to sites of infection (or the
site of antigen presence) is a common process which occurs in
myriad disease states. The present invention is based, in part, on
the discovery that GPR43 functions as a receptor for short chain
fatty acids, such as acetate and propionate, and is responsible for
mediating PMN chemotaxis in response to such SCFAs. Accordingly,
the invention provides a mechanism for the modulation and/or
treatment of disease states which share, as a common mechanism of
action, the phenomenon of PMN cell migration. In one embodiment,
the invention provides that disease states which are characterized
by unwanted migration of immune cells, such as autoimmune diseases,
may be modulated and/or treated by administering to a patient with
such a disease an agent which inhibits a signalling activity of
GPR43, or which blocks the activation of the receptor (e.g., an
antibody which specifically binds to the GPR43 receptor).
Alternatively, the invention provides that disease states which are
characterized by insufficient immune cell migration, or diseases
caused by pathogens which must be eliminated through the
stimulation of an immune response may be modulated or treated by
administering to a patient in need thereof, an agonist of the GPR43
receptor, including, but not limited to acetate and/or propionate.
Particular diseases which may be modulated and/or treated by the
methods of the invention are indicated below. The present invention
is not limited, however, to these specific diseases, and may be
useful in the treatment of other disease states characterized by
abnormal, or insufficient immune cell migration. Accordingly, a
"PMN chemotaxis-related disease" as used herein refers to a disease
which includes as a component, migration of PMN cells toward or
away from a soluble chemotactic factor. A "PMN chemotaxis-related
disease" can be, for example, an inflammatory disease, autoimmune
diseases, IBD (Inflammatory Bowel Diseases), liver cirrhosis,
periodontal disease, and other diseases which are known to those of
skill in the art to be mediated, at least in part, by the migration
of PMN cells towards or away from a soluble chemotactic factor. A
"PMN chemotaxis-related disease" can also refer to a pathologial
condition resulting from infection by a pathogen, or from abnormal
proliferation of an endogenous pathogen in an individual.
[0304] Intestine-Related Disorders:
[0305] It is possible that products of the commensal flora promote
inflammation in the presence of an impaired mucosal barrier or
injury to the mucosa (Chadwick & Anderson, 1990), leading to
activation of the mucosal immune system in inflammatory bowel
diseases (IBD) (Chadwick et al., 2002). IBD can involve either or
both of the small and large bowel. Crohn's disease and ulcerative
colitis are the best known forms of IBD. The predominant
histopathologic feature of IBD is infiltration of acute and chronic
inflammatory cells in the affected intestine. These immune cells
can recognize and destroy intestinal cells, implicating classical
immune mechanisms in IBD pathogenesis (Perlmann & Broberger,
1963). In addition, immune cells can infiltrate intestine diffusely
in the absence of obvious morphological, clinical and endoscopic
evidence of inflammation (Fiocchi, 1998). Monocytic cells appear
also to be involved in all stages of IBD., underscoring their
importance in IBD pathophysiology (Fiocchi, 1998). In addition,
activated T lymphocytes induce mucosal damage in organ culture
(MacDonald & Spencer, 1988) and PMN are playing a key role in
the amplification of inflammation and tissue damage (Fiocchi,
1998), with a prominent neutrophil infiltration in the inflamed
colonic mucosa of patients with IBD. After migration from the
systemic circulation into the mucosal interstitial space,
neutrophils may subsequently undergo activation to produce reactive
oxygen intermediates and additional chemokines, leading to
perpetuation of the inflammatory response as well as the ultimate
mucosal injury. Because neutrophil infiltration is an integral
component of the severely-inflamed intestine with IBD, the
development of therapeutic strategies to block neutrophil migration
and activation is a highly desirable target. Indeed, treatment with
cyclosporin A, an inhibitor of migratory response of neutrophils,
improved the inflammation of IBD patients by decreasing the
inflammation due to neutrophils and lymphocytes T (Ina et al.,
2002). The concept that the normal flora somehow functions as a
modulator of physiological inflammation has been strengthened
substantially by the observations of Duchmann et al. (Duchmann et
al., 1995 & 1996). They have shown that mucosal but not
peripheral blood, mononuclear cells from patients with IBD
proliferate when exposed to autologous intestinal bacteria.
Production of factors in the colonic milieu markedly increase
production of reactive oxygen species by PMNs. Amongst these
factors, SCFA are produced by anaerobic fermentation of complex
carbohydrates in intestine (Pouteau et al., 1996; Topping &
Clifton, 2001-Eftimiadi et al., 1987), mainly acetate, propionate
and butyrate with partition as follows: acetate (60%), propionate
(25%) and butyrate (15%). The colon luminal total concentration is
around 70-100 mM (Sellin, 1999). Propionate and acetate, but not
butyrate, are potent modulators of neutrophil function (Nakao et
al., 1992) and we showed that acetate and propionate are acting on
GPR43 as agonists to modulate neutrophil activities. Therefore an
antagonist of GPR43 might decrease neutrophil activation and
inflammation in IBD.
[0306] Because of a key role of neutrophils migration and
activation in the activation of the mucosal immune system in IBD,
compounds which are antagonists of GPR43 receptor signaling may be
useful, according to the invention, to decrease neutrophil
activation and inflammation in IIBD.
[0307] Host Defense, Inflammation, Modulation of Innate Immunity
and Hematopoietic Disorders
[0308] GPR43 is expressed on leukocytes. The ligands of GPR43,
propionate and acetate, are modulating polymorphonuclear cells as
well as T lymphocytes and monocytes (Eftimiadi et al., 1991; Nakao
et al., 1992; Curi et al., 1993). But none of these effects have
been associated with the simulation of a given G-protein coupled
receptor (GPCR), although experiments with pertussis toxin and
activator/inhibitor of protein kinase C may have suggested a
GPCR-mechanism. Brunkhorst et al (1992) has suggested a GPCR
mechanism of action, for at least propionate and acetate, on a
series of PMN-activation events such as cytoskeletal F-actin
alterations, PMN polarization, F-actin localization, cytoplasmic pH
oscillation, cell shape. The present invention provides that
ligands of GPR43 could be used to modulate leukocyte activity in
different pathologies, including, but not limited to inflammatory
diseases, pathogen infection, lymphomas and leukemias to modulate
leukocyte activity.
[0309] Periodontal Diseases
[0310] Periodontal disease is the consequence of a mixed
Gram-negative infection in the gingival sulcus and has been
associated with deficits in the neutrophil response. One potential
approach to therapy is the use of biological-response modulators
that enhance the neutrophil response. Various periodontal and root
canal pathogens, such as the Bacteroides species, can produce
significant amounts of short chain fatty acids (SCFA). Accordingly,
the a GPR43 ligand, as provided by the present invention, may be
useful to modulate the neutrophil response and decrease the
symptoms of periodontal diseases.
[0311] Alcoholism
[0312] Most ethanol elimination occurs by oxidation to acetaldehyde
and acetate catalysed principally by alcohol deshydrogenase (ADH)
and aldehyde deshydrogenase (ALDH). Alcohol is eliminated from the
body almost entirely by hepatic metabolism first to acetaldehyde
and then to acetate and finally to carbon dioxide and water
following a time-course of elimination best described by
Michaelis-Menten kinetics (Fujimiya et al. 2000 Alcohol Clin Exp
Res 24: 16S-20S; see Li and Bosron, 1986 Ann Emerg Med
15:997-1004). Approximately 60%-75% of ethanol dose is converted to
acetate (Siler S Q, Neese R A, Hellerstein M K 1999 .mu.m J Clin
Nutr 70(5):928-36). Acetate can be assessed in human blood and
urine by headspace gas chromatography (Tsukamoto et al. Nihon
Arukoru Yakubutsu Igakkai Zasshi 1998 3:200-9) and represents a
marker for alcohol intake, heavy drinking, metabolic tolerance,
abuse, chronic alcoholism and alcohol withdrawal severity (Pronko
et al. 1997 Alcohol 32:761-8; Korri et al. 1985 Alcohol Clin Exp
Res 9:468-71; Nuutinen et al. 1985 Alcohol 2:623-6). After ethanol,
it increases to 19-57 mg/ml (Lundquist 1962 Nature N.degree.4815,
p579).
[0313] Chronic and even acute moderate alcohol use can increase
host susceptibility to infections caused by bacterial and viral
pathogens (i.e. Klebsiella pneumoniae (Shellito et al. 2001
25:872-81); and lung clearance of Pseudomonas aeruginosa
(Greensberg et al. 1999 Alcohol Clin Exp Res 23:735-44);
phagocytosis of staphylococcus aureus and epidermidis (Jareo et
al., 1995 Alcohol 30:311-8; Corberand et al. 1989, Alcohol Clin Exp
Res 13:542-6). Impaired host defense after alcohol exposure appears
to be linked to a combination of decreased inflammatory response,
altered cytokine production and abnormal reactive oxygen
intermediate generation and Neutrophils functions (Szabo 1999). The
sensitivity of the signaling cascade inositol phosphate
(IP)/Ca.sup.2+ response in neutrophils from healthy volunteers
after ingestion of 1% ethanol for 2 h is altered (Gann et al.,
Psychiatry Res 1999 89:189-99). Damage of PMN function by ethanol
consists of ultrastructural changes of neutrophil granules, and
further includes a reduction, redistribution and atypical
accumulation of autophagic vacuoles (Todorovic 1999, Indian J Med
Res 109:105-14; Todorovic et al. 1994, J Stud Alcohol 55:239-48),
and changes in neutrophil elastase activity (Sachs et al. 1990, Am
Rev Respir Dis 141:1249-55). These phenomena may accordingly
promote a deficit in neutrophil bactericidal activity against
germs. In addition, chronic ethanol intake modulates f-met-leu-phe
(fMLP) induced chemotactic activity and superoxide production by
neutrophils (Bautista et al. 1992 16:788-94).
[0314] Leucocyte infiltration in the liver is also one of the most
important features of alcoholic liver disease. In alcoholic
hepatitis, PMN selectively migrate to the liver (Bautista, Alcohol
2002 27:17-21; Siratori et al. 1992 J Hepatol 15:266-8).
Up-regulation of chemokines in the circulation and tissue is
associated with enhanced neutrophilic infiltration in the liver
(Bautista, Alcohol 2002 27:17-21). In cirrhotic alcoholics
chemotaxis, phagocytosis and bactericidal activity were all
significantly reduced (Laharrague et al. 1985 Ann Med Interne
(Paris) 136:210-2)
[0315] Acetate is capable of producing a fall in free fatty acid
(FFA) after ethanol ingestion, since ethanol is able to lower
circulating FFA to healthy volunteers. Increase in blood acetate
after ethanol is sufficient to explain the FFA fall even without
acidosis, acetate being known as an alkalinizing agent (Crouse J R,
Gerson C D, DeCarli L M, Lieber C S. 1968 J Lipid Res
9(4):509-12).
[0316] Accordingly, the above suggests that neutrophil function may
be impaired in chronic alcohol abusers, and therefore a ligand of
GPR43, according to the invention, may be useful to restore
neutrophil function.
[0317] Measuring Chemotaxis
[0318] PMN chemotaxis may be measured in vitro, according to the
invention, by procedures originally developed by S. Boyden in 1962.
(See, S. Boyden, J. Exp. Med. 115: pp. 453-466, 1962). Briefly, the
procedure involves placing a suspension of PMN cells and a chemical
agent in two separate chambers, which chambers are separated by a
polycarbonate filter. The PMN may, for example, be prepared from
the peripheral blood of a mammal. After a predetermined period of
time, the filter is removed and cells on the filter surface closest
to the chamber containing the cell suspension are carefully
removed. The remaining cells on the filter are then fixed and
stained. Using a high power microscope, the filter is examined and
the number of cells appearing on the underside of the filter (i.e.,
the side of the filter closest to the chamber containing the
chemical agent) are counted manually. A positive chemotactic
response is indicated by the cells having migrated or "crawled"
through the filter to the side closest to the chamber containing
the chemical agent. Because of the time required to do so,
typically the entire filter is not examined. Rather, representative
sample areas are examined and counted. According to the invention,
"PMN chemotaxis" is said to have occurred where there are at least
10% more PMN cells on the filter surface aposed to the chamber
containing the chemotactic factor when the chemotactic factor is
present in the chamber, than when the chemotactic factor is not
present.
[0319] Alternatively, PMN chemotaxis may be assessed in vivo in a
mammal by comparing the number of PMN cells at a given site or in a
given sample at two different time points. Upon appropriate
stimulus, PMN cells migrate from the peripheral blood circulation
into the connective tissue, and surrounding structures. To
determine whether chemotaxis has been modulated, for example, in
response to a candidate agent such as a modulator of GPR43
signalling activity, a connective tissue sample may be obtained
from a mammal and examined, using histological techniques well
known to those of skill in the art, to determine the number of PMN
cells present in the peripheral tissues (such as connective tissue
or lymphoid organs). The number of PMN cells present may then be
compared with the number present at a later time point (e.g., 1-5
hours, 1-5 days, or 1-5 weeks later). In one embodiment, the number
of PMN cells present in the tissues of a mammal is compared with
the number present after the administration of a candidate agent,
wherein an increase or decrease in the number of PMN cells present
in peripheral tissues following administration of the candidate
agent identifies the agent as a modulator of PMN chemotaxis.
EXAMPLES
[0320] The invention is illustrated by the following non-limiting
examples wherein the following materials and methods are employed.
The entire disclosure of each of the literature references cited
hereinafter are incorporated by reference herein.
Example 1
Cloning, Sequencing and Alignment
[0321] Specific oligonucleotide primers were synthesized on the
basis of the sequence of the GPR43 human receptor: a sense primer
5'-GCGGAATTCACCATGCTGCCGG ACTGGAAGAG-3' (SEQ ID NO: 6) and an
antisense primer 5'-CTAGTCTAG ACTGCTACTCTGTAGTGAAGTC-3' (SEQ ID NO:
7). A polymerase chain reaction (PCR) was performed on three
different spleen cDNAs using the Platinum Pfx DNA Polymerase. The
amplification conditions were as follows: 94.degree. C., 15 s;
50.degree. C., 30 s; 68.degree. C., 2 min for 35 cycles.
Amplifications resulted in a fragment of 1 kilobase containing the
entire coding sequence of the GPR43 gene. The coding sequence was
then subcloned between the EcoRI and XbaI sites of the pcDNA3
(Invitrogen) expression vector and sequenced on both strands for
each of the three cDNAs using the BigDye Terminator cycle
sequencing kit (Applied Biosystems, Warrington, Great Britain).
[0322] This 990 base pair (bp)-open reading frame was also
identified recently by Sawzdargo et al. (GenBank accession
AF024690) and reported to encode an orphan G-protein-coupled
receptor that they called GPR43. Oligonucleotide primers were
synthesized on the basis of this coding sequence published in
Sawzdargo et al. They were used in PCR starting from spleen cDNA. A
PCR product with a size compatible with GPR43 coding sequence was
inserted into the pcDNA3 expression vector and sequenced on both
strands (FIG. 1). The putative membrane-spanning domains are
underlined and numbered I to VII. The putative sites of
phosphorylation by caseine kinase is indicated in bold.
[0323] Alignment of the amino acid sequence of GPR43 (FIG. 2) with
PAR1 and other PAR related sequences was performed using the
ClustalX algorithm. The dendrogram of FIG. 2 was then constructed
using the TreeView algorithm. The figure shows the relationship of
GPR43 with Proteinase Activated Receptor (PAR)-1, -2, -3, and -4,
platelet-activating factor receptor (PAF), and G-protein coupled
receptor 42 (GPR42). The latter is always an orphan receptor.
Example 2
Tissue Distribution of GPR43 Human Receptor
[0324] GPR43 mRNA was amplified by RT-PCR in several human tissues
(FIG. 3).
[0325] Reverse transcription-polymerase chain reaction (RT-PCR)
experiments were carried out using a panel of polyA+ RNA
(Clontech). The GPR43 primers were as follows: GPR43 sense primer
(5'-ACTGGAAGAGCTCCTTGATC-3'; SEQ ID NO: 8) and GPR43 antisense
primer (5'-CAAGTATTGAACGATGATC-3'; SEQ ID NO: 9). The expected size
of the amplified DNA band was 439 bp. Two primers synthesized on
the basis of aldolase coding sequence were used as controls to
produce a product with an expected size of 443 bp: aldolase sense
primer 5'-GGCAAGGGCATCCTGGCTGC-3' (SEQ ID NO: 10) and aldolase
antisense reverse 5'-TAACGGG CCAGAACATTGGCATT-3' (SEQ ID NO: 11).
Approximately 75 ng of poly A+ RNA was reverse transcribed with
Superscript II (Life Technologies, Inc., Merelbeke, Belgium) and
used for PCR. PCR was performed using the Taq polymerase under the
following conditions: denaturation at 94.degree. C. for 3 min, 38
cycles at 94.degree. C. for 1 min, 58.degree. C. for 2 min and
72.degree. C. for 2 min. Aliquots (10 .mu.l) of the PCR reaction
were analysed by 1% agarose gel electrophoresis.
[0326] A 439 bp-band was clearly detected in peripheral blood
lymphocytes (PBL). The amplification of a fragment of aldolase
coding sequence was used as control.
[0327] The distribution of GPR43 in particular peripheral blood
cells, and other cell types was investigated further using
semi-quantitative PCR (FIG. 13). Semi-quantitative RT-PCR (TaqMan)
experiments were carried out over a range of 12 selected human
tissues using a panel of total and polyA+ RNA (Clontech, Ambion,
Biochain). Total RNA from blood cells and cell lines were prepared
with (Tripure Isolation Reagent, Boehringer Mannheim).
[0328] Semi-quantitative RT-PCR experiments were performed using
gene specific primers to human GPR43 receptor. The GPR43 receptor
primers were forward 5'-GGCTTTCCCCGTGCAGTAC-3' (SEQ ID NO: 12),
Taqman probe 5'-AGCTCTCCCGCCGGC CTCTG-3' (SEQ ID NO: 13) and
reverse 5'-CCAGAGCTGCAATCACTCCA-3' (SEQ ID NO: 14).
[0329] Primers designed to the house keeping gene GAPDH forward
5'-GAAGGTGAA GGTCGGAGTC-3' (SEQ ID NO: 15), Taqman probe
5'-AGCTCTCCCGCCG GCCTCTG-3' (SEQ ID NO: 16) and reverse
5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 17) were used to produce
reference mRNA profiles.
[0330] Strong level of GPR43 expression was found in
polymorphonuclear neutrophils (PMN). GPR43 also was detected at
lower levels in T lymphocytes and peripheral blood mononuclear
cells (PBMC) (FIG. 13). In comparison to the level of expression in
the granulocytes no significant expression could be detected in the
CNS and other peripheral tissues (data not shown).
Example 3
Screening for GPR43Ligands
[0331] CHO-K1 cells (ATCC CRL-9618 (Bethesda, Md., USA) were grown
in Nutrient Mixture HAM's F12 medium supplemented with 10% fetal
calf serum, 100 units/ml penicillin and 100 .mu.g/ml streptomycin.
A bicistronic plasmid encoding the human GPR43 was transfected into
CHO-K1 cells, using Fugene 6 (Roche Diagnostics, Mannheim,
Germany). Individual clones were selected two days after
transfection with 250 .mu.g/ml zeocin and GPR43-positive clones
were confirmed by northern blotting. Positive clones were used for
screening with a reference small molecule library containing 250
natural ligands of G protein coupled receptors at a concentration
of 1-100 .mu.M. A specific activity was obtained with acetate and
confirmed by a dose response curve. Additional related compounds
were tested using the same cells.
[0332] CHO-K1 cells transfected with the bicistronic plasmid that
does not encode the human GPR43 were used as control cells
(mock-transfected).
Example 4
Activity of SCFA on CHO-K1 Cells Expressing hGPR43
[0333] SCFA, ranging from 1 carbon to 4 carbon, were tested on
CHO-K1 cells stably expressing the human GPR43 for their ability to
inhibit the activity of adenylate cyclase stimulated with
forskolin.
[0334] The rank order of potency was as follows: C2
(acetate).gtoreq.C3 (propionate)>C4 (butyrate)>>C1
(formate), with acetate being the most potent to inhibit the enzyme
activity. All of these compounds decrease forskolin-stimulated
adenylate cyclase activity by 75% (FIG. 4).
[0335] The observed effect of acetate was totally abolished by
overnight preincubation with Pertussis Toxin (PTX), which disrupts
the coupling between the receptor and the G.quadrature.i/o subunit
(FIG. 5). The coupling pathway of the human GPR43 is therefore
preferentially Gi in the CHO-K1.
[0336] The observed effect of fatty acid was restricted to the
GPR43 expressing cells and none of the control cells, expressing
other recombinant GPCR or not, showed activity with the activators
mentioned (data not shown).
[0337] The above-mentioned results are pH-independent. That is, at
the concentration tested, the pH of reaction buffer was between
7-7.4. In addition, equipotent activity was observed with different
salts of the active SCFA, including ammonium (NH.sub.3.sup.+),
potassium (K.sup.+) and sodium (Na.sup.+) salts (see FIG. 7 for
results obtained with NH.sub.3.sup.+ acetate).
Example 5
Analysis of SCFA Activity in Membrane-Based Functional Assays
[0338] The activity of acetate was examined in a membrane-based
functional test. In this assay, the accumulation of
GTP.gamma.[.sup.35S] binding was monitored on a preparation of
membranes from CHO-K1 cells expressing the human GPR43 (FIG. 6).
The potency of the acetate was comparable to that observed in the
cell-based functional assay monitoring cAMP levels. The rank order
of potency of the SCFA tested was conserved using this
membrane-based assay. That is, the assay showed that
C2.gtoreq.C3>C4>>C1. C4 and C1 partly activate the human
receptor since the maximal response was lowest as compared to that
observed with acetate and propionate (data not shown).
[0339] The activity of acetate, propionate and related compounds
was restricted to GPR43 because these acids were not able to
stimulate any binding in ten different membrane preparations of
CHO-K1 cells expressing non related human G-protein coupled
receptors such as adenosine A1 receptor, adrenergic 2C receptor,
corticotropin-releasing factor 1 receptor, chemokine CCR3 receptor,
leukotriene LTB4 receptor, muscarinic M4 receptor, neuropeptide FF
2S receptor, opioid 3 receptor, serotonin 5-HT1A receptor, and
somatostatin sst5 receptor. All of these Gi-coupled receptors were
stimulated in the same experiment by their respective reference
ligand (data not shown).
[0340] The influence of the salt was evaluated to rule out any
direct counter-ion effect. In particular, sodium cations are known
to modulate, positively or negatively, the activity of G-protein
coupled receptors in the presence or absence of ligand. Acetate
tested as sodium or ammonium salt was equipotent in activating
GTP.gamma.[.sup.35S] binding on membranes suspended in assay buffer
containing 120 mM sodium or potassium (FIG. 7).
[0341] The activity of other SCFA and related compounds (alcohols,
aldehydes, cetone, di-acids . . . ) was evaluated using a single
concentration of each in the membrane-cell based assay. The order
of agonist potency is
acetate=propionate>n-butyrate=isobutyrate=n-valerate=caproate>>f-
ormate>>pyruvate=acetoacetate. Inactive compounds include:
C2-ethanol, acetaldehyde and oxalate; C3--malonate and acetone;
C4--DL-.beta.-hydroxybutyrate, GABA, L-glutamate, succinate; and
C6--citrate (FIG. 8).
Example 6
Effect of SCFA on Activity of GPR43 as Measured by Second Messenger
Accumulation
[0342] SCFA were able to stimulate the production of inositol
phosphates in CHO-K1 cells stably expressing human GPR43 (FIG. 9).
This activation was slightly affected by a PTX-pretreatment prior
to the stimulation, regardless of the SCFA used. The coupling of
human GPR43 is therefore dual, involving the activation of Gq
protein in addition to the above-described Gi coupling (FIG.
10).
[0343] Transient transfection of the cDNA for human GPR43 into
COS-7, CHO and HEK cells, with or without co-expression of a
chimeric Gqi protein, led to the fatty acid stimulation of the
accumulation of inositol phosphates, reflecting the activation of
Phospholipase C (FIG. 11). Control cells, transfected with Gqi only
or with cDNAs for other GPCRs, such as motilin or histamine H1
receptor, were not activated by acetate and other SCFAs (data not
shown). The accumulation of inositol phosphates was increased in
non-SCFA-treated cells transfected with Gqi and human GPR43 cDNAs,
giving evidence of the constitutive activation of the receptor in
the absence of added ligand at the time of the reaction (FIG.
11).
Example 7
Formulae and Activity of SCFA's Active on GPR43
[0344] Formulae of active compounds are presented in FIG. 12.
Structure-activity relationships (SRA) of the active compounds
showed that, when considered with the inactivity of closely
structurally-related compounds (ketones, alcohols and aldehydes),
the carboxylic moiety is required for activity, this moiety being
branched at the extremity of a carbon chain comprising 1-6 carbon
atoms, linear or not, with optimal activity for 2-3 C. A second
carboxylic moiety abolished the signal, whatever the length of the
carbon chain, as observed with oxalate (C2), malonate (C3),
succinate (C4), aspartate (C4), glutamate (C5) or citrate with 3
carboxylic moieties.
[0345] Substitutions with other functions differently modulate the
activity of compounds on the human GPR43. For example, --OH
substitution abolished the activity for a corresponding active
compound (n-butyrate is active, .beta.-hydroxybutyrate is not
active), while --NH.sub.3.sup.+ decreased activity without
abolishing it (acetate >>glycine). The combination of --OH
and --NH3.sup.+ functions as in serine (C3) also abolished the
activity. Ketone substituted compounds, such as pyruvate and
acetoacetate, also showed decreased but consistent activity as
compared to corresponding active, non-substituted compounds
(acetate and n-butyrate, respectively).
Example 8
Propionate and Acetate are able to Induce the Mobilization of
Intracellular Calcium in Human Neutrophils
[0346] The following experiments were conducted to test whether the
human polymorphonuclear (PMN) leukocytes could be activated with
acetate and propionate, since the receptor is strongly expressed in
peripheral blood cells containing mainly PMNs. Activation was
determined by the quantification of the intracellular calcium
mobilized from internal pool after activation by acetate and
propionate of the cell membrane receptor.
[0347] PMN were purified from the venous blood of healthy
volunteers. Cells were isolated according to established methods.
For intracellular calcium measurements, the cells were loaded for
30 min at room temperature with Fura-2 .mu.M (Molecular Probes).
Calcium transients were monitored by a LSB 50B spectrofluorimeter
(Perkin Elmer). Briefly, neutrophils suspensions (1.times.107
cells/ml) were incubated with 2 .mu.M Fura-2 .mu.M for 30 minutes
at 37.degree. C. The cells were then washed free of the
extracellular probe, resuspended at 5.times.106 cells/ml and
allowed to reequilibrate for 10 minutes at 37.degree. C. Cells were
then transferred to the thermostatted cuvette compartment
(37.degree. C.) of the fluorometer and the fluorescence monitored
(excitation and emission wavelengths, 340 and 510 nm
respectively).
[0348] Injection of propionate or acetate on PMN yields to an
increase of intracellular calcium as compared to basal condition.
FIG. 14 shows the kinetic plot of such an increase for varying
concentration of Na propionate. The increase of intracellular
calcium is monitored as an increase of the ratio "basal
fluorescence" over "stimulated fluorescence".
[0349] Injection with increasing concentration of propionate (FIG.
15) or acetate (FIG. 16) leads to a concentration-dependent
increase of intracellular calcium. Propionate and acetate are
equipotent (EC.sub.50=540 .mu.M and 537 .mu.M, for propionate and
acetate respectively).
[0350] The results show that propionate and acetate are able to
induce the mobilization of intracellular calcium in human
neutrophils. According to our previous results describing the
complete pharmacological characterization of GPR43 as the cell
surface target for short-chain fatty acids such as propionate and
acetate, we conclude that the observed effect on calcium
mobilization is mediated through the stimulation of the receptor of
interest. Naccache et al (J Cell Physiol 1988 July; 136(1):118-24),
Fonteriz et al (Biochem Biophys Acta 1991 Jun. 7; 1093 (1):1-6) and
Nakao et al (Infect Immun 1992 December; 60 (12):5307-11) have
described that acetate and propionate stimulate the cytoplasmic
calcium mobilization in PMN with millimolar EC50. But none of them
associated the observed response with the simulation of a given
G-protein coupled receptor (GPCR), although experiments with
pertussis toxin and activator/inhibitor of protein kinase C may
have suggested a GPCR-mechanism. Brunkhorst et al (Infection and
Immunity July 1992, vol 60, 7:2957-2968) has suggested a GPCR
mechanism of action, for at least propionate and acetate, on a
serie of PMN-activation event such as cytoskeletal F-actin
alterations, PMN polarization, F-actin localization, cytoplasmic pH
oscillation, cell shape.
[0351] We have showed that acetate and propionate were equipotent
as activator of recombinant hGPR43 expressed in recombinant
system.
[0352] We conclude therefore that our data firstly associate that
the actions of acetate and propionate on the calcium-mobilization
on human neutrophils are mediated through the activation of GPR43
solely.
Example 9
Chemotaxis Induced by SCFAs: Calcium and Chemotactic Assays on
Neutrophils
[0353] Peripheral blood mononuclear cells were purified from buffy
coats of healthy volunteers as previously described (Struyf S, De
Meester I, Scharpe S, Lenaerts J P, Menten P, Wang J M, Proost P,
Van Damme J., Eur J Immunol 1998 April; 28(4):1262-71). For
intracellular calcium measurements, the cells were loaded for 30
min at room temperature with Fura-2 AM (Molecular Probes). Calcium
transients were monitored by a LS50B spectrofluorimeter (Perkin
Elmer) as described (Grynkiewicz G, Poenie M, Tsien R Y., J Biol
Chem 1985 Mar. 25; 260(6):3440-50) at a final cell concentration of
106 cells/ml in buffer containing 125 .mu.M probenecid. Chemotaxis
was assessed in 48-well chambers using polycarbonate filter
membranes with 3 .mu.m (mesh size) (Neuroprobes, Inc.). The results
are represented as chemotactic index (FIG. 17).
[0354] Chemotactic Response of Neutrophilic Granulocytes to
SCFA.:
[0355] Freshly isolated peripheral blood neutrophils from healthy
donors were tested for their chemotactic response to sodium acetate
and propionate. Both SCFAs yielded the classical bell-shaped
dose-response curve, the optimal concentration being 10-3 M (FIG.
17). We conclude that SCFA induce chemotaxis on neutrophils. The
potency of SCFAs in neutrophil chemotaxis was inferior to that of
FMLP which was still fully active at 10-8 M. Furthermore, the
efficacy of fMLP is also superior to that of SCFA in that the
maximal chemotactic index of fMLP was on average at least 3-fold
higher (data not shown).
OTHER EMBODIMENTS
[0356] The foregoing examples demonstrate experiments performed and
contemplated by the present inventors in making and carrying out
the invention. It is believed that these examples include a
disclosure of techniques which serve to both apprise the art of the
practice of the invention and to demonstrate its usefulness. It
will be appreciated by those of skill in the art that the
techniques and embodiments disclosed herein are preferred
embodiments only that in general numerous equivalent methods and
techniques may be employed to achieve the same result.
[0357] All of the references, including patents and patent
applications, identified hereinabove, are hereby expressly
incorporated herein by reference to the extent that they describe,
set forth, provide a basis for or enable compositions and/or
methods which may be important to the practice of one or more
embodiments of the present inventions.
REFERENCES
[0358] 1. Abbrachio, M. P. and Burnstock, G. (1994) Pharmacol.
Ther. 64, 445-475. [0359] 2. Fredholm, B. B. et al.(1997) Trends
Pharmacol. Sci. 18, 79-82. [0360] 3. Webb, T. E. et al. (1993) FEBS
Lett. 324, 219-225. [0361] 4. Leon, C. et al. (1997) FEBS Lett.
403, 26-30. [0362] 5. Communi, D. et al. (1997) J. Biol. Chem. 272,
31969-31973. [0363] 6. Lustig, K. D. et al. (1993) Proc. Natl.
Acad. Sci. U.S.A. 90, 5113-5117. [0364] 7. Parr, C. E. et al.
(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3275-3279. [0365] 8.
Bogdanov, Y. et al. (1997) J. Biol. Chem. 272, 12583-12590. [0366]
9. Boyer, J. L. et al. (2000) Mol. Pharmacol. 57, 805-810. [0367]
10. Webb, T. E. et al. (1996) Mol. Pharmacol. 50, 258-265. [0368]
11. Chang, K. et al. (1995) J. Biol. Chem. 270, 26152-26158. [0369]
12. Communi, D. et al. (1996) Biochem. Biophys. Res. Commun. 222,
303-308. [0370] 13. Nicholas, R. A. et al. (1996) Mol. Pharmacol.
50, 224-229. [0371] 14. Communi, D. et al. (1995) J. Biol. Chem.
270, 30849-30852. [0372] 15. Nguyen, T. et al. (1995) J. Biol.
Chem. 270, 30845-30848. [0373] 16. Webb, T. E. et al. (1996)
Biochem. Biophys. Res. Commun. 219, 105-110. [0374] 17. Akbar, G.
K. M. et al. (1996) J. Biol. Chem. 271, 18363-18367. [0375] 18.
Yokomizo, T. et al. (1997) Nature 387, 620-624. [0376] 19. Li, Q.
et al. (1997) Biochem. Biophys. Res. Commun. 236, 455-460. [0377]
20. Janssens, R. et al. (1997) Biochem. Biophys. Res. Commun. 226,
106-112. [0378] 21. Zhang, F. L et al. (2001) J. Biol. Chem. 276
(11), 8608-8615. [0379] 22. Hollopeter, G. et al. (2001) Nature
409, 202-207. [0380] 23. Chambers, J. K. et al. (2000) J. Biol.
Chem. 275 (15), 10767-10771. [0381] 24. Wittenberger, T. et al.
(2001) J. Mol. Biol. 307, 799-813. [0382] 25. Communi, D. et al.
(1995b). Circ. Res., 76, 191-198. [0383] 26. Brooker, G. et al.
(1979) Adv. Cyclic Nucleotide Res. 10, 1-33. [0384] 27. Minamide,
L. S, and Bamburg, J. R. (1990) Anal. Biochem. 190, 66-70. [0385]
28. Erb, L. et al. (1995) J. Biol. Chem. 270, 4185-4188. [0386] 29.
Baltensperger, K. and Porzig, H. (1997) J. Biol. Chem. 272,
10151-10159. [0387] 30. Eason, M. G. et al. (1992) J. Biol. Chem.
267 (22), 15795-15801. [0388] 31. Chabre, O. et al. (1994) J. Biol.
Chem. 269 (8), 5730-5734. [0389] 32. Boyer, J. L. et al. (1993) J.
Pharmacol. Exp. Ther. 267, 1140-1146. [0390] 33. Simon, J. et al.
(2001) Br. J. Pharmacol. 132, 173-182. [0391] 34. Gudermann et al.
(1995) J. Mol. Med. 73, 51-63. [0392] 35. Lundquist F. (1960) Acta
Physiol. Scand. 175, 97 [0393] 36. Bergman E. (1990) Physiol. Rev.
70, 567-590 [0394] 37. Cummings J. H., et al. (1987) Gut 28:1221-7p
[0395] 38. Mirzabekov et al. (2000) Nature Biotechnology 18,
649-654
Sequence CWU 1
1
171993DNAHomo sapiens 1atgctgccgg actggaagag ctccttgatc ctcatggctt
acatcatcat cttcctcact 60ggcctccctg ccaacctcct ggccctgcgg gcctttgtgg
ggcggatccg ccagccccag 120cctgcacctg tgcacatcct cctgctgagc
ctgacgctgg ccgacctcct cctgctgctg 180ctgctgccct tcaagatcat
cgaggctgcg tcgaacttcc gctggtacct gcccaaggtc 240gtctgcgccc
tcacgagttt tggcttctac agcagcatct actgcagcac gtggctcctg
300gcgggcatca gcatcgagcg ctacctggga gtggctttcc ccgtgcagta
caagctctcc 360cgccggcctc tgtatggagt gattgcagct ctggtggcct
gggttatgtc ctttggtcac 420tgcaccatcg tgatcatcgt tcaatacttg
aacacgactg agcaggtcag aagtggcaat 480gaaattacct gctacgagaa
cttcaccgat aaccagttgg acgtggtgct gcccgtgcgg 540ctggagctgt
gcctggtgct cttcttcatc cccatggcag tcaccatctt ctgctactgg
600cgttttgtgt ggatcatgct ctcccagccc cttgtggggg cccagaggcg
gcgccgagcc 660gtggggctgg ctgtggtgac gctgctcaat ttcctggtgt
gcttcggacc ttacaacgtg 720tcccacctgg tggggtatca ccagagaaaa
agcccctggt ggcggtcaat agccgtggtg 780ttcagttcac tcaacgccag
tctggacccc ctgctcttct atttctcttc ttcagtggtg 840cgcagggcat
ttgggagagg gctgcaggtg ctgcggaatc agggctcctc cctgttggga
900cgcagaggca aagacacagc agaggggaca aatgaggaca ggggtgtggg
tcaaggagaa 960gggatgccaa gttcggactt cactacagag tag 9932330PRTHomo
sapiens 2Met Leu Pro Asp Trp Lys Ser Ser Leu Ile Leu Met Ala Tyr
Ile Ile1 5 10 15Ile Phe Leu Thr Gly Leu Pro Ala Asn Leu Leu Ala Leu
Arg Ala Phe 20 25 30Val Gly Arg Ile Arg Gln Pro Gln Pro Ala Pro Val
His Ile Leu Leu 35 40 45Leu Ser Leu Thr Leu Ala Asp Leu Leu Leu Leu
Leu Leu Leu Pro Phe 50 55 60Lys Ile Ile Glu Ala Ala Ser Asn Phe Arg
Trp Tyr Leu Pro Lys Val65 70 75 80Val Cys Ala Leu Thr Ser Phe Gly
Phe Tyr Ser Ser Ile Tyr Cys Ser 85 90 95Thr Trp Leu Leu Ala Gly Ile
Ser Ile Glu Arg Tyr Leu Gly Val Ala 100 105 110Phe Pro Val Gln Tyr
Lys Leu Ser Arg Arg Pro Leu Tyr Gly Val Ile 115 120 125Ala Ala Leu
Val Ala Trp Val Met Ser Phe Gly His Cys Thr Ile Val 130 135 140Ile
Ile Val Gln Tyr Leu Asn Thr Thr Glu Gln Val Arg Ser Gly Asn145 150
155 160Glu Ile Thr Cys Tyr Glu Asn Phe Thr Asp Asn Gln Leu Asp Val
Val 165 170 175Leu Pro Val Arg Leu Glu Leu Cys Leu Val Leu Phe Phe
Ile Pro Met 180 185 190Ala Val Thr Ile Phe Cys Tyr Trp Arg Phe Val
Trp Ile Met Leu Ser 195 200 205Gln Pro Leu Val Gly Ala Gln Arg Arg
Arg Arg Ala Val Gly Leu Ala 210 215 220Val Val Thr Leu Leu Asn Phe
Leu Val Cys Phe Gly Pro Tyr Asn Val225 230 235 240Ser His Leu Val
Gly Tyr His Gln Arg Lys Ser Pro Trp Trp Arg Ser 245 250 255Ile Ala
Val Val Phe Ser Ser Leu Asn Ala Ser Leu Asp Pro Leu Leu 260 265
270Phe Tyr Phe Ser Ser Ser Val Val Arg Arg Ala Phe Gly Arg Gly Leu
275 280 285Gln Val Leu Arg Asn Gln Gly Ser Ser Leu Leu Gly Arg Arg
Gly Lys 290 295 300Asp Thr Ala Glu Gly Thr Asn Glu Asp Arg Gly Val
Gly Gln Gly Glu305 310 315 320Gly Met Pro Ser Ser Asp Phe Thr Thr
Glu 325 33037PRTArtificialPeptide derived from myristoylated
alanine-rich protein kinase C sybstrate protein MARCKS 3Phe Lys Lys
Ser Phe Lys Leu1 5413PRTArtificialSrc-releted peptide 4Arg Arg Leu
Ile Glu Asp Ala Glu Tyr Ala Ala Arg Gly1 5 10511DNAUnknownNF-kB
binding element consensus sequence 5ggggactttc c
11632DNAArtificialGPR43 sense primer 6gcggaattca ccatgctgcc
ggactggaag ag 32731DNAArtificialGPR43 antisense primer 7ctagtctaga
ctgctactct gtagtgaagt c 31820DNAArtificialGPR43 sense primer
8actggaagag ctccttgatc 20919DNAArtificialGPR43 antisense primer
9caagtattga acgatgatc 191020DNAArtificialAldolase sense primer
10ggcaagggca tcctggctgc 201123DNAArtificialAldolase antisense
primer 11taacgggcca gaacattggc att 231219DNAArtificialGPR43 forward
primer 12ggctttcccc gtgcagtac 191320DNAArtificialTaqman forward
probe 13agctctcccg ccggcctctg 201420DNAArtificialTaqman reverse
probe 14ccagagctgc aatcactcca 201519DNAArtificialGAPDH forward
primer 15gaaggtgaag gtcggagtc 191620DNAArtificialGADPH Taqman
forward probe 16agctctcccg ccggcctctg 201720DNAArtificialGAPDH
Taqman reverse probe 17gaagatggtg atgggatttc 20
* * * * *
References