U.S. patent application number 10/127940 was filed with the patent office on 2003-09-25 for assay for non-peptide agonists to peptide hormone receptors.
Invention is credited to Beinborn, Martin, Kopin, Alan S..
Application Number | 20030180798 10/127940 |
Document ID | / |
Family ID | 24278490 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180798 |
Kind Code |
A1 |
Kopin, Alan S. ; et
al. |
September 25, 2003 |
Assay for non-peptide agonists to peptide hormone receptors
Abstract
The invention features a method for determining whether a
candidate compound is a non-peptide agonist of a peptide hormone
receptor. In this method, a candidate compound is exposed to a form
of the peptide hormone receptor which has an enhanced ability to
amplify the intrinsic activity of a non-peptide agonist. The second
messenger signaling activity of the enhanced receptor is measured
in the presence of the candidate compound, and compared to the
second messenger signaling activity of the enhanced receptor
measured in the absence of the candidate compound. A change in
second messenger signaling activity indicates that the candidate
compound is an agonist. An increase in second messenger signaling
activity indicates that the compound is either a full or partial
positive agonist; a decrease in second messenger signaling activity
indicates that the compound is an inverse (also termed a
`negative`) agonist.
Inventors: |
Kopin, Alan S.; (Wellesley,
MA) ; Beinborn, Martin; (Brookline, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
24278490 |
Appl. No.: |
10/127940 |
Filed: |
April 23, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10127940 |
Apr 23, 2002 |
|
|
|
09076510 |
May 12, 1998 |
|
|
|
6376198 |
|
|
|
|
09076510 |
May 12, 1998 |
|
|
|
08570157 |
Dec 11, 1995 |
|
|
|
5750353 |
|
|
|
|
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/72 20130101; G01N 33/74 20130101; C12Q 1/02 20130101; A61K
38/00 20130101; G01N 2333/72 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Goverment Interests
[0002] This invention was made in part with Government funding
under National Institute of Health grant #DK46767, and the
Government therefore has certain rights in the invention.
Claims
What is claimed is:
1. A method for determining whether a candidate compound is a
non-peptide agonist of a peptide hormone receptor, said method
comprising the steps of: (a) exposing said candidate compound to a
form of said peptide hormone receptor that has the ability to
amplify the activity of a non-peptide agonist as compared to the
corresponding wild-type receptor; and (b) measuring the activity of
said form in the presence of said candidate compound relative to
the activity of said form in the absence of said compound, a change
in said activity indicating that said candidate compound is an
agonist.
2. The method of claim 1, wherein said form of said receptor is a
mutant eceptor.
3. The method of claim 1, wherein said form of said receptor has a
higher basal activity than the basal activity of a corresponding
human wild-type receptor.
4. The method of claim 1, wherein said form of said receptor is a
constitutively active receptor.
5. The method of claim 1, wherein said form of said receptor is a
non-human receptor.
6. The method of claim 5, wherein said form of said receptor is a
non-human wild-type receptor.
7. The method of claim 1, wherein said form of said receptor is a
naturally-occurring mutant receptor.
8. The method of claim 1, wherein an increase in said activity
indicates that said candidate compound is a positive agonist.
9. The method of claim 1, wherein said positive agonist is a
partial agonist.
10. The method of claim 1, wherein a decrease in said activity
indicates that said candidate compound is an inverse agonist.
11. A method of isolating a form of a peptide hormone receptor
suitable for detecting agonist activity in a non-peptide ligand,
said method comprising: (a) exchanging a region of a functional
domain of a first peptide hormone receptor with a corresponding
region of a functional domain of a second peptide hormone receptor,
said functional domain being selected from the group consisting of
an intracellular loop and a transmembrane domain; and (b) measuring
the ability of said first peptide hormone receptor to amplify an
agonist signal relative to the ability of a corresponding wild-type
human receptor to amplify said agonist signal, a greater
amplification in said first peptide hormone receptor indicating
that said first peptide hormone receptor is suitable for detecting
agonist activity in a ligand.
12. The method of claim 11, wherein said second peptide hormone
receptor is linked to a different second messenger pathway than
said first peptide hormone receptor.
13. A method of isolating a form of a peptide hormone receptor that
amplifies the intrinsic activity of an agonist, said method
comprising: (a) constructing a series of mutant forms of said
receptor by replacing an original amino acid with a replacement
amino acid, said replacement amino acid; and (b) measuring the
ability of said first peptide hormone receptor to amplify an
agonist signal relative to the ability of a corresponding wild-type
human receptor to amplify said agonist signal, a greater
amplification in said first peptide hormone receptor indicating
that said first peptide hormone receptor is suitable for detecting
agonist activity in a non-peptide ligand.
14. The method of claim 13, wherein said replacing comprises
replacing an amino acid in an intracellular domain of said
receptor.
15. The method of claim 13, wherein said replacing comprises
replacing an amino acid in a transmembrane domain flanking an
intracellular portion of said receptor.
16. The method of claim 13, wherein said replacement amino acid is
a different charge from said original amino acid.
17. The method of claim 13, wherein said replacement amino acid is
glutamic acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/076,510, filed May 12, 1998, now U.S. Pat. No. 6,376,198, which
is a divisional of U.S. Ser. No. 08/570,157, filed Dec. 11, 1995,
now U.S. Pat. No. 5,750,353.
BACKGROUND OF THE INVENTION
[0003] This invention relates to peptide hormone receptors.
[0004] Peptide hormone receptors are important targets for drug
research because a considerable number of diseases and other
adverse effects result from abnormal receptor activity. High
affinity, high specificity, non-peptide antagonists for peptide
hormone receptors have been developed. These antagonists are
therapeutically useful for decreasing receptor activation by
hormones. Developing non-peptide agonists proved to be far more
difficult.
[0005] One peptide hormone of interest, cholecystokinin (CCK), is a
neuropeptide with two distinct receptors: CCK-A and CCK-B/gastrin
(Vanderhaeghen et al., Nature, 257:604-605, 1975; Dockray, Nature,
264:568-570, 1976; Rehfeld, J. Biol. Chem., 253:4022-4030, 1978;
Hill et al., Brain Res., 526:276-283, 1990; Hill et al., J.
Neurosci., 10:1070-1081, 1990; Woodruff et al., Neuropeptides,
(Suppl.) 19:57-64, 1991). The peripheral type receptor CCK-A is
located in discrete brain nuclei and, in certain species, the
spinal cord, and is also involved in gallbladder contraction and
pancreatic enzyme secretion. The CCK-B/gastrin receptor is most
abundant in the cerebral cortex, cerebellum, basal ganglia, and
amygdala of the brain, as well as in parietal cells of the
gastrointestinal tract. CCK-B receptor antagonists have been
postulated to modulate anxiety, panic attacks, analgesia, and
satiety (Ravard et al., Trends Pharmacol. Sci., 11:271-273, 1990;
Singh et al., Proc. Natl. Acad. Sci. U.S.A., 88:1130-1133, 1991;
Faris et al., Science, 219:310-312, 1983; Dourish et al.,
Eur.J.Pharmacol., 176:35-44, 1990; Wiertelak et al., Science,
256:830-833, 1992; Dourish et al., Science, 245:1509-1511,
1989).
SUMMARY OF THE INVENTION
[0006] Applicants have developed a systematic screening assay for
identifying non-peptide agonists specific to peptide hormone
receptors. The assay is based on applicants' recognition that a
peptide hormone receptor having the capability of amplifying the
intrinsic activity of a ligand is useful as a screening vehicle to
identify receptor-specific agonists. In addition, a receptor with a
signaling activity higher than the corresponding human wild-type
basal level of signaling activity is especially useful for
detecting a reduction in activity induced by an inverse agonist. In
both cases, the receptor amplifies the signal generated when the
ligand interacts with its receptor, relative to the signal
generated when the ligand interacts with a human wild-type
receptor. Thus, forms of a receptor with the ability to amplify
receptor signaling are useful for efficiently screening positive
and inverse non-peptide agonists to the corresponding human
wild-type form of the receptor.
[0007] Accordingly, the invention features a method for determining
whether a candidate compound is a non-peptide agonist of a peptide
hormone receptor. In this method, a candidate compound is exposed
to a form of the peptide hormone receptor which has a greater, or
an enhanced, ability to amplify the intrinsic activity of a
non-peptide agonist (hereafter an `enhanced receptor`). The second
messenger signaling activity of the enhanced receptor is measured
in the presence of the candidate compound, and compared to the
second messenger signaling activity of the enhanced receptor
measured in the absence of the candidate compound. A change in
second messenger signaling activity indicates that the candidate
compound is an agonist. For example, an increase in second
messenger signaling activity indicates that the compound is either
a full or partial positive agonist; a decrease in second messenger
signaling activity indicates that the compound is an inverse (also
termed a `negative`) agonist.
[0008] By "intrinsic activity" is meant the ability of a ligand to
activate a receptor, i.e., to act as an agonist. By `amplify` is
meant that the signal generated when the ligand interacts with the
enhanced receptor is either higher for a positive agonist, or lower
for an inverse agonist, than the signal produced when the same
ligand interacts with a corresponding non-enhanced receptor, e.g.,
a wild-type human receptor. A `non-enhanced receptor`, for the
purposes of this invention, is a wild-type human receptor for the
peptide hormone of interest. By "corresponding" is meant the same
type of peptide hormone receptor albeit in another form, e.g., a
constitutively active mutant receptor. By way of example, the
corresponding wild-type form of a constitutively active mutant
CCK-B/gastrin receptor would be a wild-type CCK-B/gastrin receptor;
the human CCK-B/gastrin receptor is the corresponding human form of
the rat CCK-B/gastrin receptor.
[0009] Examples of enhanced receptors include synthetic mutant
receptors, e.g., constitutively active mutant receptors; other
mutant receptors with normal basal activity which amplify the
intrinsic activity of a compound; naturally-occurring mutant
receptors, e.g., those which cause a disease phenotype by virtue of
their enhanced receptor activity, e.g., a naturally-occurring
constitutively active receptor; and either constitutively active or
wild-type non-human receptors, e.g., rat, mouse, mastomys, Xenopus,
or canine receptors or hybrid variants thereof, which amplify an
agonist signal to a greater extent than does the corresponding
wild-type human receptor. An enhanced receptor may, but does not
always, have a higher basal activity than the basal activity of a
corresponding human wild-type receptor. Methods for measuring the
activity of an enhanced receptor relative to the activity of a
corresponding wild-type receptor are described and demonstrated
below.
[0010] Examples of peptide hormone receptors within the scope of
the invention include, but are not limited to, receptors specific
for the following peptide hormones: amylin, angiotensin, bombesin,
bradykinin, C5a anaphylatoxin, calcitonin, calcitonin-gene related
peptide (CGRP), chemokines, cholecystokinin (CCK), endothelin,
follicle stimulating hormone (FSH), formyl-methionyl peptides,
galanin, gastrin, gastrin releasing peptide, glucagon,
glucagon-like peptide 1, glycoprotein hormones,
gonadotrophin-releasing hormone, leptin, luteinizing hormone (LH),
melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin,
parathyroid hormone, secretin, somatostatin, tachykinins, thrombin,
thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinal
polypeptide (VIP), and vasopressin. An enhanced receptor can
further embrace a single transmembrane domain peptide hormone
receptor, e.g., an insulin receptor.
[0011] An "agonist", as used herein, includes a positive agonist,
e.g., a full or a partial positive agonist, or a negative agonist,
i.e., an inverse agonist. An agonist is a chemical substance that
combines with a receptor so as to initiate an activity of the
receptor; for peptide hormone receptors, the agonist preferably
alters a second messenger signaling activity. A positive agonist is
a compound that enhances or increases the activity or second
messenger signaling of a receptor. A "full agonist" refers to an
agonist capable of activating the receptor to the maximum level of
activity, e.g., a level of activity which is induced by a natural,
i.e., an endogenous, peptide hormone. A "partial agonist" refers to
a positive agonist with reduced intrinsic activity relative to a
full agonist. As used herein, a "peptoid" is a peptide-derived
partial agonist. An "inverse agonist", as used herein, has a
negative intrinsic activity, and reduces the receptor's signaling
activity relative to the signaling activity measured in the absence
of the inverse agonist. A diagram explaining the difference between
full and partial agonists, inverse agonists, and antagonists is
shown in FIG. 1 (see also Milligan et al., TIPS, 16:10-13,
1995).
[0012] Examples of peptide hormone receptor specific peptide
agonists and non-peptide antagonists useful in the screening assay
of the invention are described below. Non-peptide ligands include,
but are not limited to, the benzodiazepines, e.g.,
azabicyclo[3.2.2]nonane benzodiazepine (L-740,093; Castro Pineiro
et al., WO 94/03437). L-740,093 S and L-740,093 R refer to the
S-enantiomer and the R-enantiomer of L-740,093, respectively. Where
the peptide hormone receptor is a CCK-A or CCK-B/gastrin receptor,
useful peptide agonists include, but are not limited to, gastrin
(e.g., sulphated ("gastrin II") or unsulphated ("gastrin I") forms
of gastrin-17, or sulphated or unsulphated forms of gastrin-34), or
cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8s), unsulphated
CCK-8 (CCK-8d), CCK-4, or pentagastrin (CCK-5)). Full agonists of
the CCK-B/gastrin receptor include, but are not limited to, CCK-8s,
and more preferably gastrin (gastrin I).
[0013] In contrast, an "antagonist", as used herein, refers to a
chemical substance that inhibits the ability of an agonist to
increase or decrease receptor activity. A `full`, or `perfect`
antagonist has no intrinsic activity, and no effect on the
receptor's basal activity (FIG. 1). Peptide-derived antagonists
are, for the purposes herein, considered to be non-peptide
ligands.
[0014] The invention also features a method of isolating a form of
a peptide hormone receptor suitable for detecting agonist activity
of a non-peptide ligand. The method involves (a) exchanging a
region of a functional domain of a first peptide hormone receptor
with a corresponding region of a functional domain of a second
peptide hormone receptor, the functional domain being selected from
the group consisting of an intracellular loop and adjacent parts of
a transmembrane domain; and (b) measuring the ability of the first
peptide hormone receptor to amplify an agonist signal relative to a
corresponding wild-type human receptor, a greater amplification in
the first peptide hormone receptor would indicate that the first
peptide hormone receptor is suitable for detecting agonist activity
in a non-peptide ligand. The corresponding region can be between
one and ten amino acids, e.g., a block of five to ten amino acids,
or up to thirty or a hundred amino acids in length. The first and
second peptide hormone receptors are preferably linked to different
second messenger pathways. Those skilled in the art know which
particular amino acids of the peptide hormone receptors are
considered to be within extracellular, intracellular (cytoplasmic),
or transmembrane regions of the receptor. For example,
extracellular, intracellular, and transmembrane regions of the
CCK-B/gastrin receptor are determined by sequence alignment with
other receptors (FIG. 2), or by hydropathy analysis (Baldwin, EMBO
J., 12:1693-1703, 1993). Conformation receptor modelling is
described further below.
[0015] Another method of isolating a form of a peptide hormone
receptor suitable for detecting agonist activity in a non-peptide
ligand involves (a) constructing a series of mutant forms of the
receptor by replacing an original amino acid with another amino
acid, i.e., a replacement amino acid; and (b) measuring the ability
of the first peptide hormone receptor to amplify an agonist signal
relative to the corresponding wild-type human receptor. An
amplification in the first peptide hormone receptor would indicate
that the first peptide hormone receptor is suitable for detecting
agonist activity in a non-peptide ligand. The replaced amino acid
can lie in an intracellular domain of the receptor or in a region
of a transmembrane domain flanking an intracellular portion of the
receptor, e.g., the intracellular domain-proximal half of the
transmembrane domain, or within, e.g., 8 or 10 amino acids of the
intracellular domain. The replacement amino acid can be of the same
type in each of the mutant constructs, or various types of amino
acids can be substituted at random. The replacement amino acid can
be of the same or a different charge from the original amino acid,
e.g., a negative amino acid can be exchanged for a positive amino
acid, a positive amino acid can be exchanged for a negative amino
acid, or a positive or negative amino acid can be exchanged for a
neutral amino acid. Preferably, the replacement amino acid is
glutamine, glutamic acid, aspartic acid, or serine.
[0016] Other terms used in the various embodiments of the invention
will be understood from the following definitions. For example, by
a "peptide hormone" is meant a polypeptide that interacts with a
target cell by contacting an extracellular receptor, i.e., a
"peptide hormone receptor". A "peptide" is used loosely herein to
refer to a molecule comprised of amino acid residues that are
connected to each other by peptide bonds. A "mutant receptor" is
understood to be a form of the receptor in which one or more amino
acid residues in the predominant receptor occurring in nature,
e.g., in a naturally-occurring wild-type receptor, have been either
deleted or replaced with a different type of amino acid residue. By
a "constitutively active receptor" is meant a receptor with a
higher basal activity level than the corresponding wild-type
receptor, where activity means the spontaneous ability of a
receptor to signal in the absence of further activation by a
positive agonist. The basal activity of a constitutively active
receptor can also be decreased by an inverse agonist. A
"naturally-occurring" receptor refers to a form or sequence of the
receptor as it exists in an animal, or to a form of the receptor
that is synonymous with the sequence known to those skilled in the
art as the "wild-type" sequence. Those skilled in the art will
understand a "wild-type" receptor to refer to the conventionally
accepted "wild-type" amino acid consensus sequence of the receptor,
or to a "naturally-occurring" receptor with normal physiological
patterns of ligand binding and signaling. A "second messenger
signaling activity" refers to production of an intracellular
stimulus (including, but not limited to, cAMP, cGMP, ppGpp,
inositol phosphate, or calcium ion) in response to activation of
the receptor, or to activation of a protein in response to receptor
activation, including but not limited to a kinase, a phosphatase,
or to activation or inhibition of a membrane channel.
[0017] "Sequence identity," as used herein, refers to the subunit
sequence similarity between two nucleic acid or polypeptide
molecules. When a given position in both of the two molecules is
occupied by the same nucleotide or amino acid residue, e.g., if a
given position (as determined by conventionally known methods of
sequence alignment) in each of two polypeptides is occupied by
serine, then they are identical at that position. The identity
between two sequences is a direct function of the number of
matching or identical positions, e.g., if 90% of the positions in
two polypeptide sequences are identical, e.g., 9 of 10, are
matched, the two sequences share 90% sequence identity. Methods of
sequence analysis and alignment for the purpose of comparing the
sequence identity of two comparison sequences are well known by
those skilled in the art. "Biological activity", as used herein,
refers to the ability of a peptide hormone receptor to bind to a
ligand, e.g., an agonist or an antagonist and to induce
signaling.
[0018] The invention provides an efficient and rapid assay for
identifying non-peptide agonists that interact with a peptide
hormone receptor. The newly identified agonists can serve as
therapeutics, or as lead compounds for further pharmaceutical
research. Systematic chemical modifications can be made; their
effects can be functionally assessed in enhanced receptors
according to the method of the invention. By following such a
development strategy the intrinsic activity of new agonists is
optimized so as to provide useful therapeutics against diseases
involving a peptide-hormone receptor.
[0019] Also embraced are the various mutant peptide hormone
receptors disclosed herein, and their respective nucleic acid
coding sequences. Plasmid manipulation, storage, and cell
transformation are performed by methods known to those of ordinary
skill in the art. See. e.g., Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY.
1988, 1995.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
DETAILED DESCRIPTION
[0021] We first briefly describe the drawings.
DRAWINGS
[0022] FIG. 1 is a schematic diagram showing the relationship
between a full or partial agonist, an inverse agonist, and an
antagonist.
[0023] FIG. 2 is an illustration showing a multiple alignment of
cloned CCK receptor deduced amino acid sequences: mastomys CCK-B
(SEQ ID NO: 1), rat CCK-B (SEQ ID NO: 2), human CCK-B (SEQ ID NO:
3), canine CCK-B (SEQ ID NO: 4), human CCK-A (SEQ ID NO: 5), rat
CCK-A (SEQ ID NO: 6), and Xenopus CCK-XL (SEQ ID NO: 7). `A` marks
the position in the hCCK-A receptor where an E to Q substitution
results in an increase in PD 135,158 intrinsic activity without
increasing basal receptor activity. `B` marks the position in the
hCCK-B receptor where an L to either S or E substitution results in
an increase in basal activity. The corresponding L to S in the
hCCK-A receptor does not result in an increase in basal activity.
`C` marks the position in the hCCK-B receptor where a V to E
substitution results in an increase in basal activity. The
corresponding I to E substitution in the human CCK-A receptor does
not result in an increase in basal activity. The numbering shown is
generic; each receptor is different based on deletions or
insertions.
[0024] FIG. 3 is a bar graph showing that the intrinsic activity of
peptide, peptide-derived and non-peptide ligands at the wild-type
CCK-B/gastrin receptor (top panel) is amplified in a constitutively
active receptor mutant (bottom panel).
[0025] FIG. 4 is an illustration of inositol phosphate production
by the non-peptide agonist L-740,093. Top panel: L-740,093 S
stimulated inositol phosphate production in COS-7 cells expressing
a constitutively active human CCK-B/gastrin receptor. Bottom panel:
YM022 antagonizes the partial agonist activity induced by 10 nM
L-740,093-S.
[0026] FIG. 5 is an illustration of the inositol phosphate
production of the non-peptide agonist L-740,093-R. Top panel:
L-740,093 R inhibits basal inositol phosphate production in COS-7
cells expressing a constitutively active human CCK-B/gastrin
receptor. Bottom panel: The inverse agonist activity induced by 10
nM L-740,093 R is partially abolished by YM022 in a
concentration-dependent fashion.
[0027] FIG. 6 is a comparison of intrinsic activities of
CCK-B/gastrin receptor ligands utilizing the wild-type and the
constitutively active receptors. Values for all compounds follow a
logarithmic-linear correlation (r2=0.93).
[0028] FIG. 7 is a competition binding curve showing the extent of
.sup.125I-CCK-8 receptor binding. Binding of .sup.125I-CCK-8 to
COS-7 cells, transiently transfected with hCCK-B-pcDNAI is shown in
the presence of increasing concentrations of CCK-8, gastrin I, and
CCK-4 (part A) and L-364,718 and L-365,260 (part B).
[0029] FIG. 8 is a graph showing second messenger signaling (i.e.,
mobilization of intracellular calcium) in COS-7 cells that express
the recombinant human brain CCK-B receptor with (part A, left
panel) and without (part A, right panel) the addition of the
calcium chelator, EGTA. This is paralleled by an increased
production of inositol phosphate (part B).
[0030] FIG. 9 is a schematic representation of the seven
transmembrane (TM) domain structure of the human CCK-B/gastrin
receptor. The C-terminal domain of the third intracellular loop is
highlighted in black.
[0031] FIG. 10 is a bar graph of basal inositol phosphate
accumulation in COS-7 cells transfected with wild-type
CCK-B/gastrin receptor (WT), or with one of two constitutively
active mutants (Mut. 1, Mut.2).
[0032] FIG. 11 is a bar graph showing a functional comparison of
the CCK receptors human CCK-A (hCCK-A), human CCK-B (hCCK-B), dog
CCK-B (dCCK-B), mouse CCK-B (mCCK-B), and the mastomys CCK
receptor.
* * * * *
[0033] Recent drug development efforts have led to the discovery of
many small molecules which competitively block G-protein coupled
peptide hormone receptors. In contrast, very few non-peptide
ligands have been identified which activate this family of
receptors. Here, Applicants demonstrate that chemical modifications
of known non-peptide ligands for the CCK-B/gastrin receptor can
interconvert small molecules from antagonists to either positive
agonists or to inverse agonists. Changes in the intrinsic activity
of the ligand resulting from such modifications were detectable
because Applicants designed a screening assay which employed a
constitutively active mutant of the human CCK-B/gastrin receptor
(.sup.325L.fwdarw.E). Several peptide, `peptoid` and
benzodiazepine-based nonpeptide ligands were tested in this assay,
and evaluated for their abilities to activate the recombinant
wild-type or constitutively active mutant receptor, respectively.
Whereas full agonists had similar signaling efficacy in both
receptors when compared to the intrinsic activity of the peptide
agonist CCK-8s, the effect of ligands with lesser intrinsic
activity was logarithmically amplified by the constitutively active
mutant receptor. The prototype benzodiazepine-derived non-peptide
`antagonist` L-365,260 barely increased basal activity of the
wild-type CCK-B/gastrin receptor, but was identified as a partial
agonist using the .sup.325L.fwdarw.E mutant. Minor chemical
modification of L-365,260 resulted in compounds which were pure
antagonists (YM022), partial agonists (L-740,093 S) or inverse
agonists (L-740,093 R). The drug discovery process for novel
non-peptide agonists, including those with reverse intrinsic
activity, should be guided by using enhanced receptors, e.g.,
constitutively active mutant receptors, in the screening assay so
as to expedite identification of potential lead compounds.
[0034] I. Working Example
[0035] The following example demonstrates the usefulness of an
enhanced peptide hormone receptor to screen for non-peptide
agonists.
[0036] Using a constitutively active mutant of the human
CCK-B/gastrin receptor it was discovered that several
benzodiazepine-based putative non-peptide `antagonists` had
detectable intrinsic activity when binding to this receptor.
[0037] The constitutively active CCK-B/gastrin receptor mutant
.sup.325L.fwdarw.E was transiently overexpressed in COS-7 cells.
The fact that it was constitutively active was evident from
ligand-independent production of inositol phosphate; the wild-type
receptor, in contrast, exhibits only ligand-dependent inositol
phosphate production. Both mutant and the wild-type receptors
induced similar inositol phosphate production when maximally
stimulated with the peptide agonists CCK-8s or gastrin I (FIG. 3).
In contrast, only the mutant CCK-B/gastrin receptor allowed
detection of the different degrees of intrinsic activities of three
benzodiazepine-derived compounds, L-740,093 R, YM022 and L-365,260.
Each of these compounds were previously considered prototype
non-peptide antagonists of the wild-type CCK-B/gastrin receptor
(Castro Pineiro et al., WO 94/03437; Lotti et al., Eur. J.
Pharmacol., 162:273-280, 1989; Nashida et al., J. Pharmacol. Exp.
Ther., 270:1256-61, 1994; Nashida et al., J. Pharmacol. Exp. Ther.,
269:725-31, 1994).
[0038] The non-peptide compound L-365,260 had 62% efficacy when
compared to the full agonist CCK-8s, and was on that basis
identified as a partial agonist in the .sup.325L.fwdarw.E
constitutively active mutant receptor (FIG. 3, right section). In
fact, close re-examination of this compound's function in the
wild-type CCK-B/gastrin receptor also revealed a barely detectable,
yet significant, increase in inositol phosphate production that had
not been seen with the other non-peptide compounds.
[0039] From the above results it was concluded that minor changes
in the chemical groups attached to the benzodiazepine backbone can
result in marked alterations in intrinsic activity of small
non-peptide compounds. The stereochemistry of
benzodiazepine-derived CCK receptor ligands is another feature
which can alter binding affinity as well as receptor selectivity
(Showell et al. J. Med. Chem. 37:719-721, 1994).
[0040] The following additional observations confirmed that
differences in ligand stereochemistry determine the functional
properties of the CCK-B/gastrin receptor specific compounds. For
example, it was noted that L-740,093 S was almost a full agonist in
the .sup.325L.fwdarw.E CCK-B/gastrin receptor mutant (FIG. 3). When
tested with the human wild-type CCK-B/gastrin receptor, L-740,093 S
functions as a partial agonist (25% efficacy compared with CCK-8s).
As such, L-740,093 S is the first known non-peptide agonist for the
CCK-B/gastrin receptor. The mirror image of L-740,093 S, L-740,093
R, has properties opposite to those of the S enantiomer. L-740,093
R reduces the basal activity of the constitutively active receptor
almost to wild-type levels. To confirm the functional
classification of CCK-B/gastrin receptor non-peptide ligands, basic
pharmacologic principles were tested to determine whether they
applied to interactions between the CCK-B/gastrin receptor and the
benzodiazepine-derived agonists and antagonists. Of the compounds
tested, YM022 came closest to being a `perfect` antagonist, with
almost no intrinsic activity on either the wild-type or the
constitutively active CCK-B/gastrin receptor. In both the wild-type
and the constitutively active receptors, YM022 blocked CCK-8s
induced inositol phosphate production with almost identical
affinity, reflected by similar pA2 values (9.78 and 9.37,
respectively). Consistent with the functional classification of
L-740,093 S as a non-peptide agonist, the inositol phosphate
production induced by this compound could be blocked by YM022
(pA2=9.54; FIG. 4). YM022 was also able to attenuate the inverse
agonist activity of L-740,093 R on the constitutively active
CCK-B/gastrin receptor (FIG. 5). In a concentration-dependent
manner, YM022 partially restored basal activity to the
constitutively active receptor which had been inhibited by 20 nM
L-740,093 R. The fact that basal activity was not restored
completely is explained by the fact that YM022 itself is a weak
inverse agonist in this mutant rather than a pure receptor
antagonist.
[0041] The pA2 value measures the functional affinity of a
competitive antagonist. In contrast to IC.sub.50 values (50%
inhibitory concentration), pA.sub.2 values are independent of which
agonist concentrations are used to measure antagonist affinities.
Ideally, pA.sub.2 values should also be independent of what
specific agonist compounds are tested to assess antagonist
affinities. The pA2 value is defined as the negative logarithm of
the specific antagonist concentration which shifts the agonist
concentration-response curve by a factor of two to the right. In
other words, in the presence of a given antagonist concentration,
one would need twice as much agonist as would be required in the
absence of antagonist to induce the same effect. pA.sub.2 values of
competitive antagonists are typically assessed by Schift plots, but
can also be measured by simplified `null` methods (Lazareno et al.,
Trends in Pharmacol. Sci., 14:237-239, 1993).
[0042] In addition to non-peptide ligands, the constitutively
active mutant receptor amplified the intrinsic activity of
peptide-derived partial agonists (`peptoids`; Horwell et al., Eur.
J. Med. Chem., 30 Suppl.:537S-550S, 1995; Horwell et al., J. Med.
Chem., 34:404-14, 1991). The peptoids used in the following
experiments were derived from sequential modification of CCK-4. Two
prototype `peptoid` compounds, PD 135,158 and PD 136,450, were
converted from partial agonists in the wild-type to almost full
agonists in the constitutively active CCK-B/gastrin receptor. Thus,
peptide-derived as well as non-peptide compounds have increased
efficacy on the constitutively active versus the wild-type
CCK-B/gastrin receptor. Despite these marked alterations in
efficacy, the ratio of wild-type versus mutant receptor affinities,
as determined by .sup.125I-CCK-8 competition binding experiments,
fell within a two-fold range (Table 1A). There was no apparent
correlation between the intrinsic activity of CCK-B/gastrin
receptor ligands and potency shifts between the wild-type and the
constitutively active receptors (Table 1B).
[0043] Precedent with the constitutively active CCK-B/gastrin
receptor illustrates a new strategy using mutant receptors as a
`magnifying glass` to screen for non-peptide leads with some degree
of intrinsic activity. It should be noted that the constitutively
active .sup.325L.fwdarw.E mutant reliably predicted the intrinsic
activity that a compound would possess when stimulating the
wild-type receptor (FIG. 6). This was true over the spectrum of
peptide, `peptoid`, and non-peptide ligands tested.
[0044] The intrinsic activity (percent maximal stimulation of
inositol phosphate formation) of all compounds was tested at
concentrations that were at least 100-fold higher than the
corresponding receptor affinities.
[0045] The intrinsic activity of L-740,093 S was comparable to that
observed for the `peptoid` ligand PD 135,158, a compound that has
been recently demonstrated to be a partial agonist in vivo (Ding et
al., Gastroenterology, 109:1181-87, 1995).
1TABLE 1 A) .sup.125I CCK-8 binding affinities of tested ligands
Wild- type receptor .sup.325L .fwdarw. E Mutant Ratio Ki(nM) Ki(nM)
(Wild-type/Mutant) Compound Gastrin I 1.35 .+-. 0.28 0.80 .+-. 0.16
1.69 CCK-8s 0.12 .+-. 0.01 0.07 .+-. 0.01 1.71 PD 135,158 2.25 .+-.
0.61 1.01 .+-. 0.19 2.23 PD 136,450 0.99 .+-. 0.1 0.59 .+-. 0.12
1.68 L-740,093 R 0.19 .+-. 0.02 0.18 .+-. 0.04 1.06 YM022 0.07 .+-.
0.01 0.08 .+-. 0.01 0.88 L-364,718 150 .+-. 42 170 .+-. 34 0.88
L-365,158 7.16 .+-. 0.87 7.83 .+-. 1.51 0.91 L-740,093 S 19.5 .+-.
1.5 16 .+-. 1.4 1.22
[0046]
2 B) Signaling potencies of tested ligands Wild- type receptor
.sup.325L .fwdarw. E Mutant Ratio (Wild- IC50(nM) 95% C.I. IC50(nM)
95% C.I. type/Mutant) Compound Gastrin I 0.24 (0.12-0.48) 0.20
(0.06-0.69) 1.20 CCK-8s 0.14 (0.11-0.19) 0.15 (0.08-0.30) 0.93 PD
135,158 1.05 (0.29-3.83) 1.01 (0.21-4.80) 1.04 PD 136,450 0.36
(0.04-3.35) 0.58 (0.23-1.46) 0.62
[0047] II. Receptor Binding and Activity Assays
[0048] A. Receptor Binding Assays: The binding of a ligand to a CCK
receptor, e.g., the CCK-A or the CCK-B/gastrin receptor, can be
measured according to the following example. In this example, the
binding affinity of a ligand to the human CCK-B/gastrin receptor is
measured.
[0049] COS-7 cells (1.5.times.10.sup.6) were plated in 10-cm
culture dishes (Nunc) and grown in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum in a 5% CO.sub.2, 95% air
incubator at 37.degree. C. After an overnight incubation, cells
were transfected (Pacholczyk et al., Nature 350:350-354, 1991) with
5-7 .mu.g of a pcDNA I expression vector containing hCCKB
(HCCKB-pcDNA I). Twenty-four hours after transfection cells were
split into 24-well dishes (2.times.10.sup.4 cells/well) (Costar).
After an additional 24 hours, competition binding experiments were
performed in Hank's buffer supplemented with 25 mM
phenylmethylsulfonyl fluoride (PMSF). Twenty pM of .sup.125I CCK-8
(DuPont-New England Nuclear) was used as radioligand. Equilibrium
binding occurred after incubation for 80 min. at 37.degree. C. Cell
monolayers were then washed three times, hydrolyzed in 1 N NaOH,
and the amount of radioactivity to the receptor was quantified.
Unlabeled agonists (e.g., CCK-8s, unsulphated CCK-8 (CCK-8us),
gastrin I, CCK-4 (Peninsula)) and antagonists (L364,718 and
L365,260 (Merck)) were tested over the concentration range of 0.1
pM to 10 .mu.M. All binding experiments were repeated three to five
times.
[0050] The competition data were analyzed using computer software
which is specifically designed for the purpose of radioligand
binding assays (Inplot 4.0, GraphPad, San Diego, Calif.). Analyses
of competition and saturation binding data can also be performed
using computerized non-linear curve fitting (McPherson, G. A., J.
Pharmacol Methods, 14:213-28, 1985).
[0051] The affinities of all agonists and antagonists were
confirmed by repeating the above assay using Chinese hamster ovary
(CHO) cells stably transfected with human CCK-B/gastrin receptor
cDNA. This CHO cell line was established by transfecting a
hCCKB-pcDNAI Neo expression vector (Invitrogen) into CHO cells
using a standard lipofection protocol (Bethesda Research
Laboratories) followed by G418 selection.
[0052] Where binding parameters are determined in isolated plasma
membranes, binding can be performed, e.g., for 60 min. at
22.degree. C. (Kopin et al., Proc. Natl. Acad. Sci. USA,
89:3605-09, 1992). Separation of bound and free radioligand can be
achieved by receptor-binding filtermat filtration (Klueppelberg, U.
G., et al., 1989, Biochemistry 28:3463-8).
[0053] In order to compare the binding specificity of CCK-B/gastrin
mutant receptors of the invention with the binding specificity
typical of wild-type CCK-B/gastrin receptors see Matsumoto et al.
(Am J. Physiol, 252:G143-G147, 1987) and Lee et al. (J. Biol.
Chem., 268(11):8164-69, 1993).
[0054] Comparison of binding affinity to that of a wild-type human
CCK-B receptor: A base line value for binding of a radiolabelled
ligand to a human wild-type receptor, e.g., the human CCK-B/gastrin
receptor was determined (see Lee et al., J. Biol. Chem.,
268(11):8164-69, 1993). Agonist affinities of the human brain
CCK-B/gastrin receptor expressed in COS-7 cells were characterized
(FIG. 7). The structurally related agonists CCK-8s, gastrin I, and
CCK-4 all competed in a concentration-dependent manner for binding
of .sup.125I-CCK-8 to COS-7 cells expressing the recombinant
receptor. The calculated lC.sub.50 values for CCK-8s, gastrin I,
and CCK-4 are 0.14, 0.94, and 32 nM respectively (FIG. 7, part A).
Similar .sup.125I-CCK-8 competition curves were assessed with
L-364,718 and L-365,260 (FIG. 7, part B), and revealed IC.sub.50
values of 145 and 3.8 nM, respectively. Untransfected cells showed
no displaceable binding.
[0055] B. Receptor Signaling Activity Assays
[0056] Binding of an agonist to a CCK receptor elicits an increase
in the intracellular calcium concentration and in
phosphatidylinositol hydrolysis.
[0057] Measurement of [Ca.sup.2+]: Forty-eight hours after
transfection with hCCKB-pcDNAI, COS-7 cells were loaded with the
Ca.sup.2+ fluorophore fura-2 in modified Krebs-Ringer bicarbonate
buffer. Changes in the fluorescence emission ratios (340:380 nm)
after stimulation of cells with 10.sup.-7M CCK-8s or 10.sup.-6M
gastrin I were measured as previously described (Rajan et al.,
Diabetes, 38:874-80, 1989). Extracellular calcium can be chelated
with EGTA (2.5 mM) to confirm that a gastrin-induced increase in
[Ca.sup.2+] originates primarily from intracellular [Ca.sup.2+]
pools.
[0058] Measurement of inositol phosphate Metabolites: COS-7 cells
transfected with hCCKB-pcDNAI were cultured in inositol-free
Dulbecco's modified Eagle's medium (DMEM, GIBCO) which was
supplemented with 10 .mu.Ci/ml [.sup.3H]myo-inositol (ARC) for 24
hours prior to analysis. After 1 hour of equilibration in modified
Krebs-Ringer bicarbonate, the cells were stimulated with 10.sup.-7M
CCK-8s for 10 seconds and harvested in methanol-HCl. The aqueous
phase was extracted with chloroform, lyophilized, and analyzed for
inositol 1,4,5-triphosphate (Ins-1,3,4,5-P.sub.3) and inositol
1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P.sub.4) by strong
anion-exchange high performance liquid chromatography (Auger et
al., Cell, 57:167-75, 1989).
[0059] Comparison of signaling activity to that of a wild-type
human CCK-B receptor: A baseline level of human wild-type CCK-B
receptor second messenger signaling activity was measured in
response to CCK-8s stimulation of COS-7 cells expressing the
receptor (FIG. 8; see Lee et al., J. Biol. Chem., 268(11):8164-69,
1993). CCK-8s (10.sup.-7M) triggered a marked increase in free
cytosolic calcium, [Ca.sup.2+].sub.i (FIG. 8, part A, left panel).
There was no change in free cytosolic calcium in cells transfected
with the empty expression vector, pcDNAI. After chelation of
extracellular calcium (1.5 mM Ca.sup.2+ in the buffer) by 2.5 mM
EGTA, addition of CCK-8s (10.sup.-7 M) still transiently increased
[Ca.sup.2+], (FIG. 8, part A right panel), suggesting that the
initial peak of the CCK-induced increase in [Ca.sup.2+].sub.i
originated primarily from intracellular Ca.sup.2+ pools. The arrows
indicate the addition of CCK-8s (0.1 .mu.M) or EGTA (2.5 mM). The
pattern of [Ca.sup.2+].sub.i response suggests that the binding of
CCK-8s to the recombinant receptor triggers intracellular signaling
through activation of phospholipase C. This was confirmed by
measurement of inositol phosphate metabolites in hCCKB-pcDNA
1-transfected COS-7 cells 10 seconds after CCK-8s stimulation (FIG.
8, part B). This time point was chosen because it immediately
precedes the CCK-8-induced [Ca.sup.2+].sub.i peak. CCK-8s
(10.sup.-7M) increased the level of Ins-1,4,5-P.sub.3 by 453% over
control, unstimulated hCCKB-pcDNA 1-transfected COS-7 cells (n=3,
p<0.001). The level of Ins-1,3,4,5-P.sub.4, an immediate
metabolite of Ins-1,4,5,-P.sub.3, also increased by 186% over
control (n=3, p<0.01).
[0060] A simplified method for measuring total inositol phosphate
content: While the above method specifically assesses
Ins(1,4,5)P.sub.3 content, a simplified screening method can be
used to test for the total concentration of inositol phosphate; the
simplified method does not distinguish between specific isoforms.
(This method was used to measure inositol phosphate generation for
the experiments shown in FIGS. 3, 4, 5, 6, and 10.)
[0061] COS-7 cells transfected with receptor cDNA-pcDNAI were
cultured in inositol-free, serum-free Dulbecco's modified Eagle's
medium (DMEM, GIBCO), supplemented with 3 .mu.Ci/ml
.sup.3H-myo-inositol (NEN, 45-80Ci/mmol), for 18 hours prior to
analysis. The cells were then washed twice with DMEM/10 mM
LiCl.sub.2 and twice with phosphate-buffered saline/10 mM
LiCl.sub.2. After stimulation with putative agonists in
phosphate-buffered saline 10/mM LiCl.sub.2 for 30 minutes at
37.degree. C., cells were scraped in ice-cold methanol. Lipids were
extracted with chloroform (Pfeiffer et al., FEBS Lett.,
204:352-356, 1986). The upper phase was analyzed for inositol
phosphates by strong anion exchange chromatography, using Dowex
1-X8 columns (BIORAD) and differential elution with water/60 mM
ammonium fomate/2 M ammonium fornate. Eluted radioactivity was
measured by liquid scintillation counting, and inositol phosphate
content was expressed as a percentage of total
.sup.3H-radioactivity applied to the columns.
[0062] Further information on the second messenger pathways linked
to the native parietal cell gastrin receptor can be obtained in the
following references: Muallem, S. et al., 1984, Biochim Biophys
Acta 805:181-5; Chew, C. S. et al., 1986, Biochim Biophys Acta
888:116-25; Roche,S. et al., 1991, FEBS Letts., 282:147-51.
[0063] In addition to inositol phosphate production, second
messenger signaling activity can be measured according to, e.g.,
cAMP, cGMP, ppGpp, or calcium ion production, or using as
indicators, e.g., intracellular pH, pH-sensitive dyes, or
expression of a reporter gene, e.g., a luciferase gene, or
measuring channel activity or cell depolarization or
hyperpolarization by electrophysiological techniques.
[0064] III. Suitable Peptide Hormone Receptors with the Ability to
Amplify the Intrinsic Activity of a Non-peptide Agonist
[0065] The screening assay of the invention can be performed using
peptide hormone receptors that have a higher activity than the
corresponding human wild-type receptor. An enhanced basal activity
amplifies the intrinsic activity of ligands, and is useful for
detecting either activation of the receptor by a partial agonist,
or inhibition by an inverse agonist. Receptors that do not have an
enhanced basal activity relative to the corresponding wild-type
receptor, but still amplify the intrinsic activity of a partial
agonist, are also useful.
[0066] Examples of peptide hormone receptors that are useful for
screening non-peptide agonists include various forms of the
receptors that interact with the following peptide hormones (along
with references for their respective wild-type amino acid
sequences): amylin, angiotensin, bombesin, bradykinin, C5a
anaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP),
chemokines, cholecystokinin (CCK), endothelin, follicle stimulating
hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin
releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein
hormones, gonadotrophin-releasing hormone, leptin, luteinizing
hormone (LH), melanocortins, neuropeptide Y, neurotensin, opioid,
oxytocin, parathyroid hormone, secretin, somatostatin, tachykinins,
thrombin, thyrotrophin, thyrotrophin releasing hormone, vasoactive
intestinal polypeptide (VIP), and vasopressin. An enhanced receptor
can further embrace a single transmembrane domain peptide hormone
receptor, e.g., an insulin receptor. The wild-type amino acid
sequences of the above peptide hormone receptors is available in,
and/or referenced in, Watson and Arkinstall, The G-Protein Linked
Receptor, Academic Press, NY., 1994.
[0067] Forms of a peptide hormone receptor that are capable of
amplifying the intrinsic activity of an agonist include, but are
not limited to, the following forms of receptors:
[0068] 1. Mutant peptide hormone receptors that are capable of
amplifying the intrinsic activity of partial agonists.
[0069] An example is given of a mutant human CCK-A receptor that
enhances the intrinsic activity of the partial `peptoid` agonist PD
135,158, yet causes no apparent increase in agonist-independent
basal receptor activity, is the mutant CCK-A receptor pMHA35.
pMHA35 was made by replacing amino acids 138-ERY-140 of the human
wild-type CCK-A receptor with QRY in the vector pcDNAI. (See FIG. 2
for an illustration of the wild-type CCK-A receptor amino acid
sequence.)
[0070] 2. CCK-A receptors in which one or more of residues
.sup.138E, .sup.305L, and .sup.312I are replaced with any other
amino acid residue, e.g., a serine, aspartic acid, glutamine, or
glutamic acid residue.
[0071] 3. CCK-B/gastrin receptors in which one or more of residues
.sup.151E, .sup.325L, and .sup.332V are replaced with any other
amino acid residue, e.g., a serine, aspartic acid, glutamine, or
glutamic acid residue.
[0072] 4. Naturally-Occurring Mutant Receptors, including but not
limited to naturally-occurring constitutively active mutant
receptors, that are associated with a disease or other adverse
phenotype, e.g., a phenotype that results from a constitutively
active naturally-occurring mutant receptor. Examples include, but
are not limited to, the following peptide hormone receptors:
[0073] a) Point mutations in the luteinizing hormone (LH) receptor
gene are responsible for some incidences of precocious puberty.
Mutant receptors of the invention can be constructed by altering
the following amino acid residues of the LH receptor: the alanine
residue at position 568 to another amino acid, e.g., to a valine
(Latronico et al., J. Clin. Endo. & Meta., 80(8):2490-94,
1995); the asparagine residue at position 578 to another amino
acid, e.g., to a glycine or a tyrosine (Kosugi et al., Human Mol.
Genet., 4(2):183-88, 1995; Laue et al., Proc. Natl. Acad. Sci. USA,
92(6):1906-10, 1995); the Met residue at position 571 to another
amino acid, e.g., to an Ile, or the Thr residue at 577 to another
amino acid residue, e.g., to an Ile (Kosugi et al., supra; Laue et
al. supra); the Ile residue at position 542 to another amino acid,
the Asp residue at position 564 to another amino acid, the Cys
residue at position 581 to another amino acid, or the Asp residue
at position 578 to another amino acid (Laue et al., supra); amino
acid residues within transmembrane helices 5 or 6, e.g., in the
intracellular domain-proximal portion of transmembrane helix 6, or
in intracellular loop 3 (Laue et al. supra).
[0074] Also embraced are mutations at the corresponding residues of
the follicle stimulating hormone (FSH) receptor and the thyroid
stimulating hormone (TSH) receptor (Latronico et al., supra).
[0075] b) A naturally-occurring constitutively active parathyroid
(PTH) receptor results from a His to Arg substitution at conserved
position 223 (Schipani et al., Science, 268:98-100, 1995). A
constitutively active mutant G-LP1 receptor can be constructed by
substituting alternative amino acids at the corresponding residues
in related receptors, e.g., substituting another amino acid for the
homologue His in the glucagon-like peptide 1 (G-LP1) receptor. A
similar change in any of the receptors related to PTH or G-LP1 by
amino acid homology including, but not limited to, secretin,
vasoactive intestinal polypeptide, glucagon, G-LP1, and
calcitonin.
[0076] Non-peptide positive or inverse agonists identified in a
screening assay employing any of the above-listed naturally
occurring mutant receptors can be therapeutically useful against a
corresponding adverse phenotype.
[0077] 5. Strategies to identify synthetic mutant receptors.
[0078] Deletional analysis defines intracellular receptor domains
important in second messenger signaling: Recombinant CCK-A and
CCK-B/gastrin receptors are both coupled to phospholipase-C
activation. Applicants hypothesized that the third intracellular
loop of the CCK-B/gastrin receptor would include residues that are
important in second messenger signaling. To test this hypothesis, a
series of deletion mutations located in the third intracellular
loop, each lacking between six and 55 amino acids, were expressed
in COS-7 cells and tested for [.sup.125I]CCK-8 binding and .sup.3H
inositol phosphate formation. Deletion of a twelve amino acid
segment in the carboxy-terminal end of the third intracellular loop
resulted in normal affinity for CCK-8s, but caused a 90% reduction
of maximal inositol phosphate formation; all other receptors in
this series signaled normally. The region containing the twelve
amino acids that proved to functionally important was then screened
for constitutively active point mutations, as described below.
[0079] Strategy 1: Domain swapping with cAMP generating receptors
results in constitutive receptor activity: A method for rapidly
identifying constitutively active mutant receptors relies on
exchanging functional domains between two receptors, the domains
being, e.g., approximately 5-10 amino acids in length. One of the
two receptors is a form of the receptor which is the main template
of the desired mutant receptor, e.g., a wild-type receptor; the
second receptor is a different peptide hormone receptor from the
first. Candidate receptors are coupled to different signal
transduction pathways, e.g., a signal via a same or different
second messenger pathways, yet are closely related in their amino
acid sequence. These criteria are based on the idea that stretches
of amino acids which function normally in their native context can
confer agonist-independent signaling when transplanted into a
closely related receptor which is linked to a different
second-messenger signaling pathway.
[0080] The domain swapping strategy was used to identify
constitutively active mutants of the CCK-B/gastrin receptor. A
series of short segments in the third intracellular loop were
sequentially replaced with homologous amino acid sequence from the
vasopressin 2 receptor, which is the receptor most nearly identical
in sequence to hCCK-B. Vasopressin 2 is also a good candidate for
swapping domains with the CCK-B/gastrin receptor because it is,
different from the latter, linked to the adenylate cyclase
signaling pathway.
3 309 transmembrane domain VI 359 LT APGPGSGSRP TQAKLLAKKR
VVRMLLVIVV LFFLCWLPVY SANTWR AFD (SEQ ID NO: 8) AHVSA [MH40] (SEQ
ID NO: 9) SA [MH128] (SEQ ID NO: 10) S [MH156] (SEQ ID NO: 11) E
[MH162] (SEQ ID NO: 12)
[0081] When tested, a five amino acid substitution (QAKLL (SEQ ID
NO: 13) to AHVSA (SEQ ID NO: 14)) into the homologous position of
the CCK-B/gastrin receptor resulted in constitutive activity of the
CCK-B/gastrin receptor. The QAKLL (SEQ ID NO: 13) to AHVSA (SEQ ID
NO: 14) substitution caused an increased level of basal inositol
phosphate formation to 290% of the wild-type CCK-B/gastrin receptor
(FIG. 9, Mutant 2). In addition, mutations causing constitutive
activity include replacement of LL to SA, L to S, and L to E.
[0082] Strategy 2: Glutamic acid scanning mutagenesis identifies
constitutively active receptors: In addition to, or as a substitute
for, deletion analysis or domain swapping, mutant receptors can be
made using a process Applicants have named `amino acid scanning
mutagenesis.` Amino acid scanning mutagenesis involves sequentially
replacing each amino acid found in either an intracellular loop or
in the half of the transmembrane domain flanking the intracellular
portion of the receptor. An experimental option is to change the
charge of the amino acid, e.g., to exchange a negative for a
positive amino acid, a positive for a negative amino acid, or a
positive or negative amino acid for a neutral amino acid. Another
option would be to exchange each amino acid, e.g., each neutral
amino acid, with another neutral amino acid.
[0083] In the case of the CCK-B/gastrin receptor, deletion analysis
was initially used to define a functionally important twelve amino
acid segment within the third intracellular loop which was
important for second messenger signaling. Subsequently, each of the
neutral amino acids within the 12 this segment was replaced
sequentially with another amino acid, preferably with glutamic
acid. The scanning analysis technique revealed that one of the
glutamic acid substitutions caused a 228% increase in the
basal-level of inositol phosphate accumulation, relative to the
wild-type value, in transiently transfected COS-7 cells (FIG. 9,
Mutant 1).
[0084] In this example, applicants focused on the region limited to
the carboxy end of the third intracellular (IC) loop and the
portion of the sixth transmembrane domain which flanks the third IC
loop. Glutamic acid residues (E) were introduced in place of
neutral amino acid residues.
4 309 359 LT APGPGSGSRP TQAKLLAKKR VVRMLLVIVV LFFLCWLPVY SANTWR AFD
(SEQ ID NO: 15) .vertline. .vertline..vertline.
.vertline..vertline. E E E EE (SEQ ID NO: 16)
[0085] Constitutively active receptors include an amino acid
replacement of .sup.323A.fwdarw.E (MH31, SEQ ID NO: 17),
.sup.324K.fwdarw.E (MH131, SEQ ID NO:18), .sup.325L.fwdarw.E
(MH162, SEQ ID NO: 19), .sup.327A.fwdarw.E (MH13, SEQ ID NO: 20),
.sup.331V.fwdarw.E (MH130, SEQ ID NO: 21), .sup.332V.fwdarw.E
(MH129, SEQ ID NO: 22), and .sup.331VV.fwdarw.EE (MH72),
respectively, all in pcDNAI vectors, as described above.
[0086] FIG. 9 is a schematic representation of the seven
transmembrane (TM) domain structure of the human CCK-B/gastrin
receptor. The C-terminal domain of the third intracellular loop,
which is crucial for intracellular signaling, is highlighted in
black. Within this segment, two mutations were found to confer
constitutive activity on the receptor. One of the mutations was
constructed by glutamic acid substitution scanning (Mutant 1;
MH129, SEQ ID NO: 22); a second mutation was constructed by domain
swapping (Mutant 2; MHl 62, SEQ ID NO: 19). A bar graph showing the
basal inositol phosphate accumulation in COS-7 cells, which had
been transfected with the wild-type CCK-B/gastrin receptor or with
two different constitutively active mutants, is shown in FIG.
10.
[0087] Strategy 3: A third method for making a mutant receptor is
to align the receptor of interest with a known constitutively
active mutant receptor, including, but not limited to, peptide
hormone, biogenic amine, rhodopsin, or other G-protein coupled
receptors. An example of such an alignment is shown in FIG. 2.
Generally, mutations which result in constitutive activity in the
known mutant can be introduced into the corresponding position of
the receptor of interest. Examples of known constitutively active
mutant receptors include, but are not limited to, the follicle
stimulating hormone (FSH) receptor, the thyroid stimulating hormone
(TSH) receptor, and the luteinizing hormone receptor, e.g., a 568
Ala to Val mutation in the LH receptor (Latronico et al., J. Clin.
Endo. & Meta., 80(8):2490-94, 1995).
[0088] This method, based on alignment, was employed to construct a
CCK-A mutant receptor. A multiple alignment map was made which
included the human and rat CCK-A sequences, the mastomys, rat,
human, and canine CCK-B/gastrin receptor, and a Xenopus CCK-A/CCK-B
intermediate receptor (CCK-XL; FIG. 2). Based on this map,
conserved amino acids 138-ERY-140 of the CCK-A receptor were
replaced with amino acids QRY, based on a known constitutively
active rhodopsin mutant with enhanced transducin activation (Arnis
et al., J. Biol. Chem., 269:23879-81, 1994). The altered amino acid
residues are positioned in transmembrane domain III and flank the
second intracellular loop. Although the basal level of signaling
was not increased, the intrinsic activity of the non-peptide ligand
PD 135,158 was significantly increased.
[0089] Strategy 4: Additional mutant receptors can be made by
sequentially deleting intracellular portions of the receptor, and
looking for an increase in basal activity, or for overactivity of a
partial agonist, relative to the wild-type receptor.
[0090] Wild-type Receptors with Enhanced Basal Activity: Peptide
hormone receptors useful in the method of the invention can include
non-human receptors which have the ability to amplify the intrinsic
activity of non-peptide agonist than does the corresponding human
wild-type receptor, or which have a higher basal level of activity
than does the human wild-type receptor.
[0091] In FIG. 11, basal levels of inositol phosphate production
were measured for human CCK-A (hCCK-A), human CCK-B (hCCK-B), dog
CCK-B (dCCK-B), mouse CCK-B (mCCK-B), and the mastomys CCK receptor
(FIG. 11, part A), and expressed relative to the basal level of
hCCK-B.
[0092] Single experiments were also performed for the rat
CCK-B/gastrin receptor and for the related Xenopus CCK receptor
(Table 2). The human .sup.325L to E mutant served as a positive
control (n=-14).
5 TABLE 2 basal (% of CCK-8s stimulated receptor human basal) (%
human basal) rat CCK-A 77 684 Xenopus CCK 74 442 .sup.325L to E
CCKA 231 .+-. 7 771 .+-. 36
[0093] The wild-type human CCK-A and CCK-B/gastrin receptors
induced only insignificant changes of basal inositol phosphate
production in COS-7 cells (as compared to control cells transfected
with the empty plasmid vector, pcDNAI). Similarly, the wild-type
rat CCK-A and canine CCK-B/gastrin receptors, as well as the
closely related Xenopus CCK receptor all appeared more or less
functionally silent in the basal state. In contrast, the wild type
mouse CCK-B/gastrin receptor and its homologue from mastomys
natalensis significantly increased basal inositol phosphate
production in COS-7 cells over pcDNAI controls. When compared with
the slight basal activity of the wild type human CCK-B/gastrin
receptor, it was estimated that the basal activities of the wild
type mouse and mastomys homologues were 7- and 11-fold higher,
respectively. For comparison, the .sup.325L-E mutant of the human
CCK-B/gastrin receptor appeared to be at least 16-fold more active
than the human wild type receptor in its basal state. It should be
noted that the described species differences in basal activities
were clearly not related to different degrees of receptor
expression, since the maximal response to stimulation with CCK-8s
was comparable for all tested receptors (positive control).
[0094] IV. Therapeutic Use
[0095] The ability to pharmacologically modulate wild-type or
constitutively active receptor activity opens the door for a new
class of clinically useful drugs. Enhanced receptors will enable
the discovery of novel drugs directed at a broad spectrum of
diseases. Constitutively active mutants of the thyrotropin,
luteinizing hormone, and parathyroid hormone receptors are already
known to occur in nature (see above) and might provide a starting
point for non-peptide agonist/inverse agonist screening. For
example, drugs which silence constitutively active thyroid
stimulating hormone receptors, which are implicated in the etiology
of thyroid adenomas, could be used to inhibit tumor growth.
Similarly, in patients with constitutively active luteinizing
hormone receptors, inverse agonists could delay the onset of
precocious puberty.
[0096] Further information on peptide hormone receptor amino acid
sequences, receptor-specific agonists and antagonists, receptor
conformation, pharmacology, receptor-encoding genes, animal models
for subsequent follow-up studies, and database accession numbers
can be obtained from: Watson and Arkinstall, The G-Protein Linked
Receptor, Academic Press, NY., 1994; see also, Kolakowski, L. F.,
"The G Protein-Coupled Receptor Database", World-Wide-Web Site,
GCRDB-WWW.
OTHER EMBODIMENTS
[0097] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
[0098] Other embodiments are within the following claims.
Sequence CWU 1
1
* * * * *