WO2004037985A2

WO2004037985A2 - Modulation of g protein coupled receptor activity

Info

Publication number: WO2004037985A2
Application number: PCT/US2003/033480
Authority: WO
Inventors: Lennart Olsson; Tatjana Naranda; Stephen D. Yanofsky
Original assignee: Receptron, Inc.
Priority date: 2002-10-21
Filing date: 2003-10-21
Publication date: 2004-05-06
Also published as: AU2003277460A1; AU2003277460A8; WO2004037985A3

Abstract

Methods and reagents for modulating the activity of G protein coupled cell surface receptors are provided. Receptor activity modulation occurs when exogenous bioactive compounds are contacted with the receptor activation domain. The modulation can be an increase in receptor activity or a decrease in ligand-induced receptor activity. Also provided are methods for identifying and screening for bioactive compounds.

Description

PATENT APPLICATION MODULATION OF G PROTEIN COUPLED RECEPTOR ACTIVITY

FIELD OF THE INVENTION [0001] The present invention relates to methods and reagents for modulating cell surface receptor activity. The invention also relates to assays for identification of agents that modulate receptor activity and compositions containing such agents. The invention finds application in the fields of biology and medicine.

BACKGROUND OF THE INVENTION [0002] Signaling molecules such as neurotransmitters, protein hormones, cytokines and growth factors bind to specific receptors on the surface ofthe target cells they influence. These signaling molecules (ligands) bind the cell surface receptors, resulting in one or more intracellular signals that alter the behavior of target cells.

[0003] A number of medical disorders arise from defects in receptor signaling. For example, medical disorders may result from ligand deficient states (ligand is not present or is present at a level inadequate to stimulate a receptor response) and ligand resistance (receptor does not bind the ligand or is not activated by ligand binding). Approaches to treating disorders resulting from inadequate receptor signaling include administration of exogenous ligand to replace or supplement endogenous levels. For example, calcitonin (ligand for calcitonin receptor) can be administered for treatment of hypercalcemia. However, administration of such therapeutic ligands to patients has a number of disadvantages, including expense and inconvenience for the patient.

[0004] In other cases, activation of a receptor has undesired effects. For example, activation of receptors by proinflammatory cytokines play key roles in several disease conditions. In these and similar cases, it may be desirable to antagonize the effects of these ligands. [0005] Thus, methods for modulating the activity of receptors and for identifying receptor- modulating agents will be of significant medical benefit.

SUMMARY OF THE INVENTION [0006] In one aspect, the invention provides a method for modulating the activity of a G protein coupled cell surface receptor by contacting the receptor with an agent that binds the receptor activation domain and modulates receptor activity. A method for modulating the activity of cell surface G protein coupled receptor (GPCR) by contacting the receptor with an exogenous compound that binds in the activation domain. In an embodiment, the exogenous compound is a peptide. In embodiments, the G protein coupled receptor is a glucogon-like- peptide-1 receptor, a calcitonin receptor or a prostaglandin E2 receptor. In embodiments, the G protein coupled receptor is human. In a related aspect, methods for screening for exogenous compounds that module the activity of a G protein coupled cell surface receptor are provided. [0007] In one aspect, the invention provides a method for modulating the activity ofthe calcitonin receptor (CTR2-receptor or CTR2-R). In another aspect, the invention provides a method for modulating the activity ofthe glucagon-like peptide-1 receptor (GLP-1 receptor or GLP-1R). In another aspect, the invention provides a method for modulating the activity ofthe prostaglandin E2 receptor (EP2-receptor or EP2-R).

[0008] In an aspect, the invention provides a method for modulating the activity of calcitonin receptor by contacting the receptor with an exogenous compound that binds in the activation domain, where the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:2. In an embodiment, the effect ofthe exogenous compound is an increase in receptor activity. In an embodiment, the exogenous compound can increase receptor activity in the absence of ligand. In an embodiment, the effect ofthe exogenous compound is a decrease in receptor activity. In an embodiment, the contacting takes place in vitro. In an embodiment, the contacting occurs in the presence of calcitonin. In an embodiment, the exogenous compound is an oligopeptide. In an embodiment, the oligopeptide has at least about 8 contiguous residues of a sequence substantially similar to, or identical to, SEQ ID NO:2. In an embodiment, the calcitonin receptor is human.

[0009] In an aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a calcitomn receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:2. In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a calcitonin receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO:2 by a) contacting the receptor with a candidate agent and an activation- domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:2; and b) determining the binding ofthe agent or the oligopeptide to the activation domain, where the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity. In an embodiment, the candidate agent or oligopeptide is labeled. [0010] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a calcitonin receptor by identifying a compound that binds in a receptor activation domain of sequence SEQ ID NO:2, and assaying the ability ofthe compound to modulate activity ofthe cell surface receptor. In an embodiment, the ability ofthe agent to increase or stimulate receptor activation is assayed. In an embodiment, the ability ofthe agent to decrease or inhibit receptor activation is assayed.

[0011] In an aspect, the invention provides an isolated oligopeptide that binds the calcitonin receptor activation domain. In an embodiment, the oligopeptide comprises at least 8 contiguous amino acids of SEQ ID NO:2. In an embodiment, the oligopeptide increases activation of a calcitonin receptor. In an embodiment, the oligopeptide decreases activation of a calcitonin receptor. The invention also provides a pharmaceutical composition comprising an aforementioned oligopeptide in a sterile form and a pharmaceutically acceptable excipient. [0012] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of calcitonin receptor activation by administering to a patient an exogenous bioactive compound that binds the calcitonin receptor activation domain and which increases receptor activity when contacted with the receptor. In a different embodiment, the exogenous bioactive compound decreases receptor activity. In an aspect, the invention provides a method for treating a condition in a patient characterized by a deficiency of calcitonin by administering to a patient an exogenous bioactive compound that binds in a receptor activation domain and which increases receptor activity.

[0013] In an aspect, the invention provides a method for modulating the activity of GLP-1 receptor by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:l. In an embodiment, the effect ofthe exogenous compound is an increase in receptor activity. In an embodiment, the exogenous compound can increase receptor activity in the absence of ligand. In an embodiment, the effect ofthe exogenous compound is a decrease in receptor activity. In an embodiment, the contacting takes place in vitro. In an embodiment, the contacting occurs in the presence of GLP-1. In an embodiment, the exogenous compound is an oligopeptide. In an embodiment, the oligopeptide has at least about 8 contiguous residues of a sequence substantially similar to, or identical to, SEQ ID NO:l. In an embodiment, the GLP-1 receptor is human. [0014] In an aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:l. [0015] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO:l by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:l; and b) determining the binding ofthe agent or the oligopeptide to the activation domain, wherein the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity. In an embodiment, the method includes the step of contacting the receptor with receptor ligand. In an embodiment, the candidate agent or oligopeptide is labeled.

[0016] In related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor by identifying a compound that binds in a receptor activation domain having the sequence SEQ ID NO:l and assaying the ability ofthe compound to modulate activity ofthe cell surface receptor. In an embodiment, the ability ofthe agent to increase receptor activation is assayed. In an embodiment, the ability ofthe agent to decrease receptor activation is assayed.

[0017] In an aspect, the invention provides an isolated oligopeptide that binds the GLP-1 receptor activation domain. In an embodiment, the oligopeptide comprises at least 8 contiguous amino acids of SEQ ID NO: 1. In an embodiment, the oligopeptide increases activation of a GLP-1 receptor. In an embodiment, the oligopeptide decreases receptor activation. [0018] In an aspect, the invention provides a pharmaceutical composition comprising an aforementioned oligopeptide in a sterile form and a pharmaceutically acceptable excipient. [0019] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level (e.g., deficiency) of GLP-1 receptor activation by administering to a patient an exogenous bioactive compound that binds in the GLP-1 receptor activation domain and which increases receptor activity.

[0020] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of GLP-1 receptor activation by administering to a patient an exogenous bioactive compound that binds in the GLP receptor activation domain and which decreases receptor activity when contacted with the receptor. [0021] In an aspect, the invention provides a method for modulating the activity of prostaglandin E2 receptor (EP2-R) by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:3. In an embodiment, the EP2-R receptor is human. In an embodiment, the effect ofthe exogenous compound is an increase in receptor activity. In an embodiment, the exogenous compound can increase receptor activity in the absence of ligand. In an embodiment, the effect ofthe exogenous compound is a decrease in receptor activity. In an embodiment, the contacting takes place in vitro. In an embodiment, the contacting occurs in the presence of prostaglandin E2. In an embodiment, the exogenous compound is an oligopeptide, e.g., an oligopeptide that comprises at least about 8 contiguous residues of a sequence substantially similar to, or identical to, SEQ ID NO:3.

[0022] In an aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of an EP2-R receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:3. [0023] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of an EP2-R receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO: 3 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:3; and b) determining the binding ofthe agent or the oligopeptide to the activation domain, wherein the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity. In an embodiment, the method includes the step of contacting the receptor with receptor ligand. In an embodiment, the candidate agent or oligopeptide is labeled.

[0024] In related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of an EP2-R receptor by identifying a compound that binds in a receptor activation domain having the sequence SEQ ID NO: 3 and assaying the ability ofthe compound to modulate activity ofthe cell surface receptor. In an embodiment, the ability ofthe agent to increase activation is assayed. In a different embodiment, the ability ofthe agent to decrease activation is assayed.

[0025] In an aspect, the invention provides an isolated oligopeptide that binds the EP2-R receptor activation domain. In an embodiment, the oligopeptide comprises at least 8 contiguous amino acids of SEQ ID NO:3. In an embodiment, the oligopeptide is an agonist ofthe EP2-R receptor. In a different embodiment, the oligopeptide is an antagonist of ligand-induced EP2-R receptor-activation.

[0026] In an aspect, the invention provides a pharmaceutical composition comprising an aforementioned oligopeptide in a sterile form and a pharmaceutically acceptable excipient. [0027] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of EP2-R receptor activation by administering to a patient an exogenous bioactive compound that binds in the EP2 receptor activation domain and which increase receptor activity when contacted with the receptor.

[0028] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of EP2-R receptor activation by administering to a patient an exogenous bioactive compound that binds in the EP2-R receptor activation domain and which decreases receptor activity.

BRIEF DESCRIPTION OF THE DRAWINGS [0029] Figure 1 shows the stimulatory effect of GLP-1 receptor derived peptide on insulin secretion in the glucose tolerance test.

[0030] Figure 2 shows the stimulatory effect of GLP-1 receptor derived peptide on cAMP production in recombinant COS-7 cells expressing the GLP-1 receptor. [0031] Figure 3 shows the stimulatory effect of GLP-1 receptor derived peptide on cAMP production in recombinant HEK cells expressing the GLP-1 receptor. Left panel: cAMP response to GLP-1. Right panel: cAMP response to GLP-1R peptide.

[0032] Figure 4 shows that GLP-1 and GLP-1 receptor derived peptide act synergistically to stimulate receptor activity in recombinant HEK cells expressing the GLP-1 receptor. "Pep" or "P" refer to Left panel: cAMP response to GLP-1. Right panel: cAMP response to the GLP-1 receptor derived peptide and "G" refers to GLP-1.

[0033] Figure 5 A depicts the stimulatory response of salmon calcitonin (natural ligand) on cAMP production in T47-D cells.

[0034] Figure 5B shows the agonist effect of calcitonin receptor derived peptide (CTRp) on cAMP production in T47-D cells.

[0035] Figure 6 demonstrates the specificity of CTRp for the calcitonin receptor. SK-N- BE(2) cells that produce cAMP in the presence of prostaglandin E2, did not produce cAMP when exposed to CTRp. [0036] Figure 7 shows prostaglandin E2 induction of cAMP in SK-N-BE(2) cells. [0037] Figure 8 shows inhibition of prostaglandin E2 induction of cAMP by receptor derived peptide EP2p.

DETAILED DESCRIPTION OF THE INVENTION [0038] In one aspect ofthe invention, methods and compositions for modulating activity of a G protein coupled cell surface receptor are provided, as well as methods for identification of agents that modulate cell surface receptor activity. As used herein, "modulation" can be an increase in receptor activation or a decrease in ligand-induced receptor activation. An increase in receptor activation can occur in the presence of a receptor ligand or in the absence of a receptor ligand. The modulatory agents, sometimes referred to herein as "exogenous bioactive compounds" are compounds that bind an extracellular portion ofthe cell surface receptor, termed the "activation domain."

[0039] Activation domains are described generally in U.S. patent no. 6,333,031, which is incorporated herein by reference. The activation domain of a receptor occupies a site distinct from the ligand binding site. As used herein the "ligand binding site" refers to the region of a receptor that binds the natural ligand ofthe receptor (and may also bind homologs or analogs of the natural ligand). Thus, the exogenous bioactive compounds used in the practice ofthe present invention bind an extracellular region ofthe receptor that is not directly involved in binding of the natural ligand to the receptor, i.e., a region that does not normally make contact with the ligand. Further, binding to the activation domain ofthe receptor usually does not substantially change the KD ofthe binding ofthe natural receptor ligand to the ligand binding site. As used herein, "ligand" refers to compounds that bind the ligand binding site and activate the receptor. It has been demonstrated that both antagonistic and agonistic effects can be mediated by the same receptor activation domain, depending on the specific exogenous compound bound to the site. Without intending to be bound by a particular mechanism, it is likely that the specific interaction ofthe exogenous compound with the residues ofthe activation domain determine whether binding results in activation or inhibition. Thus, similar screening assays can be used to identify both agonists and antagonists of receptor activity.

[0040] Table 1, infra, provides the sequences of activation domains corresponding to the receptors for GLP-1, calcitonin and prostaglandin E2. The activation domain sequences were identified generally according to the methods described in U.S. pat. no. 6,333,031 and by cross- species sequence comparisons between human receptors and nonhuman homologs. The receptors listed in Table 1 are G Protein Coupled (seven-transmembrane segment) Receptors (GPCRs or 7TM receptors). Stimulation of these receptors leads to signal transduction through G protein activation leading to production of c AMP. G Protein Coupled Receptors are described in Trettel et al., 2003, JBiol Chem. 278:40980; Rios et al, 2001, Pharmacol Ther..92:71; Dean et al., 2001, JMed Chem. 44:4595.

TABLE 1

[0041] The gene and amino acid sequences, activities and other characteristics of human GPCRs receptors and homologs from other animals are well known in the art. Sequences of GPCRs are found in the scientific literature and databases, or can be determined by rountine methods. For example, the GLP-1, calcitonin and prostaglandin E2 receptors are described in scientific literature (see, e.g., Gorn et al., 1992, "Cloning, characterization, and expression of a Human Calcitonin Receptor from an Ovarian Carcinoma cell line" J Clin. Invest. 90:1726-1735; Rolf et al., 1994, "Cloning and characterization of an abundant subtype ofthe Human Calcitonin Receptor" Molecular Pharmacology 46, 246-255; Graziano et al., 1993, "Cloning and functional expression of a human glucagons-like peptide-1 receptor" Biophys Res Commun. 196:141-146; Dillon et al., 1993, "Cloning and functional expression ofthe human glucagon- like peptide-1 (GLP-1) receptor." Endocrinology 133:1907-10; Songzhu et al., 1993, "Cloning and expression ofthe EP2 subtype of human receptors for prostaglandin E2" Biochem. And Biophys. Res. Comm. 197:263-270) and in databases, e.g. a human GLP-1 receptor is described as SwissProt accession number P43220, a human calcitonin receptor is described as SwissProt accession number P30988, and a human Prostaglandin EP2 receptor is described as SwissProt accession number P43116.

[0042] The receptors used in methods ofthe invention generally are mammalian, e.g., human, primate (including nonhuman primates), rodent (mice, rats, hamsters, guinea pigs), cow, sheep, pig, horse, and others. Receptors that may be used include those with the complete sequence of a naturally occurring receptor (including naturally occurring alleles and variants, e.g., naturally occurring mammalian or human alleles) as well as recombinantly expressed variants, and portions of receptors (e.g., a receptor extracellular domain, or an activation-domain containing fragment comprising at least about 50 or at least about 100 amino acid residues). Suitable receptors for use in the methods ofthe invention include isolated receptor proteins and activation domain-containing fragments (e.g., for use in binding assays). Suitable receptors also include receptors expressed on the surface of cells (e.g., often for activity assays). Suitable cells include those that normally express GLP-1 receptors (e.g., without limitation HIT-T15, PANC- 1, and PC12) and/or calcitonin receptors (e.g., without limitation human mammary carcinoma cell line T47-D) and/or EP2 receptors (e.g., without limitation SK-N-BE(2), a human neuroblastoma cell line) as well as cells in which the receptors are recombinantly expressed. It will be understood that cells that express endogenous receptor can also be engineered to recombinantly express (e.g., overexpress) the same or a different receptor. Methods for recombinant expression of polypeptides are well known in the art. See, e.g., Sambrook and Russel, 2001, Molecular Cloning: A Laboratory Manual, third edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The polynucleotide sequences of GLP-1, calcitonin and prostaglandin E2 receptors are also known (e.g., sequences for human receptors) or easily determinable. For expression, typically, polynucleotides encoding the receptor are used in expression vectors containing include transcriptional and/or translational control signals (e.g., transcriptional regulatory element, promoter, ribosome-binding site, and ATG initiation codon). DNA encoding the receptor or receptor fragment is inserted into DNA constructs capable of introduction into and expression in an in vitro host cell, such as a bacterial (e.g., E. coli, Bacillus subtilus), yeast (e.g., Saccharomyces), insect (e.g., Spodoptera frugiperda), or mammalian cell systems. Numerous examples of mammalian cell culture systems are known (e.g., HEK cells, CHO cells, BaF3 cells). Useful human and nonhuman cell lines are widely available, e.g., from the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108.

[0043] In one aspect, the invention provides a method for modulating the activity of a GLP- 1 cell surface receptor, calcitonin cell surface receptor, or prostaglandin E2 receptor, respectively, by contacting the receptor with an exogenous bioactive compound that binds in the activation domain ofthe receptor, where the activation domain has a sequence identical or substantially similar to SEQ. ID. NO: 1 , 2, or 3, respectively, and where the binding results in a response in the signaling pathway ofthe receptor, i.e., modulates receptor activity. As noted, modulation of receptor activity can include an increase in receptor activation, referred to as agonist activity. One type of agonist activity is synergistic activity, in which receptor activity is stimulated by contact with both the receptor ligand and the exogenous bioactive compound that binds in the activation domain, and in which stimulation is additive or is synergistic. As noted, certain activation-domain binding compounds ofthe invention can activate the receptor in the absence of any ligand. Conversely, modulation of receptor activity can include inhibition of receptor activation, or antagonist activity. Thus, by "antagonist" herein it is meant compounds that bind the receptor activation site but do not activate the receptor, and for which ligand- induced receptor activity is inhibited or blocked. By "binding in the activation domain" is meant that the exogenous compound binds the receptor through interaction with some, but not necessarily all, amino acid residues in the activation domain.

[0044] Also shown in Table 1 are exemplary receptor derived peptide sequences (exemplary RDPSs). As is discussed below, the exemplary RDPSs, their homologs, fragments, and variants, are used to modulate activity of corresponding receptors and to identify still other modulatory agents. The RDPSs shown in Table 1 are "GLP-lp" having the sequence set forth as SEQ ID NO:l, "CTRp " having the sequence set forth as SEQ ID NO:2, and "EP2p" having the sequence set forth as SEQ ID NO:3.

[0045] For clarity, each receptor activation sequence and each exemplary receptor derived peptide sequence (RDPS) listed in Table 1 is referred to as "corresponding to," or being the "cognate" of, the receptor from which its sequence is derived. In Table 1, cognate pairs of receptors and activation/peptide sequences are presented in the same row. Thus, for example, (i) the GLP-1 receptor activation sequence (SEQ ID NO: 1) and (ii) the GLP-1 receptor are a cognate pair. Similarly, (i) a receptor derived peptide that modulates the activity ofthe GLP-1 receptor and which has sequence similarity to SEQ ID NO: 1, and (ii) the GLP-1 receptor are a cognate pair.

Exogenous Bioactive Compounds

[0046] As noted above, receptor modulatory agents are sometimes referred to herein as "exogenous bioactive compounds." Specifically, "exogenous bioactive compound" as used herein, refers to a compound that (1) is not produced endogenously by the cell or organism, i.e., it is artificially introduced to the cell or organism; (2) binds amino acid residues in the activation site of a receptor (e.g., as determined by competition assays); and (3) modulates receptor activity. Exogenous bioactive compounds include oligopeptides described in detail herein, as well as other compounds described herein. Further, exemplary assays for identifying bioactive compounds are described hereinbelow. In an aspect, the invention provides a complex comprising a receptor activation domain and an exogenous bioactive compound (e.g., oliogpeptide) described herein.

[0047] A variety of different classes of molecules can act as exogenous bioactive compounds, including oligopeptides, other biomolecules (e.g., saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs) and small organic compounds (e.g., having a molecular weight of more that about 50 and less than about 10,000 daltons, often more than about 100 and less than about 2,500 daltons). Usually candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and often at least two ofthe functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more ofthe above functional groups. In one embodiment, the exogenous bioactive compound is other than a protein or peptide.

[0048] In one embodiment, the exogenous bioactive compound is an oligopeptide. As used herein, "oligopeptide" is used interchangeably with "peptide" and "polypeptide" and refers to a polymer of amino acids. In an embodiment, the oligopeptide comprises a sequence identical or substantially similar to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3 (Table 1), or to an activation domain-binding subsequence thereof. For example, the oligopeptide compound that binds an activation domain and modulates receptor activity may comprise at least 8 contiguous residues of a sequence in Table 1, and often at least 10, at least 12, at least 15, or at least 20 residues.

[0049] As used herein, the term "substantially similar" refers to oligopeptide sequences that may be identical to the receptor activation domain (e.g., SEQ ID NO:l, 2, or 3 or that may have a degree of similarity to the receptor activation domain (e.g. SEQ ID NO: NO:l, 2, or 3) sufficient to allow binding ofthe oligopeptide to its cognate receptor activation domain resulting in modulation of receptor activity.

[0050] In some embodiments, the length ofthe peptide is fewer than about 60 amino acids, more usually fewer than about 40 amino acids, more usually fewer than 30 amino acids. [0051] An oligopeptide that is substantially similar to a receptor activation domain and which modulates activity of a receptor may differ from the receptor activation domain by amino acid substitutions, insertions, or deletions as compared to the activation domain. For example, the oligopeptide substantially similar to SEQ. ID. NO:l, 2, or 3 may include additional residues, e.g., at the amino - or carboxy terminus of SEQ. ID. NO:l, 2 or 3, or an activation domain- binding subsequence thereof. In embodiments, the peptide will contain least 8 amino acids, at least about 12 amino acids, at least about 15 amino acids, at least about 18 amino, at least about 21 amino acids, or at least about 24 amino acids identical to a sequence of SEQ. ID. NO:l, 2, or 3. In some embodiments, a substantially similar oligopeptide will have an amino acid sequence at least about 60% identical to one of SEQ. ID. NO:l, 2, or 3, at least about 70% identical, often at least about 80% identical, sometimes at least about 90% identical. Sequence identity between two peptide sequences (e.g., SEQ ID NO: 1 and a substantially similar sequence) can be easily determined by inspection. Alternatively, algorithms such as the Best Fit sequence program described by Devereux et al, 1984, Nucl. Acid Res. 12:387-95, can be used (typically using default settings).

[0052] As noted, peptides suitable for use as exogenous bioactive compounds ofthe invention may have amino acid substitutions, insertions, or deletions as compared to sequences of SEQ. ID. NO:l, 2, or 3. In one embodiment, amino acid substitutions are made. In one embodiment the number of changes will not be more than about 30%, sometimes not more than about 20%, sometimes not more than about 10%, ofthe number of amino acids in the activation domain, although in some instances higher numbers of alterations may be made. In one embodiment, with reference to the RDPSs shown in Table 1, not more than about five, alternatively not more than about three substitutions or deletions will be made. In general, it is preferable that residues critical for biological activity are either not altered or are conservatively altered (e.g., to conserve charge). Examples of conservative alterations include (but are not limited to) the substitutions shown in Table 2. Critical residues may be elucidated using known mutagenesis techniques followed by activity or binding assays, e.g., using scanning mutagenesis techniques, wherein single amino acid residues within the oligopeptide are modified by substitution with an aliphatic amino acid, e.g., serine, alanine, glycine, valine, and the like.

TABLE 2

Original Residue Exemplary Substitutions

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp Gly Pro His Asn, Gin He Leu, Nal Leu He, Val Lys Arg, His Met Leu, He Phe Met, Leu, Tyr Ser Thr Thr Ser Tip Tyr Tyr Trp, Phe Val He, Leu

[0053] Oligopeptide exogenous bioactive compounds can contain naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0054] In one embodiment modifications that are made do not substantially alter the biological activity ofthe oligopeptide, i.e., the modification does not prevent binding ofthe oligopeptide to its cognate receptor and does not destroy the modulatory activity. [0055] Alternatively, variants in which biological function has been modified can be selected for. The substitutions which in general are expected to produce the greatest changes in oligopeptide properties are those in which a nonconservative substitution is made in a critical residue, e.g., (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. [0056] Oligopeptides can be made by chemical synthesis, through recombinant means, or any other synthesis method. Usually, the oligopeptides are prepared in accordance with conventional techniques, such as synthesis (for example, use of a Beckman Model 990 peptide synthesizer or other commercial synthesizer). Peptides may be produced directly by recombinant methods (see Sambrook et al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y., 1989) or as a fusion protein, for example, to a protein that is one of a specific binding pair, allowing purification ofthe fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at a site engineered to yield the desired peptide (see for example Driscoll et al. (1993) J. Mol. Bio. 232:342-350). [0057] In addition to modifications within the peptides, they may also contain additional sequences. For example, the oligopeptides may be extended to: 1) provide convenient linking sites, e.g., cysteine or lysine; 2) to enhance stability; 3) to provide for ease of purification, e.g., epitope or purification tags (e.g., His₆); 4) to alter the physical characteristics, e.g., solubility, charge, etc.; 5) to add residues (e.g., tyrosine) to facilitate radioactive or other labeling; or 6) to stabilize the conformation. The oligopeptides may be joined to non- wild-type flanking regions as fused proteins, joined either by linking groups or covalently linked through cysteine (disulfide) or peptide linkages. The oligopeptide may be linked through a variety of bifunctional agents, such as maleimidobenzoic acid, methyidithioacetic acid, mercaptobenzoic acid, S- pyridyl dithiopropionate, and the like. The oligopeptides may be joined to a single amino acid at the N- or C-terminus of a chain of amino acids, or may be internally joined. For example, the subject peptides may be covalently linked to an immunogenic protein, such as keyhole limpet hemocyanin, ovalbumin, and the like, to facilitate antibody production to the subject oligopeptides.

[0058] As noted, the oligopeptides may be shorter than those depicted in Table 1, i.e., residues from either the N- or C-terminus ofthe oligopeptide may be deleted with the retention of biological activity, preferably full biological activity. In some cases, internal residues may be removed from the oligopeptide. Generally, this will be done by sequentially removing residues and assaying for the ability to bind to the activation domain of a receptor. Once binding has been established, activation may be evaluated.

[0059] Alternatively, the subject oligopeptides may be expressed in conjunction with other peptides or proteins, so as to be a portion ofthe chain, either internal, or at the N- or C-terminus. Various post-expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation, such that the subject peptide will be bound to a lipid group at one terminus, and will be able to be inserted into a lipid membrane, such as a liposome.

[0060] ^* The subject oligopeptides may also be modified by the addition of chemical moieties or groups. For example, the oligopeptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The subject oligopeptides may also be combined with other proteins, such as the Fc of an IgG isotype to enhance complement binding, or with a toxin, such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain. The oligopeptides may be linked to antibodies for site directed action. For conjugation techniques, see, e.g., U.S. Pat. Nos. 3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105; and 4,043,989, which are incorporated herein by reference. As outlined herein, the oligopeptides may be labeled as well.

[0061] Oligopeptides ofthe invention can be modified to increase stability, enhance pharmacological properties (half-life, absorption, potency, efficacy) and the like. Exogenous bioactive compounds also include nonpeptide compounds (peptide analogs) structurally similar to the RDPSs shown in Table 1, sometimes referred to known as "peptide mimetics" or "peptidomimetics" (Fauchere, 1986, Adv. Drug Res. 15: 29; Veber and Freidinger, 1985, TINS p.392; and Evans et al., 1987, J. Med. Chem 30: 1229). For example, useful peptidomimetics may be structurally similar to a oligopeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: --CH2NH--, --CH2S--, --CH2 — CH2 --, -CH.dbd.CH~(cis and trans), --COCH2 --, ~CH(OH)CH2 --, and -CH2 SO--, by methods known in the art and further described in the following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185 (-CH2 NH-, CH2 CH2 --); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249 (-CH2 --S); Harm, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (~CH~CH~, cis and trans); Almquist, R. G. et al., J Med Chem (1980) 23:1392-1398 (-OCH2 --); Jennings-White, C. et al., Tetrahedron Lett (1982) 23:2533 (-OCH2 --); Szelke, M. et al., European Appin. EP 45665 (1982) CA: 97:39405 (1982) (~CH(OH)CH2 -); Holladay, M. W. et al, Tetrahedron Lett (1983) 24:4401-4404 (-- C(OH)CH2 --); and Hruby, V. J., Life Sci (1982) 31:189-199 (-CH2 ~S--); each of which is incorporated herein by reference. One useful non-peptide linkage is --CH2 NH~. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad- spectrum of biological activities), reduced antigenicity, and others.

Binding and Activity Assays

[0062] Identification of receptor activation domain sequences, as well as determination of the effect of binding of agents to activation domains (see Examples), permits the design of screening assays for agents that bind the activation domain sequences and modulate receptor activity (exogenous bioactive compounds). Initial screening or validation may be carried out using binding assays, with subsequent determination ofthe effect ofthe agent on receptor activity. Alternatively, assays that determine the effect ofthe agent on receptor activity can be carried out without antecedent binding assays.

[0063] It will be appreciated that assays for activation domain binding and modulatory activity are useful in at least two different, but related, contexts. In one context, screening assays are carried out in which a plurality of assay mixtures are run in parallel with different candidate agents (e.g., high throughput screening assays). Usually candidate agents are assayed at different concentrations to obtain a differential response to the various concentrations. Such assays can be used to identify new exogenous bioactive compounds. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

[0064] In a second related context, the assays can be used to assess activity of particular variants of compounds known to bind a receptor activation sequence and modulate receptor activity, such as the receptor derived peptides described in the Examples, infra. For example, the effect of amino acid substitutions, insertions, or deletions in SEQ ID NO:l on the agonist activity ofthe peptide on GLP-1 receptor activity, SEQ ID NO:2 on the agonist activity ofthe peptide on CTR2 receptor activity, SEQ ID NO:3 on the antagonist activity ofthe peptide on EP2 receptor activity, can be readily assessed. Variants, for example, may be selected to develop exogenous bioactive compounds with enhanced half-life or other desirable properties. [0065] Useful assays will be apparent to those of skill in the art guided by the instant disclosure, and include assays described below as well as those described in U.S. Pat. No. 6,333,031.

Binding Assays

[0066] Agents capable of modulating surface receptor activity can be identified by first screening for the ability to bind an activation sequence of a receptor listed in Table 1. Some embodiments ofthe various assays described herein utilize human cell surface receptors, although other mammalian receptors may also be used, including receptors from rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. Included within the definition of cell surface receptors are proteins having amino acid substitutions, insertions, or deletions ofthe naturally occurring sequence. Furthermore, included within the definition of cell surface receptors are proteins having portions of cell surface receptors; that is, either the full-length receptor may be used, or functional portions thereof. Thus, in one embodiment, binding to a candidate agent to an oligopeptide having a sequence identical to or substantially similar to SEQ ID NO: 1, 2 or 3 or a receptor binding fragment or receptor binding variant thereof is determined to identify compounds that bind the activation domain and potentially modulate receptor activity.

[0067] In one variation, the assay comprises combining a cell surface receptor having a receptor activation of a calcitonin, GLP-1, or prostaglandin E2 receptor, and a candidate bioactive agent, and determining the binding ofthe candidate agent to the activation domain. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (e.g., phosphorylation assays), and the like. In one variation, the candidate bioactive agent is labeled, and binding determined directly. In one approach, all or a portion of the cell-surface receptor is attached to a solid support, a labeled candidate agent (for example a fluorescent label) is added, excess and unbound reagent is removed, and the presence ofthe label on the solid support is determined. Various blocking and washing steps may be utilized as is known in the art. Alternatively, the candidate agent can be immobilized. [0068] Another way to assess binding of an agent to an activation domain uses competitive binding assays to detect competition between (i) the agent and (ii) a competitor moiety that binds the receptor activation domain, for binding to a receptor activation domain listed in Table 1. Thus, in one variation, the method comprises combining a polypeptide comprising a calcitonin-, GLP-1 -, or EP2- receptor activation domain as listed in Table 1, a candidate bioactive agent, and a competitor moiety, and determining the binding ofthe candidate agent to the activation domain. For example, in one assay the ability ofthe agent to interfere with (i.e., compete with) the binding of an oligopeptide having a sequence shown in Table 1 and its cognate receptor is determined. Exemplary competitor moieties that bind a receptor activation domain for use in competition assays include oligopeptides having a sequence of SEQ. ID. NO:l, 2 or 3, or a receptor binding fragment or variant thereof (e.g., an oligopeptide having a sequence identical or substantially similar to one of SEQ. ID. NO:l, 2 or 3). For use in the assay, examples of polypeptides that comprise a cell surface receptor activation domain as listed in Table 1, as described above, include an oligopeptide having a sequence identical or substantially similar to one of SEQ. ID. NO:l, 2 or 3, a full-length receptor comprising an activation domain as listed in Table 1 (either isolated or expressed by a cell), and a fragment of the receptor comprising a portion ofthe extracellular portion ofthe receptor. [0069] In one variation of these assays, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor moiety (e.g., oligopeptide) is added first to the receptor for a time sufficient to allow binding. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4°C-40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 2 hours will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

[0070] In another variation, the competitor moiety (e.g., oligopeptide) and the candidate bioactive agent are added together. Usually the candidate bioactive agent is added first, and usually in excess. Non-binding ofthe competitor moiety is an indication that the candidate bioactive agent is binding to the activation domain and thus may be capable of modulating receptor activity. Either component can be labeled.

[0071] In a particularly useful variation, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor moiety (e.g., oligopeptide). The absence of binding by the competitor moiety may indicate that the bioactive agent is bound to the receptor with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence ofthe label on the support, coupled with a lack of competitor moiety binding, may indicate that the candidate agent is capable of binding to the activation domain and modulating receptor activity. [0072] In another variation, the methods comprise combining a cell surface receptor and a competitor moiety (e.g., oligopeptide) as described herein, to form a test mixture. The candidate bioactive agent is added to the test mixture, and the binding ofthe candidate bioactive agent to the activation domain ofthe receptor is determined. In this embodiment, either or both ofthe competitor moiety or the candidate bioactive agent is labeled, with preferred variations utilizing labeled oligopeptides, such that displacement ofthe label indicates binding by the candidate bioactive agent.

[0073] In another variation, the methods comprise differential screening to identity bioactive agents that bind a receptor activation domain. In this embodiment, the methods comprise combining a cell surface receptor and a competitor moiety (e.g., oligopeptide) that binds the activation sequence in a first sample. A second sample comprises a candidate bioactive agent, a cell surface receptor and a competitor moiety. The binding ofthe competitor moiety is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding the activation domain. That is, if the binding ofthe competitor moiety is different in the second sample relative to the first sample, the agent is capable of binding the activation domain.

[0074] A variety of assay formats will be apparent to those of skill. In one variation ofthe methods herein, the purified cell surface receptor or candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which peptide or receptor can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, or nitrocellulose, Teflon™, and the like. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding ofthe peptide or other protein is not crucial so long as it is compatible with the reagents and overall methods ofthe invention, maintains the activity ofthe peptide and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the receptor is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, and the synthesis ofthe receptor on the support surface. Following binding ofthe peptide or receptor, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein. In another variation, cell lines that overexpress the cell surface receptor are used to screen for candidate bioactive agents.

[0075] In another variation a homogeneous LANCE assay format, or similar solution phase assay, is used to identify bioactive molecules. In the LANCE type assay, all the reagents are in a solution (in contrast to solid or semi-solid plate assay). Briefly, a receptor of interest (e.g., GLP- 1, calcitonin, or prostaglandin) is incubated with biotinylated peptide that specifically binds the modulation domain ofthe receptor under conditions in which a complex between receptor and the peptide is formed. A Europium-labeled anti-receptor antibody is allowed to bind the complex and streptavidin-APC conjugate is allowed to bind to biotinylated peptide. The formation ofthe complex is detected by signal emission which occurs as a result of close proximity between Streptavidin-APC conjugate (that binds to biotinylated peptide) and Eu+- labeled anti- receptor antibody. Thus, if the complex between receptor and peptide is formed, strong signal emission occurs. Lack of signal indicates that anti-receptor antibody and streptavidin conjugate are not in a close proximity, generally because the receptor/peptide complex has not been formed. When used as a screening assay, a competition assay between the biotinylated peptide and candidate bioactive molecules (e.g., from a library of compounds) is usually carried out by adding the candidate bioactive molecule before the formation ofthe peptide-receptor complex. An absence or diminution of signal indicates competition for the modulation site between the biotinyated peptide and the candidate bioactive molecule. [0076] "Labeled," as used herein in the context of binding and activity assays refers to a compound is either directly or indirectly labeled with a compound that provides a detectable signal, e.g., radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, and the like. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal. In some variations, only one ofthe components is labeled. For example, the oligopeptides may be labeled at tyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using ¹²⁵I for the oligopeptides, for example, and a fluorophor for the candidate agents.

[0077] A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin and detergents, which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency ofthe assay, such as protease inhibitors, nuclease inhibitors, and anti-microbial agents, may be used. The mixture of components may be added in any order that provides for the binding. An oligopeptide that binds the activation-domain of a GLP-1, calcitonin or prostaglandin E2 receptor can be referred to as an "activation-domain binding oligopeptide." Examples of such oligopeptides include oligopeptides having the sequence of SEQ ID NO:l, 2 or 3 and others identified using the binding assays described herein.

Receptor Activity Assays

[0078] Methods for detecting modulation of activity of a receptor listed in Table 1 are known in the art. As used herein, "receptor activity" has its usual meaning in the art and refers to the biological function associated with binding of natural ligand ofthe cell surface receptor. Modulation of receptor activity can refer to an increase in the activity (in the presence or absence ofthe natural ligand) or a decrease in natural-ligand induced activity (generally measured in the presence of activating amounts of natural ligand or ligand analogs that bind the ligand binding site, such as homologs from other species, variants, and the like). [0079] As used herein, the terms "natural ligand," "naturally-occurring ligand," and "endogenous ligand" are used interchangeably, and refer to ligand that is natively produced by the organism expressing the subject receptor, and which binds the ligand binding site ofthe receptor. For example, glucagon-like peptide-1 is the natural ligand for the GLP-1 receptor, calcitonin is natural ligand for the calcitonin receptor, and prostaglandin E2 is the natural ligand for the prostaglandin E2 receptor. Natural ligands are examples of "activating ligands." Other activating ligands are compounds that compete with the natural ligand for binding to the receptor ligand binding site, and which when bound by the receptor cause receptor activation. [0080] As will be appreciated by those in the art, the specific biological function will vary depending on the identity ofthe receptor. Generally ligand binding results in a conformational change in the receptor. Thus, in some embodiments, activation of a receptor is detectable as a conformational change either within the receptor, or as a result of monomeric receptors becoming multimeric, which allows the receptor to facilitate signaling. In some cases, the conformational change results in receptor phosphorylation, receptor association with another cell biomolecule (e.g., protein), and/or phosphorylation of another cell protein. Methods for detecting changes in receptor conformation, receptor-biomolecule association, receptor phosphorylation or phosphorylation of other biomolecules in the receptor signaling pathway are well known. Illustrative assays are described in the Examples, infra. Receptor activation can also be detected as a downstream effect mediated by ligand binding to the receptor such as stimulation of insulin levels in plasma (see Example 1, infra), changes in concentration of intracellular biomolecules such as cAMP (see, e.g., Examples 2 and 3, infra), cell shape changes, stimulation or inhibition of cell proliferation, and a variety of other measures of receptor activation. Suitable activation assays can be carried out in vitro (e.g., in immortalized cells lines or primary cell cultures) or in vivo (e.g., in non-human animal models, or human subjects).

Applications

[0081] The exogenous bioactive compounds, e.g., oligopeptides, ofthe present invention are used in methods of modulating receptor activity. Such modulation finds use in screening assays, studies ofthe mechanism of action of receptor ligands, therapeutic uses, and other uses that will be apparent to one of ordinary skill in the art.

[0082] In an aspect the invention provides a method for modulating activity of a cell surface receptor by contacting a mammalian cell surface receptor listed in Table 1 and a compound that binds the activation sequence ofthe receptor (set forth in Table 1 for the human homolog). In one respect, the exogenous compounds may act as receptor agonists, as seen with, e.g., GLP-lp and CTRp (examples of agents that bind the receptor activation sequences of GLP-1 R or CTR). Thus, for example, in an aspect the invention provides a method for agonizing activity a cell surface receptor by contacting a mammalian cell surface receptor and an exogenous compound where the receptor and the exogenous compound are, without limitation, (a) a GLP-1 R and an oligopeptide comprising a sequence, identical to, or substantially similar to SEQ ID NO:l; (b) a GLP-1 R and a compound that competes with an oligopeptide of SEQ ID NO:l for binding the GLP-1 R; (c) a CTR and an oligopeptide comprising a sequence identical to, or substantially similar to, SEQ ID NO:2; (d) a CTR and a compound that competes with an oligopeptide of SEQ ID NO:2 for binding the CTR. In one respect, the exogenous compounds may act as receptor antagonists, as seen with, e.g., EP2p (an example of an agent that binds the receptor activation sequences of EP2-R). Thus, for example, in an aspect the invention provides a method for antagonizing activity a cell surface receptor by contacting a mammalian cell surface receptor and an exogenous compound where the receptor and the exogenous compound are, without limitation, (a) a EP2-R and an oligopeptide comprising a sequence identical to, or substantially similar to, SEQ ID NO:3; or (b) a EP2-R and a compound that competes with an oligopeptide of SEQ ID NQ:3 for binding the EP2-R.

[0083] It will be appreciated that modulation of each of the GLP- 1 -R, CTR, and EP2-R has a variety of therapeutic benefits. In general, any of a variety of diseases, symptoms, and conditions mediated, at least in part, by activation ofthe GLP-1, calcitonin or prostaglandin EP2 receptors, can be treated by administering agents that modulate receptor activity. In this context, "treatment" is an approach for obtaining beneficial or desired results, such as alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total).

[0084] Depending on the type of disorder, administration ofthe pharmaceutical composition can serve to enhance the cellular response to endogenous or exogenous ligand, e.g., in ligand resistant states, to replace endogenous ligand, e.g., in ligand deficient states, or to antagonize the effects of ligand (e.g., in cases in which expression of endogenous ligand is detrimental to the subject). Thus, in one embodiment, the exogenous compound contacts the receptor in the presence ofthe ligand which normally activates the receptor. As above, there may be endogenous ligand present, or exogenous ligand added in addition to the exogenous compound. [0085] In an aspect the invention provides a method for modulating activity of a cell surface receptor by contacting a mammalian cell surface receptor listed in Table 1 and a compound that binds the activation sequence set forth in Table 1 for the receptor. In one respect, the exogenous compounds behave as agonists and activate receptor activity. Exemplary receptor agonists of the invention include GLP-lp and CTRp (examples of agents that bind the receptor activation sequences ofthe GLP-1 receptor and the calcitonin receptor, respectively). In one respect, the exogenous compounds behave as antagonists and inhibit receptor activity. Exemplary receptor antagonists ofthe invention include EP2p (an example of an agents that binds the receptor activation sequences ofthe prostaglandin E2 receptor). Thus, for example, in an aspect the invention provides a method for modulating a cell surface receptor by contacting a mammalian cell surface receptor and an oligopeptide where the receptor and the oligopeptide are a cognate pair and are (a) a GLP-1 receptor and an oligopeptide comprising a sequence substantially similar to SEQ. ID. NO: 1 ; or (b) a CTR2 receptor and an oligopeptide comprising a sequence substantially similar to SEQ. ID. NO:2 or (c) a EP2 receptor and an oligopeptide comprising a sequence substantially similar to SEQ. ID. NO:3.

[0086] When the exogenous compound is to be administered with exogenous ligand to enhance receptor activity, the administration may be simultaneous or sequential. In this embodiment, the level of receptor activation is greater with a combination ofthe ligand and the exogenous compound as compared with the same amount of ligand alone. In sum, for some receptors, there may be a synergistic effect, i.e., the effect of adding ligand and exogenous compound (e.g., an oligopeptide ofthe invention), may be greater than either ligand alone or exogenous compound alone. In general, the exogenous compound is added to cells to contact the receptor in the absence of exogenous ligand.

[0087] It will be recognized by those of ordinary skill that modulation of each ofthe receptors listed in Table 1 has a variety of therapeutic benefits. For example, in Example 1, insulin secretion is demonstrated to increase upon binding of GLP-lp to the activation domain ofthe GLP-1 receptor. The data thus show that GLP-lp is a receptor agonist. As the effect of GLP-lp is to increase insulin secretion in response to glucose, patients with impaired glucose tolerance (type II diabetes mellitus or IDDM) may benefit from treatment with this peptide as well as with other exogenous compounds that compete with GLP-lp for binding to the receptor. [0088] Administration of agents that increase activity of the calcitonin receptor can be used to stimulate the bone restoring activity of osteoclasts, treatment of osteoperosis, impaired glucose tolerance, type I and II diabetes, hypercalcemia, and other applications for which calcitonin would be beneficial. Administration of agents that increase activity ofthe GLP-1 receptor can be used to treat impaired glucose tolerance, type I and II diabetes, obesity, and hypercalcemia. Administration of agents that increase activity ofthe prostaglandin E2 receptor can be used to reduce pain and inflammation, including treatment of osteroperosis. [0089] Depending on the type of disorder, administration ofthe pharmaceutical composition can serve to enhance the cellular response to endogenous or exogenous ligand, e.g., in ligand resistant states, to replace endogenous ligand, e.g., in ligand deficient states, or to antagonize the effects of ligand (e.g., in cases in which expression of endogenous ligand is detrimental to the subject). Thus, in one embodiment, the exogenous compound contacts the receptor in the presence ofthe ligand which normally activates the receptor. As above, there may be endogenous ligand present, or exogenous ligand added in addition to the exogenous compound. Dosages and Formulations of Exogenous Compounds

[0090] An exogenous compound may be formulated as a pharmaceutical composition by combining the compound with a pharmaceutical carrier or diluent and optionally other compounds that enhance therapeutic utility and/or facilitate storage and administration. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Thus, in one aspect the invention provides pharmaceutical compositions that include exogenous compounds ofthe invention along with pharmaceutically acceptable excipients, carrier or diluent and optionally other compounds that enhance therapeutic utility and/or facilitate storage and administration. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Pharmaceutically acceptable excipients are well known in the art and include sterile water for pharmaceutical use, isotonic solutions such as saline and phosphate buffered saline, physiological saline, PBS, and dextrose solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents. Other excipients suitable for administration to a human patient known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (19th edition, 1995, Gennavo, ed.). The pharmaceutical compositions are typically sterile (i.e., manufactured or formulated as a sterile composition) and optionally can be prepared in compliance with all Good Manufacturing Practice (GMP) regulations ofthe U.S. Food and Drug Administration. [0091] The exogenous bioactive compounds ofthe invention may be administered in a physiologically acceptable carrier to a host. The agents may be administered in a variety of ways, e.g., orally, parenterally (e.g., by intravascular infusion or injection at an epidermal, subcutaneous, intramuscular, or intraperitoneal site), topically, transdermally, or by transmucosal absorption. Depending upon the manner of introduction, the agents may be formulated in a variety of ways.

[0092] The formulation of bioactive agent will vary depending upon the purpose ofthe formulation, the particular mode employed for modulating the receptor activity, the intended treatment, and the like. The concentration of therapeutically active agents in the formulation may vary from about 0.1-100 wt. %. The formulation may involve patches, tablets, capsules, liposomes, time delayed coatings, injectables, or may be formulated in pumps for continuous administration. For example, formulations for injection may comprise a physiologically acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like. Water soluble preservatives which may be employed include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in individual amounts of from about 0.001% to about 5% by weight and preferably about 0.01% to about 2%. Suitable water soluble buffering agents that may be employed are alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives such as carboxymethylcellulose may be used as a carrier in amounts of from about 0.01% to about 5% by weight. The specific dosage may be determined empirically in accordance with known ways. See, for example, Harrison's Principles of Internal Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.

[0093] Generally, a therapeutically effective dose ofthe exogenous bioactive compound will be administered. A therapeutically effective dose is an amount sufficient to modulate receptor activity. For oligopeptides, this amount is usually in the range of about 0.005-40, more usually from about 0.01-20 mg/kg of host weight, and sometimes from about 0.1 to about 1 mg/kg. Administration may be as often as daily; sometimes not more than once or twice daily, or as infrequent as weekly. The host may be any mammal including domestic animals, pets, laboratory animals and primates, particularly humans. The amount will generally be adjusted depending upon the half life ofthe molecule (peptide or small molecule) where dosages in the lower portion ofthe range may be employed where the peptide has an enhanced half life or is provided as a depot, such as a slow release composition comprising particles, introduced in a matrix which maintains the peptide over an extended period of time, e.g., a collagen matrix, use of a pump which continuously infuses the peptide over an extended period of time over a substantially continuous rate, or the like. Heller (1987), Biodegradable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 1, CRC Press, Boca Raton, Fla., pp 39-90, describes encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13:850-856 describes controlled drug release from hydrophobic polymers. EXAMPLES [0094] The following examples serve to more fully describe the manner of using the above- described invention. It is understood that these examples in no way serve to limit the scope of this invention, but rather are presented for illustrative purposes. All cells were obtained from the American Type Culture Collection.

EXAMPLE 1 BIOLOGICAL ACTIVITY OF GLP-1 RECEPTOR DERIVED PEPTIDE [0095] Example 1 demonstrates that a GLP-1 receptor derived peptide ("GLP-lp") having the sequence set forth as SEQ ID NO: 1 in Table 1 acts as a GLP-1 receptor agonist and stimulates insulin secretion.

[0096] GLP-1 peptide activity was examined using the intravenous glucose tolerance test (IVGTT). In this assay, male Wistar rats were anesthetized with 65 g/kg pentobarbital and then administered a 0.5 g/kg bolus injection of a 50% glucose solution. GLP-lp (1 mg/kg) was given at -20 and 0 minutes. Blood samples were then collected at 0, 5, 10, 15, 20, 25, 30 minutes after the peptide was provided to determine the effect ofthe administered glucose and GLP-lp on plasma insulin and blood glucose concentrations.

[0097] Figure 1 demonstrates that GLP-1 peptide significantly increases insulin levels in plasma. GLUT-4 peptide (TWLGRQGPEGPSSIPPGTLTTLW SEQ ID NO: 4) was included as a control and had no effect.

[0098] GLP-1 peptide was examined in recombinant cells transiently or stably transfected with GLP-1 receptor encoding DNAs and expressing the receptor. The cell lines were established using standard procedures. For the experimental testing, cells were washed and preincubated with 1 mM IBMX for 10 min at room temperature. The cells were washed again, peptide (GLP-lRp) or hormone GLP-1 (used as a positive control) was added to the cells, and the cells were incubated 30 minutes at 37°C, 5% CO . Media was removed, and the cells were washed and lysed. cAMP levels were determined using a quantitative cAMP assay performed according to manufacturer's manual. Results for Transiently Transfected COS7 Cells

[0099] COS7 cells were transiently transfected with a GLP-1 R expression vector and cAMP production was measured upon stimulation with 50 μM GLP-1R peptide or 100 nM GLP-1. Figure 2 shows that peptide significantly increased cAMP production. The peptide had no effect on COS-7 cells transfected with an expression vector encoding GLP2R, indicating that the peptide is specific for GLP-IR (data not shown). In addition, the GLP antagonist exendin-9 blocked the effect ofthe peptide in transiently transfected COS-7 cells (data not shown). Results for Stably Transfected HEK 293 Cells

[00100] Addition of GLP-lRp to recombinant HEK 293 cells stably transfected with GLP-IR expression vector increased cAMP production to the level similar to that seen following addition of GLP-1 (see Figure 3). As shown in Figure 3, when cells were not transfected with GLP-IR, there was no GLP-lRp response. In the same cell line, GLP-lRp (peptide) and GLP-1 hormone act synergistically. Figure 4 shows that addition of peptide (4 μM) and GLP-1 (2.5 or 5 pM) caused a synergistic increase in cAMP production (activity in the presence of both is much larger than the activity seen by peptide or hormone alone). This indicates that GLP-lRp peptide can act on GLP-1 receptor in the presence and absence of natural hormone.

EXAMPLE 2 BIOLOGICAL ACTIVITY OF CALCITONIN RECEPTOR DERIVED PEPTIDE [00101] Example 2 shows that calcitonin receptor derived peptide ("CTRp") having the sequence set forth as SEQ ID NO: 2 in Table 1 is a calcitonin receptor agonist. [00102] In general, ligand binding to the calcitonin receptor stimulates G-protein mediated activation of adenylate cyclase and formation of c AMP. Biological activity ofthe calcitonin receptor was elucidated by examining the ability of T47-D (human mammary carcinoma) cells to produce cAMP in the presence of CTRp.

[00103] The assay for cAMP production was performed using the HitHunter™ Enzyme Fragment Complementation Kit (Disco verRx, Fremont, CA). Figure 5 A shows that cAMP is generated in response to stimulation with salmon calcitonin in T47-D cells. In Figure 5B, production of cAMP after exposure to CTRp is also demonstrated. Furthermore, the response to CTRp was shown to be receptor specific. Figure 6 illustrates that SK-N-BE(2) cells produce cAMP in response to prostaglandin E2 (PGE2), but do not when treated with CTR derived peptide. Therefore, the CTRp is an agonist specific for the calcitonin receptor.

EXAMPLE 3 BIOLOGICAL ACTIVITY OF EP2 RECEPTOR DERIVED PEPTIDE [00104] It has been reported that treatment of SKNBE(2) cells with 5 micromolar all-trans retinoic acid for 3-4 days dramatically up-regulates their levels ofthe EP2 form ofthe receptor. Figures 7 and 8 demonstrate the effect of binding of EP2p to the prostaglandin E2 receptor found on SK-N-BE(2) cells. Cell cultures were split and grown in media containing 5 micromolar all-trans retinoic acid for 3-4 days before being stimulated with PGE2 (for 30 minutes prior to lysis ofthe cells) and used in a cAMP assay. Figure 7 shows that the SK-N- BE(2) cells increase production of cAMP in response to prostaglandin E2. Figure 8 shows that addition of EP2p inhibited the PGE2 induced production of cAMP.

[00105] All publications, patents, patent applications, and accession numbers (including both polynucleotide and polypeptide sequences and corresponding annotations as ofthe filing and/or priority application filing dates) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference. The entire disclosure of U.S. provisional patent application no. 60/420,487, to which this application claims priority, is incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope ofthe appended claims.

Claims

1. A method of modulating the activity of a G protein coupled receptor by contacting the receptor with an exogenous compound that binds in the receptor activation domain and modulates receptor activity, wherein the receptor is a receptor for calcitonin, GLP-1 or prostaglandin E2.

2. The method of claim 1 , comprising modulating the activity of a calcitonin receptor by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:2.

3. The method of claim 2 wherein receptor activity is increased.

4. The method of claim 2 wherein the calcitonin receptor is human.

5. The method of claim 2-4 wherein the contacting takes place in vitro.

6. The method of claim 2-5 wherein the exogenous compound is an oligopeptide.

7. The method of claim 6 wherein the oligopeptide comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO:2.

8. The method of claim 2 wherein the contacting occurs in the presence of calcitonin.

9. A method for screening candidate agents for the ability to modulate activity of a calcitonin receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:2.

10. A method for screening candidate agents for the ability to modulate activity of a calcitonin receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO:2 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:2; and b) determining the binding ofthe agent or the oligopeptide to the activation domain; wherein the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity.

11. The method of claim 10 wherein the candidate agent or oligopeptide is labeled.

12 A method for screening candidate agents for the ability to modulate activity of a calcitonin receptor by identifying a compound that binds in a receptor activation domain having the sequence of SEQ ID NO:2, and assaying the ability ofthe compound to modulate activity of the cell surface receptor.

13. The method of claim 12 wherein the ability ofthe agent to stimulate receptor activation is assayed.

14. An isolated oligopeptide that binds the calcitonin receptor activation domain.

15. The oligopeptide of claim 14 wherein the oligopeptide comprises at least 8 amino acids of SEQ ID NO:2.

16. An oligopeptide of claim 14-15 that increases activation of a calcitonin receptor.

17. A pharmaceutical composition comprising an oligopeptide of claim 14-16 in a sterile form and a pharmaceutically acceptable excipient.

18. A method for treating a condition in a patient characterized by an undesired level of calcitonin receptor activation by administering to the patient an exogenous bioactive compound that binds the calcitonin receptor activation domain and which modulates receptor activity when contacted with the receptor.

19. The method of claim 1, comprising modulating the activity of a GLP-1 receptor by contacting the receptor with an exogenous compound that binds in the activation domain and can modulate receptor activity in the absence of ligand, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:l.

20. The method of claim 19 wherein receptor activity is increased.

21. The method of claim 19 wherein the GLP-1 receptor is recombinantly expressed.

22. The method of claim 19 wherein the GLP-1 receptor is human.

23. The method of claim 19 wherein the contacting takes place in vitro.

24. The method of claim 19 wherein the exogenous compound is an oligopeptide.

25. The method of claim 24 wherein the oligopeptide comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO:l. exogenous bioactive compound that binds the calcitonin receptor activation domain and which modulates receptor activity when contacted with the receptor.

26. The method of claim 19 wherein the contacting occurs in the presence of GLP- 1.

27. A method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:l.

28. A method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO: 1 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:l; and b) determining the binding ofthe agent or the oligopeptide to the activation domain; wherein the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity.

29. The method of claim 27-28 wherein the candidate agent or oligopeptide is labeled.

30. A method for screening candidate agents for the ability to modulate activity of a GLP-1 receptor by identifying a compound that binds in a receptor activation domain having the sequence SEQ ID NO:l and assaying the ability ofthe compound to modulate activity ofthe cell surface receptor.

31. The method of claim 30 wherein the ability ofthe agent to stimulate receptor activation is assayed.

32. The method of claim 30 wherein the ability ofthe agent to inhibit receptor activation is assayed.

33. An isolated oligopeptide that binds the GLP-1 receptor activation domain.

34. The oligopeptide of claim 33 wherein the oligopeptide comprises at least 8 amino acids of SEQ ID NO: 1.

35. The oligopeptide of claim 34 that is an agonist of a GLP-1 receptor.

36. A pharmaceutical composition comprising an oligopeptide of claims 33-35 in a sterile form and a pharmaceutically acceptable excipient.

37. A method for treating a condition in a patient characterized by an undesired level of GLP-1 receptor activation by administering to the patient an exogenous bioactive compound that binds the GLP-1 receptor activation domain and which increases receptor activity when contacted with the receptor.

38. The method of claim 1 , comprising modulating the activity of a prostaglandin E2 receptor by contacting the receptor with an exogenous compound that binds in the activation domain and can modulate receptor activity in the absence of ligand, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:3.

39. The method of claim 38 wherein receptor activity is increased.

40. The method of claim 38 wherein receptor activity is decreased.

41. The method of claim 38 wherein the contacting takes place in vitro.

42. The method of claim 38 wherein the prostaglandin E2 receptor is human.

43. The method of claim 38 wherein the contacting occurs in the presence of prostaglandin E2.

44. The method of claim 38 wherein the exogenous compound is an oligopeptide.

45. The method of claim 44 wherein the oligopeptide comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO:3.

46. A method for screening candidate agents for the ability to modulate activity of a prostaglandin E2 receptor by determining the binding ofthe agent to a receptor activation domain sequence having a sequence of SEQ ID NO:3.

47. A method for screening candidate agents for the ability to modulate activity of a prostaglandin E2 receptor having an activation domain comprising a sequence substantially similar to SEQ ID NO:3 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:3; and b) determining the binding ofthe agent or the oligopeptide to the activation domain, wherein the binding ofthe agent to the activation domain or a reduction in the binding ofthe oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity.

48. The method of claim 47 comprising the step of contacting the receptor with prostaglandin E2.

49. The method of claim 46-47 wherein the candidate agent or oligopeptide is labeled.

50. A method for screening candidate agents for the ability to modulate activity of a prostaglandin E2 receptor by identifying a compound that binds in a receptor activation domain having the sequence SEQ ID NO: 3 and assaying the ability ofthe compound to modulate activity ofthe cell surface receptor.

51. The method of claim 50 wherein the ability ofthe agent to increase prostaglandin E2 receptor activation is assayed.

52. The method of claim 51 wherein the ability ofthe agent to inhibit prostaglandin E2-induced receptor activation is assayed.

53. An isolated oligopeptide that binds the prostaglandin E2 receptor activation domain.

54. The oligopeptide of claim 53 wherein the oligopeptide comprises at least 8 amino acids of SEQ ID NO:3.

55. The oligopeptide of claim 53-54 that is an antagonist of a prostaglandin E2 receptor.

56. A pharmaceutical composition comprising an oligopeptide of claims 53-55 in a sterile form and a pharmaceutically acceptable excipient.

57. A method for treating a condition in a patient characterized by an undesired level of prostaglandin E2 receptor activation by administering to the patient an exogenous bioactive compound that binds in the prostaglandin E2 receptor activation domain and which modulates receptor activity when contacted with the receptor.