Assay for and uses of peptide hormone receptor agonists

#The invention relates to methods of identifying and using a compound that is an agonist to a peptide hormone receptor.

#Peptide hormone receptors are important targets for drug research because a considerable number of diseases and other adverse effects result from abnormal receptor activity. 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, the CCK-A receptor, 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, in parietal cells of the gastrointestinal tract, in ECL cells, as well as in kidney cells. 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).

#Applicants have developed a systematic screening assay for identifying an agonist specific for a peptide hormone receptor, e.g., a peptide, peptoid, or non-peptide agonist. The assay is based on applicants' recognition that a peptide hormone receptor able to amplify the intrinsic activity of a ligand, e.g., a constitutively active peptide hormone receptor, is useful as a screening vehicle for identifying a receptor-specific agonist. A receptor with a signaling activity higher than the corresponding human wild-type basal level of signaling activity is further useful for detecting the reduction in signaling 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 agonists to the corresponding human wild-type form of the receptor.

#FIG. 1 is a schematic diagram showing the relationship between a full or partial agonist, an inverse agonist, and an antagonist.

#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.

#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).

#Recent drug development efforts have yielded small molecules which competitively block G-protein coupled peptide hormone receptors by acting as antagonists. In contrast, very few non-peptide ligands have been identified which activate this family of receptors. Here, applicants demonstrate that chemical modifications of non-peptide ligands, known to act as antagonists for the CCK-B/gastrin receptor, can result in converting the ligands 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 (325L→E, MH162). Several peptide, peptoid and benzodiazepine-based nonpeptide ligands were tested in this assay, and evaluated for the ability to activate the recombinant wild-type or constitutively active mutant receptor, respectively. Full positive agonists had similar signaling efficacy in both receptors when compared to the intrinsic activity of the peptide agonist CCK-8s. The signalling efficacy of ligands with lower intrinsic activity was logarithmically amplified when a constitutively active mutant receptor was used in the assay. The prototype benzodiazepine-derived non-peptide antagonist L-365,260 barely increased basal activity of the human wild-type CCK-B/gastrin receptor, but was identified as a partial agonist using the 325L→E mutant MH162. 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). Thus, the process of discovering novel non-peptide agonists, e.g., those with positive inverse or intrinsic activity, should be expedited by using enhanced receptors, e.g., constitutively active mutant receptors, in the screening assay.

#I. Working Examples: