Characterization And Pharmacology Of Receptors For Gastrointestinal Peptides

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The pharmacology and molecular pharmacology of various gastrointestinal (GI) peptides are being investigated. Three peptide receptor families investigated during the year are those for bombesin- (Bn) related peptides, somatatostatin (SS) family and for VIP-related peptides. Bn-related peptides (GRP, NMB) interact primarily with three distinct receptors (GRP-R, NMB-R)and the orphan receptor, BRS-3 to mediate a number of effects in the GI tract and central nervous system (CNS). Using information from structure-function studies by us and others we have synthesized high affinity Bn, VIP and somatostatin analogues that were metabolically stable and that be could to cytotoxic agents such as the topoisomerase inhibitor, camptothecin (CPT), which had potent antitumor activity against various human tumor cells. We demonstrated using inactive chemically identical analogues that this tumoricidal effect was mediated by the peptide receptors expressed on the cancer cells. To identify key amino acids responsible for selective high affinity binding of various agonists for different members of the bombesin receptor family we used an analysis of the structural homologies of different receptors of this family and site-directed mutagenesis. Between the GRP receptor and BRS-3 receptor which different by >1000 in their affinity for bombesin (BN) we found 14 important amino acid differences which were then substituted in each receptor. Molecular modeling and pharmacology studies showed 7 of these were important for high affinity for Bn and contributed to the selectivity of these receptors demonstrating this is a useful approach to identify amino acid important for selectivity and affinity. A second aspect of our studies is to develop selective receptor ligands for Bn or VIP receptor subtypes that could be metabolically stable. To accomplish this for the human BRS-3 receptor, which has no selective ligands, we made N-Methyl substitutions, D-amino acid substitutions, truncations and substitutions of various amino acids in a nonselective ligand that we had discovered that interacted with this receptor with high affinity. One analog DTyr6, Apa-4 Cl, Phe13, Nle14-Bn (6-14) was found to have a marked enhanced selectivity for the human BRS3 receptor over the other receptors of this family. This analogue was found to have greater affinity and selectivity than other recently described BRS-3 selective ligands and thus should be useful to investigate its role in physiological and pathological processes. Furthermore, using our previous ligand structure function studies we were able to construct simplified analogs of the 28 amino acid peptide VIP that were metabolically stable and had high affinity and selectivity for VPAC1 receptors.