Characterization And Pharmacology Of Receptors For Gastrointestinal Peptides

各种胃肠道(GI)多肽的药理学和分子药理学正在研究中。年内研究的三个多肽受体家族分别为蛙皮素(Bn)相关多肽、生长抑素(SS)家族和血管活性肠肽(VIP)相关多肽。BN相关多肽(GRP、NMB)主要与三种不同的受体(GRP-R、NMB-R)和孤儿受体BRS-3相互作用，介导胃肠道和中枢神经系统(CNS)的一系列作用。利用我们和其他人的结构和功能研究的信息，我们合成了代谢稳定的高亲和力Bn、VIP和生长抑素类似物，这些类似物对拓扑异构酶抑制剂喜树碱(CPT)等细胞毒剂具有很强的抗肿瘤活性。我们使用非活性的化学相同的类似物证明了这种杀瘤作用是由癌细胞上表达的多肽受体介导的。为了确定与蛙皮素受体家族不同成员的不同激动剂选择性高亲和力结合的关键氨基酸，我们使用了该家族不同受体的结构同源性分析和定点突变。在GRP受体和BRS-3受体之间，它们对蛙皮素(BN)的亲和力相差1000倍，我们发现了14个重要的氨基酸差异，并在每个受体中进行了替换。分子模拟和药理学研究表明，其中7个对Bn的高亲和力是重要的，并有助于这些受体的选择性，这表明这是一种识别对选择性和亲和力重要的氨基酸的有用方法。我们研究的第二个方面是开发代谢稳定的Bn或VIP受体亚型的选择性受体配体。为了实现人BRS-3受体的这一点，我们对没有选择性配体的人BRS-3受体进行了N-甲基取代、D-氨基酸取代、截断和取代我们已经发现与该受体具有高亲和力的非选择性配体中的各种氨基酸。一个类似物DTyr6，Apa-4Cl，Phe13，Nle14-Bn(6-14)对人Brs3受体的选择性明显高于该家族的其他受体。这种类似物被发现比最近描述的其他BRS-3选择性配体具有更高的亲和力和选择性，因此应该有助于研究它在生理和病理过程中的作用。此外，利用我们先前的配基结构功能研究，我们能够构建代谢稳定、对VPAC1受体具有高亲和力和选择性的28个氨基酸的多肽VIP的简化类似物。

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.