Characterization And Pharmacology Of Receptors For Gastrointestinal Peptides

各种胃肠道（GI）肽的药理学和分子药理学正在研究中。在这一年中研究的三个肽受体家族是蛙皮素（Bn）相关肽、生长抑素（SS）家族和VIP相关肽。BN相关肽（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，以介导胃肠道和中枢神经系统（CNS）中的许多作用。利用我们和其他人的结构-功能研究的信息，我们已经合成了高亲和力的Bn，VIP和生长抑素类似物，这些类似物代谢稳定，并且可以用于细胞毒性剂，如拓扑异构酶抑制剂喜树碱（CPT），其对各种人类肿瘤细胞具有有效的抗肿瘤活性。我们使用化学性质相同的非活性类似物证明，这种杀肿瘤作用是由癌细胞上表达的肽受体介导的。 为了确定关键的氨基酸负责选择性高亲和力结合的各种激动剂的不同成员的蛙皮素受体家族，我们使用了分析的结构同源性的不同受体的这个家庭和定点诱变。 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，我们发现14个重要的氨基酸差异，然后在每个受体中取代。 分子建模和药理学研究表明，其中7个对Bn的高亲和力很重要，并有助于这些受体的选择性，证明这是一种有用的方法来鉴定对选择性和亲和力很重要的氨基酸。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。为了对没有选择性配体的人BRS-3受体实现这一点，我们在非选择性配体中进行了N-甲基取代、D-氨基酸取代、截短和各种氨基酸的取代，我们发现这些配体以高亲和力与该受体相互作用。发现一种类似物DTyr 6，阿帕-4 Cl，Phe 13，Nle 14-Bn（6-14）对人BRS 3受体的选择性显著高于该家族的其它受体。这种类似物被发现具有更大的亲和力和选择性比其他最近描述的BRS-3选择性配体，因此应该是有用的，以研究其在生理和病理过程中的作用。 此外，使用我们先前的配体结构功能研究，我们能够构建28个氨基酸的肽VIP的简化类似物，其代谢稳定并且对VPAC 1受体具有高亲和力和选择性。

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.