Development of receptor antagonists to neuropeptide Y,galanin and pancreastatin

本课题的主要目的是开发神经肽Y （NPY）、丙氨酸和胰抑素（PST）受体拮抗剂，作为治疗糖尿病、高血压和内分泌异常的药物。为此，我们首先建立了一个新的实验室，能够进行细胞培养实验、肽合成和结构测定，然后，我们进行了拮抗剂开发的合成研究。在这项研究中，我们采用了一种基于设计和合成类似物混合物以及随后从这些混合物中分离和表征受体拮抗剂的新策略来开发受体拮抗剂。利用该策略，设计并合成了一系列NPY模拟混合物。使用NYP拮抗剂试验筛选这些混合物，鉴定出含有4到10个氨基酸的受体拮抗剂。受体拮抗剂抑制NPY或PYY诱导的人红细胞白血病（HEL）细胞内钙的释放。另一方面，为了开发胰抑素（PST）受体拮抗剂，我们需要一种简单的筛选PST受体拮抗剂的生物测定方法。因此，我们首先通过分离的大鼠胰腺b细胞检测了PST对几种胰岛素分泌剂诱导的[Ca2+]i增加的影响。我们发现PST （1-100nM）剂量依赖性地抑制单个胰岛细胞中葡萄糖诱导的[Ca2+]i升高。这一观察结果使我们能够开发一种筛选PST拮抗剂的实验方法，并合成一系列可能用作PST受体拮抗剂的PST类似物。这项研究表明，模拟混合物筛选策略显着减少了以前开发受体拮抗剂或激动剂所需的时间和资源，并有望提高我们设计用于治疗用途的新药物的能力。

The principal aim of this proposal is to develop receptor antagonists to neuropeptide Y (NPY), galanin and pancreastatin (PST) as drugs against diabetes, hypertension and endocrine abnormalities. Toward this goal, we first set up a new laboratory which is capable of carrying out cell culture experiments, peptide synthesis and structural determination, and then, we carried out synthetic studies for the development of the antagonists. In this study, we employed a novel strategy for the development of receptor antagonists based on the design and synthesis of analog mixtures and the subsequent isolation and characterization of receptor anragonists from these mixtures. Using this strategy, a series of NPY analog mixtures was designed and synthesized. Screening of these mixtures using an NYP antagonist assay identified the receptor antagonists containing from 4 to 10 amino acids. The receptor antagonists inhibited the release of intracellular calcium elicited by NPY or PYY in human erythroleukemia (HEL) cells. On the other hand, to develop receptor antagonists of pancreastatin (PST), we needed a simple bioassay method for screening of receptor antagonists to PST.We therefore first examined the effects of PST on the increase in[Ca2+]i induced by several insulin secretagogues by using the dissociated rat pancreatic B-cells. We found that PST (1-100nM) dose-dependently inhibited the glucose-induced rise in[Ca2+]i insingle pancreatic islet cells. This observation allows us to develop an assay method for screening of the PST antagonists and to synthesize a series of PST analogs possibly used as receptor antagonists to PST.This study demonstrates that the analog mixture screening strategy significantly reduces the time and resources previously required for the development of receptor antagonists or agonists, and promises to enhance our ability to design new pharmacological agents for therapeutic uses.