用基于NanoBiT的新型探针研究多肽激素LEAP2识别受体GHSR1a的分子机制

LEAP2是一个高度保守的分泌多肽，2017年末有报导称其是ghrelin受体GHSR1a的非竞争性拮抗剂。然而最近我们发现LEAP2其实是一个竞争性拮抗剂，并具有不易从受体解离的特性及加入方式依赖的罕见拮抗模式。鉴于LEAP2在能量代谢中的重要调控作用及在肥胖等疾病治疗中的应用前景，我们希望用基于NanoBiT的新型探针研究其识别受体的分子机制。首先，通过丙氨酸扫描全面解析LEAP2中氨基酸残基的功能，确定影响氧化折叠、受体结合与解离、拮抗功能与模式及反向激动功能的关键残基。在此基础上，对GHSR1a胞外区一些残基进行突变，根据突变受体与突变配基的结合活力互补，确定受体与LEAP2两者间一些重要相互作用残基的对应关系，揭示两者结合的关键作用力。最后，根据上面的研究结果对LEAP2进行合理改造，提高拮抗效果。该工作有望阐明LEAP2识别受体的分子机制并获得一些新型类似物，促进其药物研发。

LEAP2 is a highly conserved secretory peptide, and was reported as a non-competitive antagonist for the ghrelin receptor GHSR1a at the end of 2017. However, we recently found that LEAP2 was actually a competitive antagonist with a slow receptor dissociation property and an unusual addition manner-dependent antagonistic function. Considering its important regulatory role in energy metabolism and its therapeutic potential for treatment of obesity and some other diseases, we plan to investigate the molecular mechanism for LEAP2 recognizing its receptor using NanoBiT-based novel probes. First, we plan to systematically analyze functionality of LEAP2 residues through alanine-scanning mutagenesis, and thus identify key residues responsible for its oxidative folding, receptor binding and dissociation, antagonistic potency and antagonistic manner, and reverse agonistic function. On this basis, we plan to conduct mutagenesis on some extracellular residues of GHSR1a and identify some important interaction pairs between those receptor residues and ligand residues according to binding activity complementation between mutant receptors and mutant ligands, and thus disclose key interactions for binding of LEAP2 with receptor GHSR1a. Finally, we plan to conduct rational design on LEAP2 according to above results and thus improve its antagonistic function. This project will elucidate the molecular mechanism for LEAP2 recognizing its receptor, obtain some novel LEAP2 analogues, and promote LEAP2 drug development.