Is there a common allosteric binding site for all GPCRs?

项目摘要 G蛋白偶联受体（GPCR）是人类免疫系统中最大的超家族。 蛋白质组有800多个成员。鉴于GPCR在信号转导中的关键作用， 在跨膜转导中，GPCR是头号药物靶点， 目前市场上约25%的药物。尽管取得了重大进展， 靶向GPCR的药物发现，许多药学上重要的GPCR仍然存在， 难以捉摸。近年来，一种新的场地已被报道利用变构位点 用于开发治疗各种疾病的药物。识别变构 然而，在全球项目完成情况报告上建立网站仍然是一个挑战。本建议旨在探讨 跨膜结构域中部以下存在共同的变构位点 与所有人类GPCR的G蛋白结合位点在空间上重叠。很长的- 本项目的长期目标是了解变构调节的机制， GPCR。 对于本提案，拟议工程将包括两个具体目标。目的一是确定 来自A、B、C类的八种代表性GPCR的变构拮抗剂和激动剂 和F，其通过计算机模拟结合到这些GPCR上的拟议的共同变构位点， 筛选和体外试验。为了这个目的，两个具有已知结构的GPCR，一个在其 非活性构象和一个处于其活性构象，将选自每一种 四种非感官GPCR类别，A、B、C和F。然后针对ZINC进行计算机模拟筛选 将使用每个结构执行数据库。对于排名靠前的化合物， 将通过计算研究结合位点的结合稳定性和结合亲和力 使用分子动力学模拟。最后，对于那些排名靠前的对手和 将进行激动剂、体外活性研究和位点特异性诱变研究。 目的二是提高GLP-1 R变构激动剂的选择性和效价 通过基于结构的分子设计和实验测试。这一目标将得到实现 1）获得GLP-1 R和GCGR两者的活性结构并运行分子 动力学模拟; 2）比较GLP-1 R模拟的构象快照 与GCGR的构象进行比较，并确定GLP-1 R的一种构象， 与所有GCGR构象在拟议变构位点的结构差异; 3） 将GLP-1 R变构激动剂对接至已鉴别GLP-1 R中的变构位点 从LAS步骤和虚拟组合库设计中进行构象生成 优化的化合物; 4）将最后一步中排名靠前的化合物重新对接到相同的化合物 GCGR构象上与GLP-1 R构象最相似的变构位点;和5） 体外分析测试来自最后步骤的最低等级的化合物。 鉴于这一共同结合点的战略位置， 可以为生物学研究和靶向治疗发现提供有用的场所 各种GPCR。

Project Summary G-protein coupled receptors (GPCRs) represent the largest superfamily of human proteome with more than 800 members. Given the key role of GPCRs in signal transduction across membranes, GPCRs are the number one drug target, accounting for approximately 25% of the current drugs in the market. Despite significant progresses in drug discovery targeting GPCRs, many pharmaceutically important GPCRs remain elusive. In recent years, a novel venue has been reported to exploit the allosteric sites on them for the development of drugs to treat various diseases. Identifying the allosteric sites on GPCRs, however, remains a challenge. This proposal aims to explore the existence of a common allosteric site below the middle of the transmembrane domain that spatially overlaps with the G-protein binding site for all human GPCRs. The long- term objective of this project is to understand the mechanism of allosteric regulations in GPCRs. For this proposal, proposed works will include two specific Aims. Aim I is to identify allosteric antagonists and agonists of eight representative GPCRs from Class A, B, C and F that bind to the proposed common allosteric site on these GPCRs through in silico screening and in vitro test. For this aim, two GPCRs with known structure, one in its inactive conformation and one in its active conformation, will be chosen from each of the four non-sensory GPCR Classes, A, B, C and F. Then in silico screening against ZINC database will be performed using each structure. For top-ranked compounds, their binding stability and binding affinity in the binding site will be computationally studied using molecular dynamics simulations. Finally, for those top-ranked antagonists and agonists, in vitro activity studies and site-specific mutagenesis studies will be carried out. Aim II is to improve the selectivity and potency of the identified GLP-1R allosteric agonist through structure-based molecule design and experimental test. This aim will be carried out by: 1) obtain the active structure of both GLP-1R and GCGR and run molecular dynamics simulations; 2) compare the conformation snapshots from GLP-1R simulations with those of GCGR and identify one conformation of GLP-1R that have the largest structural difference at the proposed allosteric site from all GCGR conformations; 3) dock the GLP-1R allosteric agonist to the allosteric site in the identified GLP-1R conformation from las step and perform in virtual combinatorial library design to generate optimized compounds; 4) re-dock the top-ranked compounds from last step to the same allosteric site on the GCGR conformation most similar to GLP-1R conformation; and 5) in vitro assay test the lowest ranked compounds from last step. Given the strategic location of this common binding site, the success of this proposal could provide a useful venue for biological studies and therapeutic discovery targeting various GPCRs.