Is there a common allosteric binding site for all GPCRs?

项目摘要 G蛋白偶联受体是人类最大的超家族 拥有800多个成员的蛋白质组。鉴于GPCRs在信号中的关键作用 跨膜转导，GPCRs是头号药物靶点，占 目前市场上约25%的药物。尽管在以下方面取得了重大进展 针对GPCRs的药物发现，许多具有重要药学意义的GPCRs仍然存在 难以捉摸。近年来，有报道称有一种新的场所可以利用变构部位。 关于他们用于治疗各种疾病的药物的开发。鉴定变构 然而，GPCR上的网站仍然是一个挑战。这项建议旨在探讨 跨膜结构域中部以下存在一个共同的变构位点 这与所有人类GPCR的G蛋白结合位点在空间上重叠。长的- 本项目的学期目标是了解变构调节的机制。 GPCRs。 对于这项建议，拟议的工作将包括两个具体目标。我的目标是确定 八种具有代表性的A、B、C类GPCRs的变构拮抗剂和激动剂 和F通过Silico结合到这些GPCR上建议的公共变构位置 筛选和体外试验。为此目的，两份结构已知的GPCR，一份在其 非活性构象和其活性构象中的一个构象，将从每个 A、B、C和F四个非感官GPCR类。然后在电子计算机中筛选锌 数据库将使用每个结构来执行。对于排名靠前的化合物，他们的 将通过计算研究结合部位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、体外活性研究和定点突变研究。 目的II是为了提高已鉴定的GLP-1R变构激动剂的选择性和效力 通过基于结构的分子设计和实验测试。这一目标将会实现 方法：1)获得GLP-1R和GCGR的活性结构，并运行分子 动力学模拟；2)比较GLP-1R模拟的构象快照 与GCGR的构象进行比较，确定GLP-1R的一个构象具有最大的 所有GCGR构象在建议的变构位置的结构差异；3) 将GLP-1R变构激动剂对接到已鉴定的GLP-1R中的变构位置 从LAS步骤整合并在虚拟组合库设计中执行生成 优化化合物；4)将排名靠前的化合物从最后一步重新对接到相同步骤 GCGR构象上与GLP-1R构象最相似的变构部位； 对最后一步中排名最低的化合物进行体外测试。 鉴于这一共同结合部位的战略位置，这一提议的成功 可以为生物学研究和治疗发现靶向提供有用的场所 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.