In Situ Synthesis Of GPCR Ligands

描述（由申请人提供）：G蛋白偶联受体（GPCR）是人类基因组中最大的膜蛋白家族，约有800个成员。通过多种效应子途径的GPCR信号传导具有深远的治疗意义，使这些受体成为目前市售药物的约40%的靶点。通过小分子配体调节GPCR活性正在成为开发抗癌治疗剂的新策略。然而，有限的结构信息阻碍了设计具有更高疗效和更低副作用的新药，以及详细了解GPCR的作用机制。许多GPCR的结构研究反过来又受到稳定GPCR并使其结晶的高亲和力配体的缺乏的限制。该项目试图使用原位点击化学方法开发一个新的平台，以发现结合GPCR的新型配体，作为结构和功能研究的工具化合物或作为药物发现的起点。包括原位点击化学在内的靶向合成方法，通过该方法将反应性片段连接到生物靶标的结合位点，在药物发现中显示出巨大的前景，但尚未应用于GPCR。挑战在于GPCR的膜处置、其高构象动力学、低稳定性和低表达水平。目前正在开发针对所有GPCR的拟议平台，最初主要关注已知的癌症靶点。在本研究的两年中，我们将重点关注smoothened（SMO）受体，它是一种孤儿受体，也是开发过程中激活的Hedgehog（Hh）信号通路中的关键信号转导子，因此是许多抗肿瘤药物在临床试验中的靶点。我们的目标是实现以下两个目标：（1）建立一个原位点击化学平台，以产生高亲和力的GPCR配体;（2）通过晶体学表征从Aim 1中选择的配体与SMO之间的相互作用。该研究将得到一个团队的支持，该团队开发了气相化学还原结构测定管道及其基础技术，自2007年以来，这些技术已导致对12种气相化学还原结构进行了结构测定。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome with approximately 800 members. GPCR signaling through multiple effector pathways has profound therapeutic implications, making these receptors the targets of ~40% of currently marketed drugs. Modulation of GPCR activity by small molecule ligands is emerging as a new strategy for development of anticancer therapeutics. However, design of new drugs with higher efficacy and lower side effects, and detailed understanding of GPCR mechanism of action are hampered by the limited structural information. Structural studies of many GPCRs are in turn limited by a scarcity of high-affinity ligands that stabilize GPCRs and enable their crystallization. The project attempts to develop a new platform using the in situ click chemistry approach to discover novel ligands that bind GPCRs to serve as tool compounds for structural and functional studies or as starting point for drug discovery. Target-guided synthesis approaches including in situ click chemistry, by which reactive fragments are joined in the binding sites of a biological target, have shown great promise in drug discovery but have not been applied to GPCRs. The challenges lie in the GPCRs' membrane disposition, their high conformational dynamics, low stability and low expression levels. The proposed platform is being developed targeting all GPCRs, with initial focus primarily on those known to be cancer targets. In the two years of this study, we will focus on smoothened (SMO) receptor which is an orphan receptor and a key signal transducer in the Hedgehog (Hh) signaling pathway activated during development, and thus is a target of a number of antitumor drugs in clinical trials. Our goal will be attained by achieving the following two aims: (1) Establish an in situ click chemistry platform to generate high-affinity GPCR ligands, (2) Characterize the interaction between selected ligands from Aim 1 and SMO by crystallography. The study will be supported by a team that had developed the GPCR Structure Determination Pipeline as well as its underlying technologies which have led to the structure determination of 12 GPCR structures since 2007.