Structure-based Drug Discovery for the GLP-1 Receptor

 说明(申请人提供)：胰升糖素样肽-1受体(GLP-1R)是B G蛋白偶联受体(GPCRs)家族的成员，是治疗2型糖尿病的极具吸引力的靶点。临床研究表明，GLP-1R的激活具有多种抗糖尿病作用，包括促进胰岛素的生物合成、增加葡萄糖依赖型胰岛素的分泌、抑制胰岛细胞的凋亡和促进胰岛细胞的新生。在过去的十年里，一些GLP-1R的多肽激动剂已经上市，但它们的临床应用受到半衰期短、副作用和缺乏口服生物利用度的限制。一种长期禁食、口服活性的GLP-1R小分子药物将极大地促进糖尿病治疗的技术水平。然而，由于缺乏关于GLP-1R的结构信息，这种小分子药物的开发长期以来一直受到阻碍。该方案的最终目的是获得高分辨的活性状态GLP-1R的晶体结构，以便于基于结构的小分子药物的发现。在第一阶段，我们将开发方法和工具来生产毫克量的高质量、活性状态的GLP-1R，用于第二阶段的结晶学研究。GPCR结晶中的一个主要挑战是获得大量处于活性状态的纯、稳定的受体蛋白，就GLP-1R而言，已知在没有额外稳定分子的情况下是不稳定的。这一提议表明，强有力的初步证据表明，在ConfometRx开发的一种新方法使我们能够在毫克量和足够的纯度下形成稳定的GLP-1R-Gs蛋白质复合体，用于结晶。我们还将产生构象选择性的纳米体，这些纳米体可能进一步稳定GLP-1R-Gs复合体，或作为G蛋白替代品来稳定结晶学的活性状态GLP-1R。第二阶段的主要焦点是活性状态GLP-1R的结晶和结构测定。我们将采取全面的策略来增加获得衍射级晶体的机会，例如在不同的脂质环境中结晶，以及通过T4溶菌酶和纳米体技术增强晶格接触。一旦我们获得了GLP1-R的晶体结构，它将使我们能够在电子筛选小分子激动剂或正变构调节剂。新型先导化合物将经过实验验证，并进行基于结构的优化。这项研究的商业化将通过向从事GLP-1R药物工作的制药公司提供数据和服务来实现。这项建议是对NIDDK的RFA-DK-14-008，“用于胰升糖素和内分泌研究的试剂”的回应。

 DESCRIPTION (provided by applicant): Glucagon-like peptide-1 receptor (GLP-1R), a member of family B G protein-coupled receptors (GPCRs), is a highly attractive therapeutic target for type 2 diabetes. It has been shown clinically that the activation of GLP- 1R has many anti-diabetic effects, including potentiating insulin biosynthesis, increasing glucose-dependent insulin secretion, inhibiting pancreatic ß-cell apoptosis and promoting ß-cell neogenesis. A few peptide agonists of GLP-1R have been brought to market in the past decade, but their clinical utility is limited by their short half-life, side effects and lack of oral bioavailability. A long asting, orally active small molecule drug for GLP-1R would significantly advance the state of the art in diabetes treatment. However, the development of such small molecule drugs has long been hindered by a lack of structural information about GLP-1R. The ultimate goal of this proposal is to obtain a high-resolution crystal structure of active-state GLP-1R in order to facilitate structue-based small molecule drug discovery. In Phase I, we will develop methods and tools to produce milligram quantities of high quality, active-state GLP-1R for crystallography studies that will be performed in Phase II. A major challenge in GPCR crystallization is to obtain large quantities of pure, stable receptor protein in its active-state, which in the case of GLP-1R is known to be unstable without additional stabilizing molecules. This proposal shows strong preliminary evidence that a novel method developed at ConfometRx enables us to form a stable GLP-1R-Gs protein complex in milligram quantities and at sufficient purity for crystallization. We will alo generate conformationally selective nanobodies that may further stabilize the GLP-1R-Gs complex or act as G protein surrogates to stabilize the active-state GLP-1R for crystallography. The major focus of Phase II is the crystallization and structure determination of active-state GLP-1R. We will pursue a comprehensive strategy to increase the chances of obtaining diffraction-quality crystals, such as crystallization in different lipid environments and crystal lattice contact enhancement by T4 lysozyme and nanobody techonologies. Once we have obtained the crystal structure of GLP1-R, it will allow us to perform in silico screening of small-molecule agonists or positive allosteric modulators. Novel lead compounds will be experimentally validated and subjected to structure-based optimization. Commercialization of this research will be achieved by providing data and services to pharmaceutical companies working on GLP-1R drugs. This proposal is in response to the NIDDK's RFA-DK-14-008, "Reagents for Glucagon and Incretin Research."