High-Throughput Screening for the Discovery of Protein-Binding Fragments

项目摘要 基于片段的药物发现计划从鉴定低分子药物开始 与目标蛋白(POI)结合较弱的化合物。POI结合片段有 然后细化和/或链接在一起以提供先导化合物。当前的方法 筛选片段文库的吞吐量很低，许多片段需要大量的 POI，通常以同位素标记的形式。这项提议将建立一种新的筛选 能够分析数百个潜在片段-蛋白质相互作用的平台 单管。我们将创建几十个荧光编码的一珠一化合物 由数百种低分子化合物组成的(OBOC)文库。他们会 与荧光标记的POI的四聚体形式进行筛选。亲和力效应 将充分稳定弱相互作用，使珠子能够通过流动进行分析 细胞学，识别那些能够保留POI的人。如有必要，距离较近 将探索标记以提高系统的灵敏度。这项技术将是 用于发现与VHL E3泛素连接酶结合的新片段。

Project Summary Fragment based drug discovery programs begin with the identification of low molecular compounds that bind weakly to the protein of interest (POI). POI-binding fragments are then elaborated and/or linked together to provide lead compounds. Current methods for screening fragment libraries have low throughput and many require large amounts of the POI, often in isotopically labeled form. This proposal will establish a novel screening platform capable of analyzing hundreds of potential fragment-protein interactions in a single tube. We will create dozens of fluorescently encoded one bead one compound (OBOC) libraries comprised of hundreds of low molecular weight compounds. They will be screened against a fluorescently labeled, tetrameric form of the POI. Avidity effects will stabilize the weak interactions sufficiently to allow the beads to be analyzed by flow cytometry, identifying those capable of retaining the POI. If necessary, proximity labeling will be explored to increase the sensitivity of the system. This technology will be employed to discover novel fragments that engage the VHL E3 Ubiquitin ligase.