Proteome-wide covalent ligand discovery in native biological systems.
Proteome-wide covalent ligand discovery in native biological systems.
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DOI：
10.1038/nature18002
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发表时间：
2016-06-23
期刊：
影响因子：
64.8
通讯作者：
Cravatt BF
中科院分区：
文献类型：
--
作者：
Backus KM;Correia BE;Lum KM;Forli S;Horning BD;González-Páez GE;Chatterjee S;Lanning BR;Teijaro JR;Olson AJ;Wolan DW;Cravatt BF
小分子是研究蛋白质功能的强大工具，可以作为新疗法的先导。然而，大多数人类蛋白质缺乏小分子配体，整个蛋白质类别被认为是“不可成药的”。基于片段的配体发现 (FBLD) 可以识别已证明难以通过复杂化合物库的高通量筛选来靶向的蛋白质的小分子探针。尽管通常追求可逆结合配体，但共价片段为小分子探针提供了替代途径，包括那些可以访问仅通过结合亲和力难以访问的蛋白质区域的探针。在这篇手稿中，我们报告了针对数千种蛋白质筛选的半胱氨酸反应性小分子片段的定量分析。在可成药蛋白质和化学探针缺乏的蛋白质中发现了超过 700 个半胱氨酸的共价配体，包括转录因子、接头/支架蛋白和未表征的蛋白质。在发现的非典型配体-蛋白质相互作用中，有一些化合物优先与前（无活性）半胱天冬酶反应。我们使用这些配体来区分人类细胞系与原代人类 T 细胞中的外源性凋亡途径，表明前者主要由 caspase-8 介导，而后者则依赖于 caspase-8 和 -10。基于片段的共价配体发现提供了可配体蛋白质组的极大扩展的图像，并提供了可以阐明天然生物系统中蛋白质功能的化合物。
Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered “undruggable” . Fragment-based ligand discovery (FBLD) can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries . Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes , including those that can access regions of proteins that are difficult to access through binding affinity alone . In this manuscript, we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T-cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and −10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.