Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.

去泛素化酶 (DUB) 是一类新兴的药物靶点，由约 100 种蛋白酶组成，可将泛素从蛋白质底物上裂解下来，从而调节许多细胞过程。缺乏选择性化学探针阻碍了对这一重要基因家族的药理学研究。 DUB 通过无数的相互作用来接合它们的同源配体。我们利用这种结构复杂性，为以 DUB 为重点的共价库定制化学多样化策略。将我们的文库与基于活性的蛋白质分析配对作为高密度初级筛选，我们识别出针对跨越四个亚家族的 23 个内源 DUB 的选择性命中。对氮杂环丁烷命中的优化产生了一种针对正在研究的 DUB VCPIP1 的探针，具有纳摩尔效力和家族内选择性。我们成功地从一个适度但专门构建的文库中识别出良好的化学起点以及跨基因家族的结构-活性关系，这对当前强调超高通量体外或虚拟筛选以应对不断扩大的化学空间范围的范例提出了挑战。