Zhang Yuanjin, Liu Zhonglin, Hirschi Marscha, Brodsky Oleg, Johnson Eric, Won Sang Joon, Nagata Asako, Bezwada Divya, Petroski Matthew D, Majmudar Jaimeen D, Niessen Sherry, VanArsdale Todd, Gilbert Adam M, Hayward Matthew M, Stewart Al E, Nager Andrew R, Melillo Bruno, Cravatt Benjamin F
Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
Medicine Design, Pfizer Research and Development, Pfizer, Inc., La Jolla, CA, USA.
Nat Chem Biol. 2025 Mar;21(3):420-431. doi: 10.1038/s41589-024-01738-7. Epub 2024 Sep 18.
More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile-cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.
在约20000个人类蛋白质编码基因中，超过一半都有旁系同源基因。化学蛋白质组学已发现许多仅存在于旁系同源蛋白质亚群中的亲电敏感半胱氨酸。在此，我们探究这种共价化合物 - 半胱氨酸相互作用是否可用于在缺乏半胱氨酸的旁系同源物中发现可结合配体的口袋。利用细胞周期蛋白CCNE2中C109的共价配体结合能力，我们将旁系同源物CCNE1中的相应残基替换为半胱氨酸（N112C），并通过基于活性的蛋白质谱分析发现，该突变体与色胺丙烯酰胺发生立体选择性和位点特异性反应。然后，我们将色胺丙烯酰胺 - CCNE1 - N112C相互作用转化为体外纳米生物发光共振能量转移（NanoBRET）和细胞内基于活性的蛋白质谱分析方法，这些方法能够鉴定出可逆抑制N112C突变体和野生型CCNE1:CDK2（细胞周期蛋白依赖性激酶2）复合物的化合物。X射线晶体学揭示了在CCNE1:CDK2界面处与N112相邻的一个隐藏的变构口袋，该口袋可结合可逆抑制剂。因此，我们的研究结果表明，通过化学蛋白质组学绘制的亲电试剂 - 半胱氨酸相互作用如何能够将对蛋白质配体结合能力的理解扩展到共价化学之外。
