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Expanding the ligandable proteome by paralog hopping with covalent probes

Authors
Yuanjin Zhang,Zhonglin Liu
Marsha Hirschi,Oleg Brodsky,Eric Johnson,Sang Joon Won,Asako Nagata,Matthew D Petroski,Jaimeen D Majmudar,Sherry Niessen,Todd VanArsdale,Adam M Gilbert,Matthew M Hayward,Al E Stewart,Andrew R Nager,Bruno Melillo,Benjamin F Cravatt,Qian Zhang,Martin Hirschi,Sang Won,Jaimeen Majmudar,A. Gilbert,Matthew Hayward,Albert Stewart,Andrew Nager,Matthew Petroski,Adam Gilbert
+25 authors
,Benjamin Cravatt
Published
Jan 20, 2024
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Abstract

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 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 provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

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