Activity-Based Acylome\nProfiling with <i>N</i>‑(Cyanomethyl)‑<i>N</i>‑(phenylsulfonyl)amides\nfor Targeted Lysine Acylation and Post-Translational Control of Protein\nFunction in Cells

Lysine acylations are ubiquitous and structurally diverse\npost-translational\nmodifications that vastly expand the functional heterogeneity of the\nhuman proteome. Hence, the targeted acylation of lysine residues has\nemerged as a strategic approach to exert biomimetic control over the\nprotein function. However, existing strategies for targeted lysine\nacylation in cells often rely on genetic intervention, recruitment\nof endogenous acylation machinery, or nonspecific acylating agents\nand lack methods to quantify the magnitude of specific acylations\non a global level. In this study, we develop activity-based acylome\nprofiling (ABAP), a chemoproteomic strategy that exploits elaborate <i>N</i>-(cyanomethyl)-<i>N</i>-(phenylsulfonyl)amides\nand lysine-centric probes for site-specific introduction and proteome-wide\nmapping of posttranslational lysine acylations in human cells. Harnessing\nthis framework, we quantify various artificial acylations and rediscover\nnumerous endogenous lysine acylations. We validate site-specific acetylation\nof target lysines and establish a structure–activity relationship\nfor <i>N</i>-(cyanomethyl)-<i>N</i>-(phenylsulfonyl)amides\nin proteins from diverse structural and functional classes. We identify\nparalog-selective chemical probes that acetylate conserved lysines\nwithin interferon-stimulated antiviral RNA-binding proteins, generating <i>de novo</i> proteoforms with obstructed RNA interactions. We\nfurther demonstrate that targeted acetylation of a key enzyme in retinoid\nmetabolism engenders a proteoform with a conformational change in\nthe protein structure, leading to a gain-of-function phenotype and\nreduced drug potency. These findings underscore the versatility of\nour strategy in biomimetic control over protein function through targeted\ndelivery and global profiling of endogenous and artificial lysine\nacylations, potentially advancing therapeutic modalities and our understanding\nof biological processes orchestrated by these post-translational modifications.