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Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules., Competing Interests: Declaration of interests B.F.C. is a scientific advisor to Vividion Therapeutics. M.A.E. holds equity in Nexo Therapeutics and serves on their scientific advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells., Competing Interests: Declaration of interests G.M.S., V.F.V., and L.R.W. are employees of Vividion Therapeutics, and B.F.C. is a founder and member of the Board of Directors of Vividion Therapeutics. G.W.Y. is a co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y.’s interests have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. A US provisional patent has been filed related to the work disclosed in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)