Search

Chemical proteomics enables the global analysis of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, remained limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these "photo-stereoprobes" interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible NanoBRET assays. Integrated phenotypic screening and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of DOS-inspired photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and facilitating the discovery and characterization of bioactive compounds in phenotypic screens., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.)

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds-termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.