Yongfeng Tao

Proteasome regulatory particles determine cellular protein degradation capacities. Yet specific ubiquitin-independent degradation targets of individual proteasome regulatory particles, and physiological roles of such pathways remain largely elusive. Here, through a chemical biology-driven approach, we identify a covalent small molecule that selectively targets a conformationally dynamic pocket in the non-catalytic proteasome regulator PSME4 and modulates tumor cell proteasome targets. Using this chemical probe together with orthogonal genetic perturbations, we show that blocking or reducing PSME4 stabilizes STAT1, restores interferon-associated transcription, enhances antigen presentation, and disrupts IL-6/STAT3 signaling, collectively reprogramming tumor inflammation and suppressing tumor growth in vivo . These findings define a PSME4-STAT1-STAT3 axis that couples proteasomal degradation to antigen presentation and cytokine signaling and provide a framework for pharmacologically targeting non-catalytic proteasome regulators to reprogram tumor immunity. More broadly, our findings reveal that proteasome composition can actively govern the balance between immunostimulatory and immunosuppressive signaling programs.

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.

5‐Methylcytosine (m⁵C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1‐7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C⁵ position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell‐active inhibitors of these enzymes are lacking. Here, we use cysteine‐directed activity‐based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross‐reactivity with other human NSUNs and exhibit good proteome‐wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2‐tRNA interactions in cancer cells, leading to a global reduction in tRNA m⁵C content. Our findings thus highlight the potential to create isotype‐selective and cell‐active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.

5‐Methylcytosine (m⁵C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1‐7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C⁵ position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell‐active inhibitors of these enzymes are lacking. Here, we use cysteine‐directed activity‐based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross‐reactivity with other human NSUNs and exhibit good proteome‐wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2‐tRNA interactions in cancer cells, leading to a global reduction in tRNA m⁵C content. Our findings thus highlight the potential to create isotype‐selective and cell‐active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.

Residual tumor burden and chemotherapy-resistance point to a need for drug leads active toward cancer stem cells. Just as natural products have led the way for current treatments, the potential for novel applications of these molecules remains high.

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

Pharmacophore-directed retrosynthesis applied to ophiobolin A led to bicyclic derivatives that were synthesized and display anticancer activity. Key features of the ultimate defensive synthetic strategy include a Michael addition/facially selective protonation sequence to set the critical C6 stereocenter and a ring-closing metathesis to form the cyclooctene. Cytotoxicity assays toward a breast cancer cell line (MDA-MB-231) confirm the anticipated importance of structural complexity for selectivity (vs MCF10A cells) while C3 variations modulate stability.

The hypercalins are dearomatized acylphloroglucinols with a pendant complex cyclopentane ring that exhibit activity against several cancer cell lines. We report the first total synthesis of (+)‐hypercalin C employing a convergent strategy that enabled the dissection of the essential structural features required for the observed anticancer activity. A strategic disconnection involving an unusual C –C Suzuki–Miyaura coupling with an α‐bromo enolether also revealed an unexpected C−H activation. This strategy targeted designed analogues along the synthetic route to address particular biological questions. These results support the hypothesis that hypercalin C may act as a proton shuttle with the dearomatized acylphloroglucinol moiety being essential for this activity.

Identifying the covalent targets of a natural product-derivative in cancer cells.