Redirecting the pioneering function of FOXA1 with covalent small molecules.

Pioneer transcription factors (TFs) exhibit a specialized ability to 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 small molecules that stereoselectively and site-specifically bind the pioneer TF, 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 covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary 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.

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders.

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.

Expanding the ligandable proteome by paralog hopping with covalent probes.

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.

Covalent Targeting As a Common Mechanism for Inhibiting NLRP3 Inflammasome Assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines, including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remain poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit the inflammasome assembly and activity. From this screen, we identify and profile inflammasome inhibition of 20 new covalent compounds across nine different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks the activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular cross-linking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, further support the continued development of electrophilic compounds that target reactive cysteine residues on NLRP3 to regulate its activation and activity.

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2.

5-Methylcytosine (m5 C) 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 m5 C 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.

Assigning functionality to cysteines by base editing of cancer dependency genes.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Collateral lethality between HDAC1 and HDAC2 exploits cancer-specific NuRD complex vulnerabilities.

Transcriptional co-regulators have been widely pursued as targets for disrupting oncogenic gene regulatory programs. However, many proteins in this target class are universally essential for cell survival, which limits their therapeutic window. Here we unveil a genetic interaction between histone deacetylase 1 (HDAC1) and HDAC2, wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal deletions that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic disruption or dTAG-mediated degradation, we show that targeting HDAC2 suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2-NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders that could leverage HDAC1/2 synthetic lethality to target NuRD vulnerabilities. Altogether, we identify HDAC1/2 collateral synthetic lethality as a potential therapeutic target and reveal an unexplored mechanism for targeting NuRD-associated cancer dependencies.

Dimethyl fumarate inhibits ZNF217 and can be beneficial in a subset of estrogen receptor positive breast cancers.

PURPOSE: The oncogenic factor ZNF217 promotes aggressive estrogen receptor (ER)+breast cancer disease suggesting that its inhibition may be useful in the clinic. Unfortunately, no direct pharmacological inhibitor is available. Dimethyl fumarate (DMF) exhibits anti-breast cancer activities, in vitro and in pre-clinical in vivo models. Its therapeutic benefits stem from covalent modification of cellular thiols such as protein cysteines, but the full profile of molecular targets mediating its anti-breast cancer effects remains to be determined. METHODS: ER+breast cancer cells were treated with DMF followed by cysteine-directed proteomics. Cells with modulated ZNF217 levels were used to probe the efficacy of DMF. RESULTS: Covalent modification of ZNF217 by DMF identified by proteomics was confirmed by using a DMF-chemical probe. Inhibition of ZNF217's transcriptional activity by DMF was evident on reported ZNF217-target genes. ZNF217 as an oncogene has been shown to enhance stem-like properties, survival, proliferation, and invasion. Consistent with ZNF217 inhibition, DMF was more effective at blocking these ZNF217-driven phenotypes in cells with elevated ZNF217 expression. Furthermore, partial knockdown of ZNF217 led to a reduction in DMF's efficacy. DMF's in vivo activity was evaluated in a xenograft model of MCF-7 HER2 cells that have elevated expression of ZNF217 and DMF treatment resulted in significant inhibition of tumor growth. CONCLUSION: These data indicate that DMF's anti-breast cancer activities in the ER+HER2+models, at least in part, are due to inhibition of ZNF217. DMF is identified as a new covalent inhibitor of ZNF217.

Proteomic discovery of chemical probes that perturb protein complexes in human cells.

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.

Remodeling oncogenic transcriptomes by small molecules targeting NONO.

Much of the human proteome is involved in mRNA homeostasis, but most RNA-binding proteins lack chemical probes. Here we identify electrophilic small molecules that rapidly and stereoselectively decrease the expression of transcripts encoding the androgen receptor and its splice variants in prostate cancer cells. We show by chemical proteomics that the compounds engage C145 of the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress an array of cancer-relevant genes and impair cancer cell proliferation. Surprisingly, these effects were not observed in cells genetically disrupted for NONO, which were instead resistant to NONO ligands. Reintroduction of wild-type NONO, but not a C145S mutant, restored ligand sensitivity in NONO-disrupted cells. The ligands promoted NONO accumulation in nuclear foci and stabilized NONO-RNA interactions, supporting a trapping mechanism that may prevent compensatory action of paralog proteins PSPC1 and SFPQ. These findings show that NONO can be co-opted by covalent small molecules to suppress protumorigenic transcriptional networks.

TMEM164 is an acyltransferase that forms ferroptotic C20:4 ether phospholipids.

Ferroptosis is an iron-dependent form of cell death driven by oxidation of polyunsaturated fatty acid (PUFA) phospholipids. Large-scale genetic screens have uncovered a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Understanding of the enzymes involved in PUFA-ePL production, however, remains incomplete. Here we show, using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions and targeted lipidomics, that the uncharacterized transmembrane protein TMEM164-the genetic ablation of which has been shown to protect cells from ferroptosis-is a cysteine active center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to produce PUFA ePLs. Genetic deletion of TMEM164 across a set of ferroptosis-sensitive cancer cell lines caused selective reductions in C20:4 ePLs with minimal effects on C20:4 diacyl PLs, and this lipid profile produced a variable range of protection from ferroptosis, supportive of an important but contextualized role for C20:4 ePLs in this form of cell death.

Glutathione supports lipid abundance in vivo.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor.

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

Covalent Targeting of Splicing in T Cells.

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1.

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.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Selective inhibitors of SARM1 targeting an allosteric cysteine in the autoregulatory ARM domain.

The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.