Global targeting of functional tyrosines using sulfur-triazole exchange chemistry.

Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.

Direct Target Site Identification of a Sulfonyl-Triazole Covalent Kinase Probe by LC-MS Chemical Proteomics.

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.

Deciphering T Cell Immunometabolism with Activity-Based Protein Profiling.

As a major sentinel of adaptive immunity, T cells seek and destroy diseased cells using antigen recognition to achieve molecular specificity. Strategies to block checkpoint inhibition of T cell activity and thus reawaken the patient's antitumor immune responses are rapidly becoming standard of care for treatment of diverse cancers. Adoptive transfer of patient T cells genetically engineered with tumor-targeting capabilities is redefining the field of personalized medicines. The diverse opportunities for exploiting T cell biology in the clinic have prompted new efforts to expand the scope of targets amenable to immuno-oncology. Given the complex spatiotemporal regulation of T cell function and fate, new technologies capable of global molecular profiling in vivo are needed to guide selection of appropriate T cell targets and subsets. In this chapter, we describe the use of activity-based protein profiling (ABPP) to illuminate different aspects of T cell metabolism and signaling as fertile starting points for investigation. We highlight the merits of ABPP methods to enable target, inhibitor, and biochemical pathway discovery of T cells in the burgeoning field of immuno-oncology.

Next-Generation Cell-Active Inhibitors of the Undrugged Oncogenic PTP4A3 Phosphatase.

Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.

Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics.

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.

CRISPR Screen Reveals BRD2/4 Molecular Glue-like Degrader via Recruitment of DCAF16.

Molecular glues (MGs) are monovalent small molecules that induce an interaction between proteins (native or non-native partners) by altering the protein-protein interaction (PPI) interface toward a higher-affinity state. Enhancing the PPI between a protein and E3 ubiquitin ligase can lead to degradation of the partnering protein. Over the past decade, retrospective studies of clinical drugs identified that immunomodulatory drugs (e.g., thalidomide and analogues) and indisulam exhibit a molecular glue effect by driving the interaction between non-native substrates to CRBN and DCAF15 ligases, respectively. Ensuing reports of phenotypic screens focused on MG discovery have suggested that these molecules may be more common than initially anticipated. However, prospective discovery of MGs remains challenging. Thus, expanding the repertoire of MGs will enhance our understanding of principles for prospective design. Herein, we report the results of a CRISPR/Cas9 knockout screen of over 1000 ligases and ubiquitin proteasome system components in a BRD4 degradation assay with a JQ1-based monovalent degrader, compound 1a. We identified DCAF16, a substrate recognition component of the Cul4 ligase complex, as essential for compound activity, and we demonstrate that compound 1a drives the interaction between DCAF16 and BRD2/4 to promote target degradation. Taken together, our data suggest that compound 1a functions as an MG degrader between BRD2/4 and DCAF16 and provides a foundation for further mechanistic dissection to advance prospective MG discovery.

Modulation of PKM activity affects the differentiation of TH17 cells.

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-ß1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function.