An Oncometabolite Isomer Rapidly Induces a Pathophysiological Protein Modification.

Metabolites regulate protein function via covalent and noncovalent interactions. However, manipulating these interactions in living cells remains a major challenge. Here, we report a chemical strategy for inducing cysteine S-succination, a nonenzymatic post-translational modification derived from the oncometabolite fumarate. Using a combination of antibody-based detection and kinetic assays, we benchmark the in vitro and cellular reactivity of two novel S-succination "agonists," maleate and 2-bromosuccinate. Cellular assays reveal maleate to be a more potent and less toxic inducer of S-succination, which can activate KEAP1-NRF2 signaling in living cells. By enabling the cellular reconstitution of an oncometabolite-protein interaction with physiochemical accuracy and minimal toxicity, this study provides a methodological basis for better understanding the signaling role of metabolites in disease.

Bioorthogonal oncometabolite ligation.

Dysregulated cellular metabolism is an emerging hallmark of cancer. Improved methods to profile aberrant metabolic activity thus have substantial applications as tools for diagnosis and understanding the biology of malignant tumors. Here we describe the utilization of a bioorthogonal ligation to fluorescently detect the TCA cycle oncometabolite fumarate. This method enables the facile measurement of fumarate hydratase activity in cell and tissue samples, and can be used to detect disruptions in metabolism that underlie the genetic cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The current method has substantial utility for sensitive fumarate hydratase activity profiling, and also provides a foundation for future applications in diagnostic detection and imaging of cancer metabolism.

A chemoproteomic portrait of the oncometabolite fumarate.

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Photoinducible Oncometabolite Detection.

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.