Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a central enzyme in glycolysis that regulates the Warburg effect in cancer cells. In addition to its role in metabolism, GAPDH is also implicated in diverse cellular processes, including transcription and apoptosis. Dysregulated GAPDH activity is associated with a variety of pathologies, and GAPDH inhibitors have demonstrated therapeutic potential as anticancer and immunomodulatory agents. Given the critical role of GAPDH in pathophysiology, it is important to have access to tools that enable rapid monitoring of GAPDH activity and inhibition within a complex biological system. Here, we report an electrophilic peptide-based probe, SEC1, which covalently modifies the active-site cysteine, C152, of GAPDH to directly report on GAPDH activity within a proteome. We demonstrate the utility of SEC1 to assess changes in GAPDH activity in response to oncogenic transformation, reactive oxygen species (ROS) and small-molecule GAPDH inhibitors, including Koningic acid (KA). We then further evaluated KA, to determine the detailed mechanism of inhibition. Our mechanistic studies confirm that KA is a highly effective irreversible inhibitor of GAPDH, which acts through a NAD+-uncompetitive and G3P-competitive mechanism. Proteome-wide evaluation of the cysteine targets of KA demonstrated high selectivity for the active-site cysteine of GAPDH over other reactive cysteines within the proteome. Lastly, the therapeutic potential of KA was investigated in an autoimmune model, where treatment with KA resulted in decreased cytokine production by Th1 effector cells. Together, these studies describe methods to evaluate GAPDH activity and inhibition within a proteome, and report on the high potency and selectivity of KA as an irreversible inhibitor of GAPDH.
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