The mechanism of the GAPDH-catalyzed reaction involves nucleophilic attack of the active-site cysteine, C152, on the aldehyde group of G3P, followed by subsequent hydride transfer to NAD +, resulting in the formation of a covalent enzyme-substrate complex. GAPDH catalyzes the conversion of glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3 BPG) in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD +). 6,7 As a result, GAPDH inhibition has been identified as a promising strategy for the treatment of cancer and autoimmune diseases. 5 Furthermore, overexpression of GAPDH has been reported in a variety of cancers and is associated with a poor survival rate. However, flux control analysis identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a rate-limiting step in Warburg metabolism. 4 Therapeutic strategies for targeting Warburg metabolism have largely focused on targeting hexokinase and phosphofructokinase, enzymes that traditionally regulate glycolytic flux. 3 Another hexokinase inhibitor, 2-deoxyglucose (2-DG), reduced joint inflammation and immune-cell activation in an autoimmune model. Recently, d-mannoheptulose, an inhibitor of hexokinase, was shown to enhance anti-tumor effects and reduce growth of breast cancer cells by inducing apoptosis. As a result, glycolytic enzymes have emerged as promising therapeutic targets for cancer and immunomodulation. 1,2 Compared to normal cells, cancer cells and activated immune cells are more dependent on glycolysis than oxidative phosphorylation for ATP production. This metabolic reprogramming is known as the Warburg effect. In order to support the biosynthetic demands of rapidly dividing cells, energy metabolism is reprogrammed to increase glucose uptake and lactic-acid fermentation, independent of oxygen supply. 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.ĭysregulation of glycolysis is a hallmark of cancer and other diseases. 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. 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. Our mechanistic studies confirm that KA is a highly effective irreversible inhibitor of GAPDH, which acts through a NAD +-uncompetitive and G3P-competitive mechanism. We then further evaluated KA, to determine the detailed mechanism of inhibition. 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). 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. 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. Dysregulated GAPDH activity is associated with a variety of pathologies, and GAPDH inhibitors have demonstrated therapeutic potential as anticancer and immunomodulatory agents. In addition to its role in metabolism, GAPDH is also implicated in diverse cellular processes, including transcription and apoptosis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a central enzyme in glycolysis that regulates the Warburg effect in cancer cells.
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