Whereas NAD linked cGPDH can interconvert DHAP and glycerol 3-phosphate, the ubiquinone-linked reaction catalyzed by mGPDH is irreversible and displays product inhibition by DHAP. Additionally, lipid metabolites such as free fatty acids and acyl CoAs, inorganic ions such as sulfate and phosphate, and several heavy metal ions also inhibit FAD-linked GPDH activity. Importantly, many of these are not cell-permeable and none is selective for mGPDH. Other proposed small-molecule inhibitors of mGPDH activity such as polyborates, indomethacin, doxorubicin, or diazoxide are also not selective. Therefore, a need remains for selective and cell-permeant inhibitors of mGPDH to elucidate the role of this widely-expressed enzyme under 1474110-21-8 appropriate physiological conditions. Here we describe the identification and characterization of a novel class of smallmolecule inhibitors of mGPDH activity that are cellpermeant, potent, and highly selective for mGPDH. The effects of small-molecule inhibitors on respiration were tested in plate-attached skeletal muscle mitochondria using a Seahorse XF24 Analyzer according to published protocols. Briefly, mitochondria were attached to Seahorse assay plates by centrifugation in a mannitol and sucrose-based medium containing 0.3% BSA without or with 250 nM free calcium at pH 7.0. Compounds were titrated to 80 mM on each of four parallel plates with media containing one of the following substrates: 10 mM pyruvate and 0.5 mM malate; 5 mM glutamate and 5 mM malate; 5 mM succinate and 4 mM rotenone; or 16.7 mM glycerol phosphate and 4 mM rotenone with 250 nM free calcium. Compounds were added in these media just prior to loading the assay plate into the Seahorse instrument. Motivated by the observation that subtle changes in structure 1332295-35-8 resulted in changes to both the potency and selectivity of our novel mGPDH inhibitors, we tested an additional 18 compounds structurally related to the top hits in our primary screen to identify structural features that determined the relative potency and selectivity for inhibition of H2O2 production by mGPDH. These 20 compounds were retested for effects on eight assays of site-specific H2O2 production and fo
