Abstract
Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and K i values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.
Highlights
Mitochondrial sn-glycerol 3-phosphate dehydrogenase is an important link between cytosolic and mitochondrial energy transduction. mGPDH is a ubiquinone-linked flavoprotein embedded in the outer leaflet of the mitochondrial inner membrane that transfers reducing equivalents directly from glycerol 3-phosphate into the electron transport chain [1,2]
Neither glyceraldehyde 3-phosphate nor dihydroxyacetone phosphate (DHAP) is oxidized by mitochondria [14] and they may be useful as selective inhibitors of mGPDH in isolated mitochondria
Consistent with their known effects on mGPDH enzymatic activity [2,13,14,15], both glyceraldehyde 3-phosphate and DHAP inhibited the rate of H2O2 production from mGPDH (Fig. 2A)
Summary
Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH3; EC 1.1.5.3; gene symbol GPD2) is an important link between cytosolic and mitochondrial energy transduction. mGPDH is a ubiquinone-linked flavoprotein embedded in the outer leaflet of the mitochondrial inner membrane that transfers reducing equivalents directly from glycerol 3-phosphate into the electron transport chain [1,2]. Its oxidation to dihydroxyacetone phosphate (DHAP) by mGPDH and the subsequent reduction of DHAP back to glycerol 3phosphate by a distinct soluble GPDH (cytosolic GPDH; cGPDH; EC 1.1.1.8; gene symbol GPD1) regenerates NAD+ consumed during glycolysis (Fig. 1A) This cyclic process of transferring reducing equivalents from cytosolic NADH into the mitochondrial respiratory chain is known as the glycerol phosphate shuttle. The active site of mGPDH faces the mitochondrial intermembrane space, as does its calcium-sensitive EF-hand domain that lowers the Km for glycerol 3-phosphate as physiological levels of free calcium rise [1,2,4,5] This orientation is thought to allow mGPDH to coordinate cytosolic and mitochondrial metabolism during periods of high activity and, not surprisingly, mGPDH is expressed most highly in tissues with variable energy demands including thermogenic brown fat, type II skeletal muscle fibers, brain, sperm and pancreatic b-cells [6,7]. Ablation of cGPDH as well as mGPDH prevents compensatory responses in glycerol 3-phosphate metabolism, causes dramatic changes in metabolic profiles, and is lethal within one week of birth [12]
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