A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration

Abstract

We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells.

Fig 1. In vitro MDH2 inhibitory activity of compounds.

^aMDH2 enzyme activity was determined by oxaloacetate-dependent NADH oxidation assays. Values are the means of three experiments. ^bN.D. measns non-determined. ^c‘–’ means no effect.

Fig 2. Inhibition of MDH2 activity by compound 7.

(A) Effect of compound 7 on MDH2 activity. In vitro MDH2 activity was determined by oxaloacetate-dependent NADH oxidation assays. Statistical significance: *P < 0.05 and **P < 0.01, compared with untreated control. (B) Kinetic study of MDH2 by compound 7. Double-reciprocal plot of the inhibitory effect of compound 7 against NADH-dependent MDH2 activity. Concentration of compound 7 (in μM): 0 (closed circle), 1.25 (open circle), 2.5 (closed triangle), and 5 (open triangle). (C) Molecular docking study of MDH2 compound 7. Docking simulation showing the binding of compound 7 in the NADH binding site of MDH2 (PDB ID code 2DFD). The numbers of amino acid residues are based on PDB structure.

Fig 3. Inhibition of mitochondrial respiration by compound 7.

(A) Effect of compound 7 on mitochondrial respiration. OCR was measured using XF24 extracellular flux analyzer by adding oligomycin (1 μM), FCCP (0.5 μM), and rotenone (1 μM)/antimycin A (1 μM) to compound 7-treated HCT116 cells. (B) Effect of compound 7 on intracellular oxygen tension. HCT116 cells were treated with compound 7 for 6 h under hypoxia condition. The intracellular oxygen tension was detected by hypoxia-sensitive probe MAR (0.5 μM). The scale bar indicates 200 μm. The fluorescence intensity of MAR probe was shown by vertical bars. Statistical significance: **P < 0.01, compared with hypoxia control.

Fig 4. Inhibition of hypoxia-induced HIF-1α activation and tumor growth by compound 7.

(A) Effect of compound 7 on HIF-1α activation. The hypoxia-induced transcriptional activation of HIF were determined by a cell-based HRE reporter gene assay. HCT116 cells stably expressing HRE-luciferase reporter gene were incubated for 12 h with or without drugs under normoxic or hypoxic conditions. After removing the supernatant, luciferase activity was measured. Statistical significance: **P < 0.01, compared with untreated control. (B) Effects of compound 7 on hypoxia-induced HIF-1α accumulation. The protein levels in compound 7-treated cells were detected by immunoblot analysis in HCT116 cells. (C) Effects of compound 7 on expression of mRNA level of HIF-1α target genes. The mRNA levels were detected by RT-PCR analysis. (D) Inhibitory effects of compound 7 on HIF-1α accumulation in various cancer cells.

Fig 5. Reduction of intracellular ATP content and fatty acids by compound 7.

(A) Effect of compound 7 on ATP level. After treatment of HCT116 cells with compound 7 for 6 hr, intracellular ATP content was measured using a luciferase-based assay system. (B) Activation of ATP-related signaling by compound 7. The protein levels in compound 7-treated cells were detected by immunoblot analysis. (C) Suppression of level of intracellular fatty acids by compound 7. HCT116 cells were incubated with compound 7 for 48 hr, and then intracellular fatty acids were stained with Nile Red. Statistical significance: *P < 0.05 and **P < 0.01, compared with untreated control.

Fig 6. In vivo anti-tumor effects of compound 7 in HCT116 xenograft model.

(A) Inhibition of tumor growth by compound 7. Total 5 × 10⁶ HCT116 cells were subcutaneously inoculated in the right flank of female nude mice. Drugs were administered intraperitoneally once a day for 14 days after reaching a tumor volume of 80–100 mm³. (B) Images of xenografts and tumor weight in control group and compound 7-treated group at the end of the experiment. Statistical significance: **P < 0.01, compared with vehicle control.