Figure 6

CPI-613 induces reactive oxygen species-mediated glutathionylation and inhibition of tumor cell α-ketoglutarate dehydrogenase. (A) KGDH E2 enriched in CPI-613-treated cells following capture of glutathionylated proteins using biotin-switch (text). KGDH glutathionylation suppressed by co-treatment with 250 μM NAC. (B) Cells were treated with 240 μM CPI-613 then exposed to 100 mM NEM followed by 100 mM DTT, chemically reversing redox modifications. Western blot analysis of native KGDH lipoate indicates increased levels of NEM-protected residues in CPI-613-treated samples. Thus, KGDH lipoate sulfurs are a target of drug-induced redox modification. (C) α-ketoglutarate produces robust in vitro NAD⁺ reduction and this process is significantly inhibited in CPI-613-treated cells. This inhibition is relieved by 10 mM DTT treatment of the lysates. Thus, KGDH from CPI-613-treated cells is inhibited by redox modification. (D) CPI-613 (2 hours at 240 μM) selectively inhibits mitochondrial ATP production driven by PDH and KGDH substrates, pyruvate and glutamine, but not driven by fatty acid oxidation. (E) 500 μM NAC increases carbon flux through KGDH, suggesting a role for H₂O₂ in KGDH regulation in H460 tumor cells. NAC treatment strongly reverses the inhibition of KGDH activity by CPI-613. The magnitude of this effect (4.7-fold) is greater than the increase in carbon flux in untreated cells (2.3-fold), indicating that NAC acts to reverse CPI-613 inhibition of KGDH activity in addition to its effects on KGDH regulation in absence of drug. (F) Proposed model of mechanism of action of CPI-613 on KGDH. CPI-613 may ‘misinform’ existing tumor cell KGDH redox autoregulation (shaded block arrow), increasing this ROS signal generated by the E3 subunit (including from the reverse reaction, NADH oxidation), resulting in E2-subinit redox modification and KGDH inhibition. All results are representative of at least three independent experiments. Error bars represent SEM. DMSO, dimethyl sulfoxide; DTT, dithiothreitol; KGDH, α-ketoglutarate dehydrogenase; NAC, N-acetylcysteine; NEM, N-ethylmaleimide.