Epoxycyclohexenedione-Type Compounds Make Up a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier

Abstract

Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). This study investigated the mechanism of inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs upon being incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration in AAC without forming intermolecular S-S linkages. When all solvent-accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has an alkyl chain that is shorter than those of AMM-59 and -120, was significantly less efficient than that of the two compounds. On the basis of these results, the mechanism underlying the aggregation of AAC induced by ECHDs is discussed.