A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence

// Ju-Yi Hsieh 1, 2, * , Shao-Yu Li 1, * , Wen-Chen Tsai 1, 2 , Jyung-Hurng Liu 3, 4 , Chih-Li Lin 5 , Guang-Yaw Liu 2 , Hui-Chih Hung 1, 3, 4 1 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan 2 Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan 4 Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan 5 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Hui-Chih Hung, e-mail: hchung@dragon.nchu.edu.tw Guang-Yaw Liu, e-mail: liugy@csmu.edu.tw Keywords: allosteric inhibitor, selective inhibitor, non-competitive inhibition, mutagenesis analysis, cellular senescence Received: February 20, 2015 Accepted: May 06, 2015 Published: May 19, 2015 ABSTRACT Here, we found a natural compound, embonic acid (EA), that can specifically inhibit the enzymatic activity of mitochondrial NAD(P) + -dependent malic enzyme (m-NAD(P)-ME, ME2) either in vitro or in vivo . The in vitro IC 50 value of EA for m-NAD(P)-ME was 1.4 ± 0.4 μM. Mutagenesis and binding studies revealed that the putative binding site of EA on m-NAD(P)-ME is located at the fumarate binding site or at the dimer interface near the site. Inhibition studies reveal that EA displayed a non-competitive inhibition pattern, which demonstrated that the binding site of EA was distinct from the active site of the enzyme. Therefore, EA is thought to be an allosteric inhibitor of m-NAD(P)-ME. Both EA treatment and knockdown of m-NAD(P)-ME by shRNA inhibited the growth of H1299 cancer cells. The protein expression and mRNA synthesis of m-NAD(P)-ME in H1299 cells were not influenced by EA, suggesting that the EA-inhibited H1299 cell growth occurs through the suppression of in vivo m-NAD(P)-ME activity EA treatment further induced the cellular senescence of H1299 cells. However, down-regulation of the enzyme-induced cellular senescence was not through p53. Therefore, the EA-evoked senescence of H1299 cells may occur directly through the inhibition of ME2 or a p53-independent pathway.