A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence
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Ju-Yi Hsieh1,2,*, Shao-Yu Li1,*, Wen-Chen Tsai1,2, Jyung-Hurng Liu3,4, Chih-Li Lin5, Guang-Yaw Liu2, Hui-Chih Hung1,3,4
1Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
2Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
3Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan
4Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan
5Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
*These authors have contributed equally to this work
Hui-Chih Hung, e-mail: [email protected]
Guang-Yaw Liu, e-mail: [email protected]
Keywords: allosteric inhibitor, selective inhibitor, non-competitive inhibition, mutagenesis analysis, cellular senescence
Received: February 20, 2015 Accepted: May 06, 2015 Published: May 19, 2015
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 IC50 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.
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