title: A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence.
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 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.
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan.
authors.forename: Ju-Yi
authors.initials: JY
authors.lastname: Hsieh
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Shao-Yu
authors.initials: SY
authors.lastname: Li
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan.
authors.forename: Wen-Chen
authors.initials: WC
authors.lastname: Tsai
authors.affiliation: Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan.|Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan.
authors.forename: Jyung-Hurng
authors.initials: JH
authors.lastname: Liu
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Chih-Li
authors.initials: CL
authors.lastname: Lin
authors.affiliation: Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan.
authors.forename: Guang-Yaw
authors.initials: GY
authors.lastname: Liu
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan.|Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan.
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009284:Naphthols / Q000494:pharmacology*; D020836:Protein Structure, Quaternary; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009284:Naphthols; D054852:Small Molecule Libraries; D009249:NADP; C004368:pamoic acid; D008291:Malate Dehydrogenase; C495394:malic enzyme 2; human
keywords: allosteric inhibitor; cellular senescence; mutagenesis analysis; non-competitive inhibition; selective inhibitor