title: Discovery of a novel inhibitor of NAD(P)(+)-dependent malic enzyme (ME2) by high-throughput screening.
abstract: OBJECTIVE
  Malic enzymes are oxidative decarboxylases with NAD(+) or NAD(P)(+) as cofactor that catalyze the conversion of L-malate to pyruvate and CO2. The aim of this study was to discover and characterize a potent inhibitor of human NAD(P)(+)-dependent malic enzyme 2 (ME2).
  
  
  METHODS
  Recombinant human ME2-His-Tag fusion protein was overexpressed in E coli and purified with Ni-NTA resin. A high-throughput screening (HTS) assay was developed to find ME2 inhibitors. Detergent Brij-35 was used to exclude false positives. The characteristics of the inhibitor were analyzed with enzyme kinetics analysis. A thermal shift assay for ME2 was carried out to verify the binding of the inhibitor with the enzyme.
  
  
  RESULTS
  An HTS system for discovering ME2 inhibitors was established with a Z' factor value of 0.775 and a signal-to-noise ratio (S/N) of 9.80. A library containing 12 683 natural products was screened. From 47 hits, NPD387 was identified as an inhibitor of ME2. The primary structure-activity relationship study on NPD387 derivatives showed that one derivative NPD389 was more potent than the parent compound NPD387 (the IC50 of NPD389 was 4.63 ± 0.36 μmol/L or 5.59 ± 0.38 μmol/L, respectively, in the absence or presence of 0.01% Brij-35 in the assay system). The enzyme kinetics analysis showed that NPD389 was a fast-binding uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.
  
  
  CONCLUSIONS
  NPD389 is a potent ME2 inhibitor that binds to the enzyme in a fast-binding mode, acting as an uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.
authors.affiliation: School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wen
authors.affiliation: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xu
authors.affiliation: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Fang-lei
authors.initials: FL
authors.lastname: Chen
authors.affiliation: School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Jing-ya
authors.initials: JY
authors.lastname: Li
authors.affiliation: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Li-hong
authors.initials: LH
authors.lastname: Hu
authors.affiliation: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D008291:Malate Dehydrogenase; C495394:malic enzyme 2; human; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)