Abstract
Aim:
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.
Conclusion:
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.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81125023 and 81021062), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2013ZX09507002), and the Shanghai Commission of Science and Technology (11DZ2292200 and 13DZ2290300). We also thank Yan-yun FU and Bei-ying QIU for their help in editing the manuscript.
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Wen, Y., Xu, L., Chen, Fl. et al. Discovery of a novel inhibitor of NAD(P)+-dependent malic enzyme (ME2) by high-throughput screening. Acta Pharmacol Sin 35, 674–684 (2014). https://doi.org/10.1038/aps.2013.189
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DOI: https://doi.org/10.1038/aps.2013.189
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