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Discovery of a novel inhibitor of NAD(P)(+)-dependent malic enzyme (ME2) by high-throughput screening.

Wen Y, Xu L, Chen FL, Gao J, Li JY, Hu LH, Li J - Acta Pharmacol. Sin. (2014)

Bottom Line: A library containing 12 683 natural products was screened.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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.

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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NPD389 was identified as a more potent inhibitor of ME2 than NPD387. (A) The structure of NPD389. (B) Dose-response curve of inhibition of ME2 by NPD389. (C) Dose-dependent inhibition of ME2 by NPD389 with 0.01% Brij-35. Error error bars represent SD. n=3.
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fig4: NPD389 was identified as a more potent inhibitor of ME2 than NPD387. (A) The structure of NPD389. (B) Dose-response curve of inhibition of ME2 by NPD389. (C) Dose-dependent inhibition of ME2 by NPD389 with 0.01% Brij-35. Error error bars represent SD. n=3.

Mentions: Through structural modification, we generated NPD389, which is a more potent inhibitor of ME2 than NPD387 (Figure 4A). The IC50 of NPD389 was 4.63±0.36 μmol/L or 5.59±0.38 μmol/L when 0.01% Brij-35 was absent from or present in the assay system, respectively (Figure 4B and 4C, Table 2).


Discovery of a novel inhibitor of NAD(P)(+)-dependent malic enzyme (ME2) by high-throughput screening.

Wen Y, Xu L, Chen FL, Gao J, Li JY, Hu LH, Li J - Acta Pharmacol. Sin. (2014)

NPD389 was identified as a more potent inhibitor of ME2 than NPD387. (A) The structure of NPD389. (B) Dose-response curve of inhibition of ME2 by NPD389. (C) Dose-dependent inhibition of ME2 by NPD389 with 0.01% Brij-35. Error error bars represent SD. n=3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814031&req=5

fig4: NPD389 was identified as a more potent inhibitor of ME2 than NPD387. (A) The structure of NPD389. (B) Dose-response curve of inhibition of ME2 by NPD389. (C) Dose-dependent inhibition of ME2 by NPD389 with 0.01% Brij-35. Error error bars represent SD. n=3.
Mentions: Through structural modification, we generated NPD389, which is a more potent inhibitor of ME2 than NPD387 (Figure 4A). The IC50 of NPD389 was 4.63±0.36 μmol/L or 5.59±0.38 μmol/L when 0.01% Brij-35 was absent from or present in the assay system, respectively (Figure 4B and 4C, Table 2).

Bottom Line: A library containing 12 683 natural products was screened.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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.

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.

Show MeSH
Related in: MedlinePlus