Screening of lactate dehydrogenase inhibitor from bioactive compounds in natural products by electrophoretically mediated microanalysis.

Lactate dehydrogenase (LDH) is a key enzyme in the glycolysis, which has been reported that the expression of LDH is elevated in a variety of cancer types and can promote tumor invasion and metastasis. Therefore, LDH has come to be an emerging therapeutic target for cancer. In this work, we described a new strategy for rapid screening of LDH inhibitors from natural products by integrating electrophoretically mediated microanalysis (EMMA), transverse diffusion of laminar flow profiles (TDLFP) and rapid pressure direction switching. LDH activity could be assayed by the quantification of the peak area of the produced β-Nicotinamide adenine dinucleotide hydrate (NAD) and the inhibitory effect on LDH was reflected by the reduction of NAD peak area. Parameters affecting CE separation and enzymatic reaction were evaluated, including the pH of background electrolyte, incubation time, methanol percentage and enzyme concentration. The Michaelis-Menten constant (K) determined on-line by EMMA method were 226.9 μM and 31.8 μM for substrates sodium pyruvate and NADH, respectively and the half-maximal inhibitory concentration (IC) for the known positive inhibitor gossypol was determined to be 9.269 μM, which was comparable with the previous literature. Then the inhibitory activity of 12 bioactive compounds from natural products on LDH was investigated by employing the developed method. Three compounds including quercetin, luteolin, ursolic acid had potential inhibitory effect on LDH. Molecular docking study was implemented and well supported the experimental results. This study provides a potential tool for the preliminary screening of LDH inhibitors from bioactive compounds in natural products by capillary electrophoresis.