Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China; State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing 10080, China.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China.
J Chromatogr A. 2021 Oct 25;1656:462554. doi: 10.1016/j.chroma.2021.462554. Epub 2021 Sep 15.
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.
乳酸脱氢酶（LDH）是糖酵解中的一种关键酶，已有报道称 LDH 在多种癌症类型中的表达升高，并能促进肿瘤侵袭和转移。因此，LDH 已成为癌症治疗的一个新靶点。在这项工作中，我们描述了一种新的策略，通过整合电泳介导的微分析（EMMA）、层流横向扩散（TDLFP）和快速压力方向切换，从天然产物中快速筛选 LDH 抑制剂。通过产生的β-烟酰胺腺嘌呤二核苷酸水合物（NAD）峰面积的定量可以测定 LDH 活性，并且 LDH 的抑制作用反映为 NAD 峰面积的减少。评估了影响 CE 分离和酶反应的参数，包括背景电解质的 pH 值、孵育时间、甲醇百分比和酶浓度。通过 EMMA 方法在线测定的米氏常数（K）分别为 226.9 μM 和 31.8 μM，用于底物丙酮酸钠和 NADH，并且已知阳性抑制剂棉酚的半最大抑制浓度（IC）确定为 9.269 μM，与以前的文献相当。然后，采用所开发的方法研究了 12 种天然产物生物活性化合物对 LDH 的抑制活性。三种化合物，包括槲皮素、木犀草素、熊果酸，对 LDH 具有潜在的抑制作用。实施了分子对接研究，并很好地支持了实验结果。这项研究为通过毛细管电泳从天然产物中的生物活性化合物中初步筛选 LDH 抑制剂提供了一种潜在的工具。
