National Center of Mass Spectrometry in Changchun & Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China; University of Science and Technology of China, Hefei, 230029, China.
National Center of Mass Spectrometry in Changchun & Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China.
Talanta. 2020 Mar 1;209:120554. doi: 10.1016/j.talanta.2019.120554. Epub 2019 Nov 13.
Lactate dehydrogenase (LDH), catalyzing the conversion of pyruvate to lactate during glycolysis, is overexpressed in cancer cells. LDH inhibitors are a promising approach for the treatment of cancer. But up till now, there is limited method for rapid screening of LDH inhibitors. Herein, the use of LDH functionalized magnetic nanoparticles as a drug discovery tool for the selective enrichment of LDH potential inhibitors from natural products was firstly reported in this study. Firstly, LDH was immobilized onto the surface of amino-modified magnetic nanoparticles via covalent binding. In order to obtain the maximum enzyme activity, the immobilization conditions including pH, time and LDH concentration were optimized. The amount of LDH immobilized on MNPs was about 49 μg enzyme/mg carrier under the optimized conditions. Subsequently, the ligand fishing assay was performed to validate the specificity and selectivity of immobilized LDH using a model mixture, which consisted of galloflavin, chlorogenic acid and verbascoside. Finally, the immobilized LDH approach combined with ultra-high performance liquid chromatography-tandem mass spectrometry technique (UHPLC-MS/MS) was applied to screen potential LDH inhibitors from two anthraquinone-rich natural products (Rhubarb and Polygonum cuspidatum). Nine and six compounds were identified from Rhubarb and Polygonum cuspidatum extracts respectively, of which three compounds were common to both. Our results have proven that LDH functionalized magnetic nanoparticles have a significant prospect for drug discovery from complex matrices.
乳酸脱氢酶（LDH）在糖酵解过程中催化丙酮酸转化为乳酸，在癌细胞中过度表达。LDH 抑制剂是治疗癌症的一种有前途的方法。但到目前为止，还没有快速筛选 LDH 抑制剂的方法。本研究首次报道了将 LDH 功能化磁性纳米粒子用作药物发现工具，用于从天然产物中选择性富集 LDH 潜在抑制剂。首先，通过共价结合将 LDH 固定在氨基修饰的磁性纳米粒子表面。为了获得最大的酶活性，优化了固定化条件，包括 pH 值、时间和 LDH 浓度。在优化条件下，MNPs 上固定的 LDH 量约为 49μg 酶/mg 载体。随后，采用模型混合物（由金缕梅黄素、绿原酸和麦角甾苷组成）进行配体钓取实验，验证固定化 LDH 的特异性和选择性。最后，将固定化 LDH 方法与超高效液相色谱-串联质谱技术（UHPLC-MS/MS）相结合，从两种蒽醌丰富的天然产物（大黄和虎杖）中筛选潜在的 LDH 抑制剂。从大黄和虎杖提取物中分别鉴定出 9 种和 6 种化合物，其中 3 种化合物在两者中均存在。我们的结果证明 LDH 功能化磁性纳米粒子在从复杂基质中发现药物方面具有重要的前景。
