Robin Shannon K D, Ansari Marc, Uppugunduri Chakradhara Rao S
Research platform of Pediatric Onco-Hematology, Department of Paediatrics, Gynaecology and Obstetrics, Faculty of Medicine, University of Geneva; Section of Biology, Faculty of Science, University of Geneva.
Research platform of Pediatric Onco-Hematology, Department of Paediatrics, Gynaecology and Obstetrics, Faculty of Medicine, University of Geneva; Onco-Hematology Unit, Department of Women, Children-Adolescents, University Hospitals of Geneva;
J Vis Exp. 2020 Oct 10(164). doi: 10.3791/61347.
Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.
谷胱甘肽S-转移酶（GSTs）是一类代谢酶，负责通过谷胱甘肽（GSH）偶联作用消除内源性或外源性亲电化合物。此外，GSTs还是参与凋亡途径的丝裂原活化蛋白激酶（MAPKs）的调节因子。在接受亲电烷化剂化疗的患者中，GSTs的过表达与治疗效果降低相关。使用GST抑制剂可能是扭转这种趋势并增强治疗效力的一种潜在解决方案。要实现这一目标，需要发现此类化合物，并建立准确、快速且简便的酶检测方法。在文献中，使用1-氯-2,4-二硝基苯（CDNB）作为底物的分光光度法是最常用的方法。然而，已报道的GST抑制实验并未提供详细描述最佳抑制检测各阶段的方案，例如CDNB的米氏常数（Km）的测量或所用酶浓度的说明，而这些都是评估受试化合物抑制效力的关键参数。因此，通过本方案，我们描述了优化的分光光度法GST酶检测的每个步骤，以筛选潜在抑制剂库。我们解释了半数最大抑制浓度（IC50）和抑制常数（Ki）的计算方法，这两个特性用于衡量酶抑制剂的效力。所描述的方法可以使用从细胞中提取的GSTs池或纯重组人GSTs来实施，即GSTα1（GSTA1）、GSTμ1（GSTM1）或GSTπ1（GSTP1）。然而，本方案不适用于GSTθ1（GSTT1），因为CDNB不是该同工型的底物。该方法用于测试姜黄素对马肝GSTs的抑制效力。姜黄素是一种具有抗癌特性的分子，在计算机对接预测后显示出对GST同工型具有亲和力。我们证明姜黄素是一种有效的竞争性GST抑制剂，IC50为31.6±3.6μM，Ki为23.2±3.2μM。姜黄素有可能与亲电化疗药物联合使用以提高其疗效。
