INTERACTIONS OF GLUTATHIONE-S-TRANSFERASE-PI WITH ETHACRYNIC-ACID AND ITS GLUTATHIONE CONJUGATE
INTERACTIONS OF GLUTATHIONE-S-TRANSFERASE-PI WITH ETHACRYNIC-ACID AND ITS GLUTATHIONE CONJUGATE
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DOI：
10.1016/0167-4838(93)90245-m
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发表时间：
1993-07-10
期刊：
影响因子：
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通讯作者：
ANSARI, GAS
中科院分区：
文献类型：
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作者：
AWASTHI, S;SRIVASTAVA, SK;ANSARI, GAS
依他尼酸是一种利尿药物，已知是谷胱甘肽S-转移酶（GST）的抑制剂，已显示可增强烷化剂类化疗药物在对烷化剂耐药的培养癌细胞中的细胞毒性。依他尼酸的这种作用可能是通过抑制GST介导的，GST与烷化剂的解毒有关。除了作为GST的抑制剂之外，依他尼酸还作为与GSH缀合的底物与GST相互作用，以产生依他尼酸-GSH缀合物。该缀合物通过酶促和非酶促形成，并且其本身是GST抑制剂。由于依他尼酸-GSH缀合物本身可能能够通过GST抑制介导烷化剂的逆转，我们合成并纯化了依他尼酸-GSH缀合物，研究了依他尼酸和缀合物抑制人肺pi类GST的动力学，并使用HPLC方法比较了缀合物的酶促和非酶促形成的动力学。我们的研究结果表明，依他尼酸-GSH缀合物是比依他尼酸更有效的人肺GST-π抑制剂（K（i）分别为1.5和11.5 μ M），并且它们的GST抑制机制是不同的（分别为竞争性和非竞争性）。体外缀合物形成的酶促和非酶促速率的比较表明，GST-pi催化依他尼酸与GSH的快速缀合，依他尼酸的浓度比几乎完全抑制GST催化的1-氯-2，4-二硝基苯缀合所需的浓度高一个数量级。然而，由于快速的非酶促反应，以及GST-pi的抑制与缀合物在反应混合物中的积累，在存在或不存在GST-pi的情况下，150分钟后形成的缀合物的总量几乎相同。这些研究结果表明，依他尼酸-GSH结合物抑制GST可能是依他尼酸逆转烷化剂耐药性的主要机制。
Ethacrynic acid, a diuretic drug known to be an inhibitor of glutathione S-transferases (GSTs), has been shown to enhance the cytotoxicity of the alkylating agent class of chemotherapeutic drugs in cultured cancer cells resistant to alkylating agents. This action of ethacrynic acid is presumably mediated by inhibition of GSTs which are implicated in detoxification of alkylating agents. In addition to being an inhibitor of GSTs, ethacrynic acid also interacts with GSTs as a substrate for conjugation with GSH to yield an ethacrynic acid-GSH conjugate. This conjugate is formed both enzymatically and non-enzymatically and itself is a GST inhibitor. Since ethacrynic acid-GSH conjugate is itself likely to be able to mediate reversal of alkylating agents through GST inhibition, we have synthesized and purified the ethacrynic acid-GSH conjugate, studied the kinetics of inhibition of human lung pi-class GST by ethacrynic acid and the conjugate, and compared the kinetics of the enzymatic and non-enzymatic formation of the conjugate using an HPLC method. Results of our studies showed that the ethacrynic acid-GSH conjugate was a more potent inhibitor of human lung GST-pi than ethacrynic acid (K(i) = 1.5 vs. 11.5 muM, respectively) and that their mechanisms for GST inhibition were distinct (competitive and non-competitive, respectively). Comparison of enzymatic and non-enzymatic rates of conjugate formation in vitro indicated that GST-pi catalyzed a rapid conjugation of ethacrynic acid with GSH at a concentration of ethacrynic acid an order of magnitude above that required to nearly completely inhibit GST catalyzed conjugation of 1-chloro-2,4-dinitrobenzene. However, because of the rapid non-enzymatic reaction, and the inhibition of GST-pi with the accumulation of the conjugate in the reaction mixture, the overall quantity of the conjugate formed after 150 min was nearly identical in the presence or absence of GST-pi. Results of these studies suggest that inhibition of GSTs by ethacrynic acid-GSH conjugate may be the main mechanism through which ethacrynic acid reverses alkylating agent resistance.