Reducing agents affect inhibitory activities of compounds: Results from multiple drug targets

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• 作者：Hyun Lee ; Jaime Torres ; Lena Truong ; Rima Chaudhuri ; Anuradha Mittal ; Michael E. Johnson ; ^{mjohnson@uic.edu}
• 关键词：Reducing agent ; Inhibitor screening ; False positives ; False negatives ; Potency comparison
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：1 April, 2012
• 卷：423
• 期：1
• 页码：46-53
• 文件大小：1359 K

摘要

High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and nonphysiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent reduced glutathione (GSH). Here, we compared the effects of three reducing agents鈥攄ithiothreitol (DTT), 尾-mercaptoethanol (尾-MCE), and tris(2-carboxyethyl)phosphine (TCEP)鈥攁s well as GSH against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high-scoring compounds (鈭?60 compounds) with the four reducing agents surprisingly produced many nonoverlapping hits. More importantly, we found that various reducing agents altered inhibitor potency (IC₅₀) from approximately 10 渭M with one reducing agent to complete loss (IC₅₀ > 200 渭M) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in an HTS is critical because this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.