About: High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and non-physiological 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: dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris-(2-carboxyethyl)-phosphine (TCEP), in addition to GSH, against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many non-overlapping hits. More importantly, we find that various reducing agents alter inhibitor potency (IC(50)) from ~10 µM with one reducing agent to complete loss (IC(50) > 200 µM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in a HTS is critical as 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.   Goto Sponge  NotDistinct  Permalink

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  • High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and non-physiological 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: dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris-(2-carboxyethyl)-phosphine (TCEP), in addition to GSH, against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many non-overlapping hits. More importantly, we find that various reducing agents alter inhibitor potency (IC(50)) from ~10 µM with one reducing agent to complete loss (IC(50) > 200 µM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in a HTS is critical as 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.
Subject
  • Pharmaceutics
  • Thiols
  • Drug discovery
  • Scientific techniques
  • Reducing agents
  • Chemical reactions
  • Primary alcohols
  • Digital television
  • Soil chemistry
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