Reducing agents affect inhibitory activities of compounds: results from multiple drug targets.
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-dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris(2-carboxyethyl)phosphine (TCEP)-as 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 (~560 compounds) with the four reducing agents surprisingly produced many nonoverlapping hits. More importantly, we found that various reducing agents altered inhibitor potency (IC(50)) from approximately 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 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.
Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607, USA.
Hyun
H
Lee
Jaime
J
Torres
Lena
L
Truong
Rima
R
Chaudhuri
Anuradha
A
Mittal
Michael E
ME
Johnson
D004229:Dithiothreitol / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry*; D016174:Hepacivirus / Q000201:enzymology; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry*; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004229:Dithiothreitol; D005978:Glutathione; D008623:Mercaptoethanol; D010447:Peptide Hydrolases; D010720:Phosphines; D011480:Protease Inhibitors; D011506:Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080938:tris(2-carboxyethyl)phosphine