Screening for Caspase-3 Inhibitors: Effect of a Reducing Agent on Identified Hit Chemotypes

¹ ChemDiv, Inc., San Diego, California.; Chemical Diversity Research Institute, Khimki, Moscow Region, Russia.
² ChemDiv, Inc., San Diego, California.
³ Chemical Diversity Research Institute, Khimki, Moscow Region, Russia.

^* To whom correspondence should be addressed. E-mail: io{at}chemdiv.com.

	Abstract

When studying cysteinyl proteases in general and caspases in particular, it is generally accepted that a reaction buffer must contain a reducing agent to prevent essential cysteinyl groups from spontaneous oxidation. Dithiothreitol (DTT) and -mercaptoethanol (-MCE) are 2 of the most broadly used reducing agents. While screening a library of small molecules against caspase-3, the authors have found that the nature of the reducing agent used, DTT or -MCE, dramatically affects screening results and leads to identification of nonoverlapping hits. Screening in DTT-containing buffer revealed few novel classes of small molecules that selectively and reversibly inhibit caspase-3 but failed to identify isatin sulfonamides recently found to be potent and selective caspase-3 inhibitors (false negatives). On the other hand, screening in the presence of -MCE failed to identify a series of hit compounds, 1,3-dioxo-2,3-dichloro-1H-pyrrolo[3,4-c]quinolines, discovered with DTT, whereas isatin sulphonamides in these conditions exhibited strong caspase-3 inhibition. In this work, the authors show that thiol-containing reducing agents can affect catalytic activity of caspase-3 and modify its thermostability in a redox-potential-independent manner. The authors speculate that the differential structural modifications of caspase-3 seen with different reducing agents represent structurally different caspase-3 conformations and are responsible for its differential sensitivity to small molecules of different chemotypes. Hence, selection of the reducing agent may dramatically affect the quality of high-throughput screening campaigns.

Key Words: caspase-3, inhibition, dithiothreitol, -mercaptoethanol, redox potential, screening

First published on July 14, 2006, doi:10.1177/1087057106289231

Journal of Biomolecular Screening 2006;11:694.

A more recent version of this article appeared on September 1, 2006


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