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

ChemDiv, Inc., San Diego, California, Chemical Diversity Research Institute, Khimki, Moscow Region, Russia

Sergei Malarchuk

ChemDiv, Inc., San Diego, California

Elena Dubrovskaya

ChemDiv, Inc., San Diego, California

Alexander Khvat

ChemDiv, Inc., San Diego, California

Sergey Tkachenko

ChemDiv, Inc., San Diego, California

Volodymyr Kysil

ChemDiv, Inc., San Diego, California

Dmitry Kravchenko

Chemical Diversity Research Institute, Khimki, Moscow Region, Russia

Alexandre Ivachtchenko

ChemDiv, Inc., San Diego, California, Chemical Diversity Research Institute, Khimki, Moscow Region, Russia

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 ß-mercaptoethanol (ß-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 ß-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 ß-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 • ß-mercaptoethanol • redox potential • screening

This version was published on September 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 6, 694-703 (2006)
DOI: 10.1177/1087057106289231


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