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J Biomol Screen. 2009 Feb;14(2):111-20. doi: 10.1177/1087057108328763. Epub 2009 Feb 4.

Defining balanced conditions for inhibitor screening assays that target bisubstrate enzymes.

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  • 1Department of Enzymology and Mechanistic Pharmacology, GlaxoSmithKline, Collegeville, PA 19426, USA.


High-throughput screening (HTS) is a common mechanism for identifying lead compounds for drug discovery efforts. Small molecules can inhibit enzymes by a variety of mechanisms, such as competitive, noncompetitive, and uncompetitive with respect to the substrate(s) of the catalytic reaction. To optimize the chances of finding the broadest diversity of inhibitor modalities during screening, one must run assays under ;;balanced'' conditions where the potency of inhibitors with various modes of action falls within a similar range. When an enzyme reaction involves more than one substrate, the definition and assessment of the apparent potency of inhibitors (IC(50)), in relation to their true potency (K(i)), can be nontrivial. This article provides a theoretical analysis, on the basis of the Cheng-Prusoff derivation, of the IC(50)/K( i) relationship of bisubstrate enzyme reactions following various sequential kinetic mechanisms, as well as the application and limitations of this information for defining optimal screening conditions for such enzymes.

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