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Journal of Biomolecular Screening
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Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists

Michael Habig

Lead Finding Platform, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

Anke Blechschmidt

Protein Structure Unit, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

Sigmar Dressler

Lead Finding Platform, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

Barbara Hess

Lead Finding Platform, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

Viral Patel

Novartis Institute for Tropical Diseases Pte Ltd, Singapore

Andreas Billich

Autoimmunity Transplantation & Inflammation, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

Christian Ostermeier

Protein Structure Unit, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland

David Beer

Novartis Institute for Tropical Diseases Pte Ltd, Singapore

Martin Klumpp

Lead Finding Platform, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland, martin.klumpp{at}novartis.com

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC50 values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms. (Journal of Biomolecular Screening 2009:679-689)

Key Words: high-throughput screening • stoichiometric inhibitors • stoichiometric binding • enzyme

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 679-689 (2009)
DOI: 10.1177/1087057109336586
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