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A Simple Assay for Detection of Small-Molecule Redox Activity

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania

Jessica Schneck

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania

Dean E. Mcnulty

Departement of Computational and Structural Chemistry, GlaxoSmithKline, Collegeville, Pennsylvania

Elsie Diaz

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania

Martin Brandt

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania

Sara H. Thrall

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania

Benjamin Schwartz

Departments of Biological Reagents and Assay Development, GlaxoSmithkline, Collegeville, Pennsylvania, Benjamin_2_Schwartz{at}gsk.com

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts. (Journal of Biomolecular Screening 2007:881-890)

Key Words: cysteine proteases • cysteine phosphatases • iron enzymes • redox cofactors • nuisance hits • cathepsin L • caspase-8

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 881-890 (2007)
DOI: 10.1177/1087057107304113


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