A simple assay for detection of small-molecule redox activity

J Biomol Screen. 2007 Sep;12(6):881-90. doi: 10.1177/1087057107304113. Epub 2007 Jun 19.

Abstract

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.

Publication types

  • Validation Study

MeSH terms

  • Cathepsin L
  • Cathepsins / metabolism
  • Chromatography, Liquid
  • Cysteine Endopeptidases / metabolism
  • Drug Evaluation, Preclinical / methods*
  • Inhibitory Concentration 50
  • Models, Biological
  • Oxidants / analysis*
  • Oxidation-Reduction*
  • Protein Processing, Post-Translational / drug effects
  • Tandem Mass Spectrometry

Substances

  • Oxidants
  • Cathepsins
  • Cysteine Endopeptidases
  • Cathepsin L