A label-free LC/MS/MS-based enzymatic activity assay for the detection of genuine caspase inhibitors and SAR development

J Biomol Screen. 2013 Sep;18(8):868-78. doi: 10.1177/1087057113492851. Epub 2013 Jun 24.


The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry-based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.

Keywords: LC/MS/MS assay; artifactual inhibition; caspases; nonpeptidic inhibitors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caspase 2 / metabolism
  • Caspase 3 / metabolism
  • Caspase 6 / metabolism
  • Caspase Inhibitors / chemistry
  • Caspase Inhibitors / pharmacology*
  • Chromatography, Liquid / methods
  • Drug Design
  • Enzyme Assays / methods*
  • Isatin / chemistry
  • Isatin / pharmacology
  • Neurodegenerative Diseases / metabolism
  • Oligopeptides / pharmacology
  • Tandem Mass Spectrometry / methods


  • Caspase Inhibitors
  • Oligopeptides
  • acetyl-aspartyl-glutamyl-valyl-aspartal
  • acetyl-valyl-isoleucyl-aspartyl-aldehyde
  • Isatin
  • Caspase 2
  • Caspase 3
  • Caspase 6