A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

Abstract

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.

A Comprehensive Exploration of Caspase Detection Methods: From Classical Approaches to Cutting-Edge Innovations

“…The emergence of MS has brought about a groundbreaking revolution in identifying, detecting, and quantifying protein expression levels and evaluating posttranslational modifications and regulation of enzyme activities [94,[129][130][131][132]. This technology has proven to be unparalleled in its sensitivity, specificity, resolution, and high-throughput screening, as well as its adaptability to achieving various experimental endpoints and platforms, allowing it to become a leading tool in protease research, including caspases.…”

“…The researchers utilized this approach to evaluate and optimize the structure-activity relationship (SAR) of various inhibitors for these two enzymes. One of the advantages of this method is its ability to avoid interferences in the detection of pharmacological activity caused by labeling the caspase substrates, which has been a drawback of fluorogenic assays that utilize rhodamine-labeled caspase substrates in particular [131,132]. An additional illustration of an MS-based technique for assessing caspase activity, particularly caspase 3, and identifying potential inhibitors involves the utilization of MALDI-TOF MS analysis to quantify the cleavage product of a peptide probe containing a caspase-3 cleavable segment (DEVD) linked with a quantifiable segment of FRGLRGFKC labeled by maleimide.…”

“…This approach provides the benefits of rapid detection, consistent quantification, efficient highthroughput capability, and simplified sample preparation. The researchers showcased the method's effectiveness in identifying caspase 3 activity in a cellular model of Parkinson's disease [131,132].…”

“…To this end, many of the common selective caspase inhibitors that have been designed based on substrate specificity profiles and are available through commercial vendors lack selectivity and show significant cross-reactivity towards other caspases [76]. The introduction of label-free LC/MS/MS-based enzymatic activity assays has been crucial in supporting the SAR studies for developing genuine caspase inhibitors of improved selectivity [131].…”