| Autor: |
Maillard MC; CHDI Management, Inc., Los Angeles, CA 90045, USA. michel.maillard@chdifoundation.org, Dominguez C, Gemkow MJ, Krieger F, Park H, Schaertl S, Winkler D, Muñoz-Sanjuán I |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of biomolecular screening [J Biomol Screen] 2013 Sep; Vol. 18 (8), pp. 868-78. Date of Electronic Publication: 2013 Jun 24. |
| DOI: |
10.1177/1087057113492851 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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