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Fluorescence Lifetime Assays: A Smart Solution for Inhibitor Profiling on Protease Panels

Hassiepen, Ulrich and Doering, Klaus and Woelcke, Julian (2009) Fluorescence Lifetime Assays: A Smart Solution for Inhibitor Profiling on Protease Panels. Screening - Trends in Drug Discovery, 10 (4). pp. 11-13. ISSN 1611-6038

Abstract

We are monitoring enzyme mediated peptide modifications, such as proteolytic cleavage, using fluorescence lifetime as the readout parameter of choice. Fluorescence lifetime (FLT) is an intrinsic parameter of the fluorescence emission, and is thus more robust against interferences compared to conventional readouts such as absorption, luminescence, or fluorescence intensity. Having set out to replace our standard fluorescence intensity assays based on dual-label peptidic substrates, we arrived at a toolbox approach to assay development for endo- as well as exopeptidases. As a probe we selected a blue-fluorescent acridone dye with a relatively long lifetime, which is affected by the spatial proximity of either one of the two natural amino acids, tyrosine and tryptophan. Labeling a protease substrate with this probe and incorporating one of the quenching amino acids on the other side of the scissile bond enabled us to monitor protease activity in vitro by quantifying the FLT of the probe. To date we have developed more than 50 assays for proteases from all four classes.

Item Type: Article
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Additional Information: Archiving status unclear?
Keywords: protease; fluorescence lifetime; assay; compound profiling; drug discovery
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Date Deposited: 13 Oct 2015 13:17
Last Modified: 13 Oct 2015 13:17
URI: https://oak.novartis.com/id/eprint/1360

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