Boettcher, Andreas, Gradoux, Nathalie, Lorthiois, Edwige, Brandl, Trixi, Orain, David, Schiering, Nikolaus, Cumin, Frederic, Woelcke, Julian and Hassiepen, Ulrich (2014) Fluorescence Lifetime-based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro. Journal of Biochemical Screening, 19 (6). pp. 870-877. ISSN 1087-05711552-454X

Abstract

Fluorescence lifetime- (FLT-) based assays have developed to highly attractive tools in drug discovery.
All recently published examples of FLT based assays describe essentially their use for monitoring enzyme-mediated peptide modifications such as proteolytic cleavage or phosphorylation/de-phosphorylation.
Here we report the development of competitive binding assays as a novel, inhibitor-centric assay principle employing the fluorescence lifetime of the acridone dye Puretime 14 (PT14) as the readout parameter.
Exemplified with two case studies on human serine proteases, the details on the rationale of the design and the synthesis of probes, i.e., active-site directed low-molecule weight (lmw) inhibitors conjugated to PT14, are provided.
Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer- (TR-FRET-) based competitive binding assays.

Item Type:	Article
Keywords:	protease, assay, inhibitor, compound profiling, fluorescence lifetime, compound interference
Date Deposited:	26 Apr 2016 23:46
Last Modified:	04 Jul 2016 23:46
URI:	https://oak.novartis.com/id/eprint/10777