Fluorescence Lifetime-Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephos...

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Bibliographic Details
Published inJournal of biomolecular screening Vol. 19; no. 6; pp. 870 - 877
Main Authors Boettcher, Andreas, Gradoux, Nathalie, Lorthiois, Edwige, Brandl, Trixi, Orain, David, Schiering, Nikolaus, Cumin, Frederic, Woelcke, Julian, Hassiepen, Ulrich
Format Journal Article
LanguageEnglish
Published United States 01.07.2014
Subjects
Acridones - chemistry
Binding, Competitive
Buffers
Catalytic Domain
Drug Discovery - methods
Fluorescence Resonance Energy Transfer
Fluorescent Dyes - chemistry
Humans
Hydrogen-Ion Concentration
Inhibitory Concentration 50
Kinetics
Lung - enzymology
Molecular Conformation
Molecular Weight
Peptides - chemistry
Protease Inhibitors - chemistry
Protein Binding
Recombinant Proteins - chemistry
Serine Proteases - chemistry
Spectrometry, Fluorescence - methods
Tryptases - chemistry
protease
compound profiling
fluorescence lifetime
inhibitor
assay
compound interference
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Summary:Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight 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-based competitive binding assays.
ISSN:2472-5552
1552-454X
DOI:10.1177/1087057114521295