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Authors: Maus, Hannah
Müller, Patrick
Meta, Mergim
Hoba, Sabrina N.
Hammerschmidt, Stefan J.
Zimmermann, Robert A.
Zimmer, Collin
Fuchs, Natalie
Schirmeister, Tanja
Barthels, Fabian
Title: Next generation of fluorometric protease assays : 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-amides) as class-spanning protease substrates
Online publication date: 14-Sep-2023
Year of first publication: 2023
Language: english
Abstract: Fluorometric assays are one of the most frequently used methods in medicinal chemistry. Over the last 50 years, the reporter molecules for the detection of protease activity have evolved from first-generation colorimetric p-nitroanilides, through FRET substrates, and 7-amino-4-methyl coumarin (AMC)-based substrates. The aim of further substrate development is to increase sensitivity and reduce vulnerability to assay interferences. Herein, we describe a new generation of substrates for protease assays based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-amides). In this study, we synthesized and tested substrates for 10 different proteases from the serine-, cysteine-, and metalloprotease classes. Enzyme- and substrate-specific parameters as well as the inhibitory activity of literature-known inhibitors confirmed their suitability for application in fluorometric assays. Hence, we were able to present NBD-based alternatives for common protease substrates. In conclusion, these NBD substrates are not only less susceptible to common assay interference, but they are also able to replace FRET-based substrates with the requirement of a prime site amino acid residue.
DDC: 540 Chemie
540 Chemistry and allied sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 09 Chemie, Pharmazie u. Geowissensch.
Place: Mainz
Version: Published version
Publication type: Zeitschriftenaufsatz
License: CC BY
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Journal: Chemistry - a European journal
Pages or article number: e202301855
Publisher: Wiley-VCH
Publisher place: Weinheim
Issue date: 2023
ISSN: 1521-3765
Publisher DOI: 10.1002/chem.202301855
Appears in collections:DFG-491381577-H

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