Next Generation of Fluorometric Protease Assays: 7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl‐amides (NBD‐Amides) as Class‐Spanning Protease Substrates

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-1,3diazol-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 substratespecific 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.

Intestinal proteases

“…Conventionally, p-nitroanilides, FRET substrates, and 7-amino-4methyl coumarin-based substrates have been used for the detection of PA. Recently, 7-nitrobenz-2-oxa-1,3diazol-4-yl-(NBD) amide-based probes were found to have improved sensitivity and less assay interference for detection of PA from a variety of proteases [9]. ABPs for neutrophilic elastases and trypsin are already being considered for in vivo imaging in animal models [10].…”

Structural Modifications of Covalent Cathepsin S Inhibitors: Impact on Affinity, Selectivity, and Permeability

“…The exchange of the different side chains between the nitrilebased inhibitors produced eight new inhibitors with affinities in the low double-digit nanomolar range or even lower, similar to the starting compounds 28 and 36, with K i values of 1.2 nM and 1.0 nM, respectively (Table 1A). 22,23 It was also observed that the tetrahydropyran derivatives and inhibitor 25 showed significantly higher K i values, compared to those of 28 and 36. Overall, the selectivity against the off-targets catB and catL remained in the same range, e.g., compound 37 retained a good selectivity of >1500 over catB, compared to 1670 for starting inhibitor 36, and even higher selectivity compared to the other starting inhibitor 28 (1054 over catB; see the SI).…”