Design, synthesis, and characterization of novel fluorogenic substrates of the proprotein convertases furin, PC1/3, PC2, PC5/6, and PC7.

Autor: Lam van TV; Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032, Marburg, Germany., Ivanova T; Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032, Marburg, Germany., Lindberg I; Department of Anatomy and Neurobiology, University of Maryland, Baltimore, MD, 21201, USA., Böttcher-Friebertshäuser E; Institute of Virology, Philipps University, Hans-Meerwein-Straße 2, D-35043, Marburg, Germany., Steinmetzer T; Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032, Marburg, Germany., Hardes K; Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032, Marburg, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology, Ohlebergsweg 12, D-35392, Giessen, Germany. Electronic address: Kornelia.Hardes@ime.fraunhofer.de.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2022 Oct 15; Vol. 655, pp. 114836. Date of Electronic Publication: 2022 Aug 11.
DOI: 10.1016/j.ab.2022.114836
Abstrakt: Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its k cat /K M value. Due to its higher k cat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled K i value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest k cat /K M values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher k cat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.
Competing Interests: Declaration of competing interest None.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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