A comprehensive guide for assessing covalent inhibition in enzymatic assays illustrated with kinetic simulations

  • Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure-activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read-out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

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Author:Elma MonsORCiD, Sander RoetORCiD, Robbert Q. KimORCiD, Monique P. C. MulderORCiD
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/35671150
Parent Title (English):Current protocols
Place of publication:Hoboken, NJ
Document Type:Article
Date of Publication (online):2022/06/07
Date of first Publication:2022/06/07
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/07/18
Tag:biochemical potency; covalent inhibition; enzyme kinetics; irreversible inhibition; simulations
Page Number:86
First Page:1
Last Page:85
This work was supported by the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510). The authors would like to thank Dr. Anthe Janssen for proof reading. In memory of Prof. Dr. Huib Ovaa, his passion for science will always be an inspiration to us.
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International