TY  - JOUR
A1  - Mons, Elma
A1  - Roet, Sander
A1  - Kim, Robbert Q.
A1  - Mulder, Monique P. C.
T1  - A comprehensive guide for assessing covalent inhibition in enzymatic assays illustrated with kinetic simulations
T2  - Current protocols
N2  - 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.
KW  - biochemical potency
KW  - covalent inhibition
KW  - enzyme kinetics
KW  - irreversible inhibition
KW  - simulations
Y1  - 2022
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/74714
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-747148
SN  - 2691-1299
N1  - 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.
VL  - 2
IS  - 6
SP  - 1
EP  - 85
PB  - Wiley
CY  - Hoboken, NJ
ER  -