Pitfalls and considerations in determining the potency and mutant selectivity of covalent epidermal growth factor receptor inhibitors

24 August 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Pursuing enzyme inhibitors with molecules that form covalent bonds with the desired target is an attractive focus in drug development that is increasing in prevalence. However, challenges arise when carrying out assessments of their time-dependent inhibitory properties as well as making correlations with values reported in the literature. Given the prominent focus on the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase in oncology, and the diverse structures and binding modes of covalent EGFR inhibitors, this perspective seeks to explore various broadly relevant factors that arise in the measurement of kinetic parameters within this class of drugs. A review of several studies indicates that variable literature potency values require investigators to include appropriate reference molecules and consistent substrate conditions for experimental consistency and proper benchmarks. The impact on covalent inhibitor potency with respect to common buffer conditions and compound liquid handling is surveyed highlighting the importance of multiple experimental variables when conducting these assays. Additionally, when assessing the potency for inhibitor selectivity in targeting EGFR mutants over wild-type (WT), it is ideal to consider ratios of true potency due to the variable ATP substrate binding affinities. The overview presented here, although most directly applicable to the tyrosine kinase inhibitor field, serves inhibitor assessments broadly by providing guided insights into conducting biochemical assays for designing and validating next-generation covalent inhibitors.


Kinase Inhibitor
Covalent Inhibitor
Kinetics Assay
Drug Discovery
Medicinal Chemistry
Targeted Therapy
Structure-Guided Drug Design
Drug Development

Supplementary materials

Supporting Information
Covalent inhibitor assay methods


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