Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors

Abstract

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure−kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility. ■ SIGNIFICANCE• The potency and selectivity of covalent enzyme inhibitors can only be determined in time-dependent activity assays (IC 50 values are inappropriate). • Kinetic values for covalent EGFR inhibitors reported in the literature are not consistent, and investigators must include benchmark compounds as important controls. • Inhibitor molecular properties, assay reagent concentrations, and dilution protocols can significantly impact potency values.

Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on SNAr Electrophiles

“…As a consequence, the potency of covalent inhibitors expressed as IC 50 values improves over time. Covalent inhibitors are thus best described by a time-independent measure of potency, namely, the second-order kinetic constant k inact / K I . ,, It is composed of the K I value, which is the concentration where the rate of covalent bond formation becomes half maximal, thus representing the noncovalent binding contribution. The first-order rate constant k inact , i.e., the maximal rate of covalent inactivation, describes how efficiently the covalent bond is formed from the prereaction complex.…”

“…Time dependence of potency is a hallmark of covalent inhibitors, which has led to different sets of criteria which should be applied for their evaluation and publication. − Most notably, covalent inhibitors typically act via a nonequilibrium 2-step binding mechanism, where an initial reversible binding event is followed by the formation of a covalent bond. As a consequence, the potency of covalent inhibitors expressed as IC 50 values improves over time.…”