Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance

Abstract

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

Keywords: capture period; cysteine oxidation; protein kinase; signaling; warhead interactions.

Fig. 1.

(A) Chemical mechanisms of irreversible enzyme inhibition. Representative covalent inhibitor with reactive MA (bracket) and the resulting EGFR adduct. (B) The postulated kinetic mechanism for two-step covalent inhibition under the special experimental conditions where the Michaelis constant for the peptide substrate, S, is very much lower than the corresponding Michaelis constant K_m,Pep. The dashed box represents the rapid equilibrium approximation for inhibitor binding and dissociation. (C) Structures of EGFR covalent inhibitors investigated in this report.

Fig. 2.

Covalent drugs and inhibitors characterization based on kinetic properties with WT (□) and L858R/T790M EGFR (●): quadrant I, low affinity and high reactivity; quadrant II, high affinity and high reactivity; quandrant III, high affinity and moderate reactivity; quadrant IV, weak affinity and moderate to low reactivity.

Fig. 3.

Specific EGFR-Cys₇₉₇ oxidation has differential effects on inhibitor and drug potencies dependent on the type of oxidation and EGFR mutation. Reversible drug affinity determined to different Cys₇₉₇ oxidation states (-SH, unoxidized; -SO₂H, sulfinylated; -SSG, glutathiolated) in (A) L858R and (B) L858R/T790M. Covalent inhibitor affinity was measured to (C) L858R and (D) L858R/T790M.