Insight into the Therapeutic Selectivity of the Irreversible EGFR Tyrosine Kinase Inhibitor Osimertinib through Enzyme Kinetic Studies.

Osimertinib is a covalent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for treating non-small cell lung cancer patients with activating EGFR mutations (Exon19del or L858R) or with the T790M resistance mutation following disease progression on first- or second-generation EGFR TKIs. The aim of this work is to rationalize and understand how osimertinib achieves mutant EGFR selectivity over the wild-type (WT) by evaluating its kinetic mechanism of action. In doing so, we developed methodologies combining steady-state and pre-steady-state kinetics to determine the covalent inactivation rates (kinact) and reversible binding affinities (Ki) for osimertinib against WT, L858R, and L858R/T790M EGFR and compared these data to the inhibition kinetics of earlier generations of EGFR TKIs. The kinact/KI values indicate osimertinib inactivates L858R and L858R/T790M with 20- and 50-fold higher overall efficiencies, respectively, compared to that for WT. The Ki and kinact values reveal that osimertinib binds 3-fold tighter to and reacts 3-fold faster with L858R than WT EGFR and binds 17-fold tighter to and reacts 3-fold faster with L858R/T790M than with the WT EGFR. We conclude that osimertinib overcomes the T790M mutation through improved affinities from stronger hydrophobic interactions with Met790 versus Thr790 and an improved rate of covalent bond formation via better positioning of the acrylamide warhead, while osimertinib targets the L858R mutation through better affinities and reactivities with the mutant in the context of differential binding affinities of the competing substrate ATP. This work highlights the importance of optimizing both reversible drug-target interactions and inactivation rates for covalent inhibitors to achieve selectivity in targeting mutant EGFRs.