Abstract:

:Osimertinib is a covalent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (Exon19del or L858R), or with T790M resistance mutation following disease progression on first- or second-generation EGFR TKIs. The aim of this work was to understand how osimertinib achieves selective inhibition of mutant EGFR relative to wildtype through 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) of osimertinib for 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 that osimertinib inactivates L858R and L858R/T790M with 20-fold and 50-fold higher overall efficiencies compared to WT, respectively. The Ki values reveal that osimertinib binds 3-fold and 17-fold tighter to L858R and L858R/T790M than to WT, respectively, while the kinact values show that osimertinib reacts 3-fold faster with L858R and L858R/T790M than with WT EGFR. We conclude that osimertinib achieves selective inhibition of L858R and L858R/T790M through improved affinities and improved rates of covalent bond formation via better positioning of the acrylamide warhead. This work highlights the importance of optimizing both reversible drug-target interactions and the inactivation rates for covalent inhibitors to achieve selectivity targeting mutant EGFR.