To further understand the functional significance of the lazertinib binding mode, we conducted biochemical assays with purified kinase domains. The three inhibitors potently inhibit EGFR mutants L858R and L858R/T790M as consistent with previous studies. Their strong potency for the T790M-containing variant is most likely due to productive binding to the T790M through intermolecular van der Waals interactions (lazertinib and osimertinib, Figure ) or methionine pi-stacking (LN2057, Figure ). LN2057 is found more potent against WT EGFR compared to osimertinib and lazertinib, most likely due to added binding affinity afforded by the imidazole-K745 H-bond (Figure ). It is also likely that enhanced binding from this H-bond direct for the observed higher potency against L858R. To compliment earlier studies, we assessed biochemical potencies for these three inhibitors against HER2. We observe that lazertinib exhibits significantly lower potency against HER2, which is proposed to limit adverse events and improve drug tolerability. For completeness, we confirmed lazertinib forms a covalent bond at Cys-805 within the HER2 purified kinase domain with LC-MS/MS (Figure ). These trends in biochemical assays showcase how structural differences between these inhibitors elicit differential effects on inhibitor potency and selectivity. Since enhanced efficacy against EGFR and diminished HER2 targeting is a proposed advantage for treatment of mutant EGFR NSCLC with lazertinib, 8 we were motivated to assess lazertinib inhibition in cellular contexts compared to osimertinib and LN2057. We first assessed dose-dependence of inhibition of EGFR(L858R/T790M) in H1975 NSCLC cells by osimertinib, lazertinib, and LN2057 by blotting for active EGFR (pY1068). After dosing 5 or 50 nM of these drugs for 2 hours, we observed that lazertinib suppressed pY1068 to a greater extent compared to equivalent dosing of osimertinib and LN2057, as consistent with previous studies (Figure ). Uniquely, lazertinib is observed to be notably less effective at inhibiting active HER2 (pY1221/1222) in BT474 HER2 overexpressing breast cancer cells, confirming the selective targeting of EGFR by lazertinib compared to osimertinib and LN2057 (Figure ). These findings are consistent with our biochemical activity assays (Table ) indicating that lazertinib potently and selectively inhibits mutant EGFR(L858R/T790M) and WT EGFR while simultaneously affording limited activity against HER2.