Structural Basis for Inhibition of Mutant EGFR with Lazertinib (YH25448)

. Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand lazertinib inhibition at the molecular level, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally-related EGFR TKIs. We observe that lazertinib binds with the novel pyrazole moiety involved in hydrogen bonds and van der Waals interactions consistent with drug potency and T790M mutant selectivity. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent against HER2 as consistent with an improved toxicity profile. The molecular basis for lazertinib inhibition of EGFR reported here highlights new strategies for structure-guided design of tyrosine kinase inhibitors.


Introduction.
Prolonged efficacy of first-generation TKIs (gefitinib and erlotinib) is eventually made limited due to drug resistance as a result of patients acquiring a second T790M "gatekeeper" mutation. 35] Despite promising indications, drug resistance to osimertinib is inevitable and caused in part by the acquisition of a third kinase domain mutation C797S that prevents formation of the potency-enabling covalent bond.More recently, osimertinib has been shown effective, and clinically-approved, as a front-line therapy in untreated patients harboring EGFR L858R and exon19del activating mutations. 6As osimertinib is the only approved third-generation EGFR TKI for L858R and exon19del EGFR mutant tumors, development efforts from Yuhan and Janssen biotech sought to produce a drug with improved medicinal chemistry properties.These efforts resulted in YH25448 (lazertinib), which is structurally related to osimertinib comprising an aminopyrimidine core and acrylamide warhead but is distinct with respect to the substituted pyrazole as well as morpholine groups (Figure 1). 7Preclinical head-to-head or in combination with the antibody Amivantamab (NCT04077463), and is currently approved to treat T790M-containing NSCLC in the Republic of Korea. 7Regardless of these improvements, drug resistance to lazertinib has been shown to be due to the acquisition of C797S mutation. 9-10Despite the improved properties and positive clinical outlook, no crystal structures have been reported detailing the molecular basis for lazertinib inhibition of mutant EGFR.
Results and Discussion.To characterize the binding mode of lazertinib in complex with the EGFR kinase domain, we determined a 2.4 Å resolution X-ray co-crystal structure of lazertinib soaked into WT EGFR crystals (Figure 2 & S1, PDB ID 7UKV).WT kinase domains crystallize in the active "ɑC-helix in" conformation due to crystal packing of the kinase domains as asymmetric dimers. 11As expected, lazertinib binds with the aminopyrimidine anchored to the hinge region by H-bonds to M793 and covalent bond formed at C797 as generally observed for third-generation TKIs. 12Importantly, the unique pyrazole group extends away from the hinge in a conformation that positions the phenyl ring toward the K745-E762 salt bridge and the N,N-dimethylmethyleneamine (methyleneamine) in a H-bond with the DFG-motif D855 carboxylate.The substituted pyrazole of lazertinib is unique among third-generation EGFR TKIs and most likely the basis for mutant-selectivity and improved medicinal chemistry properties.A comparison of our structures from the "ɑC-helix in" active WT (Figure 2) and "ɑC-helix out" inactive T790M (Figure 3) show very similar binding positions indicating that lazertinib is anchored to the EGFR kinase domain identically in both active and inactive states and independent of the Thr versus Met 790 gatekeeper residue (Figure 4A).We expect that this conformation of lazertinib is preferred as compared to the "flipped" conformation (Figure 3B) in the "ɑC-helix in" active state due to the requirement to anchor the positive methyleneamine near the K745-E762 salt bridge (Figure S3).
Recent structural studies of EGFR(T790M) co-crystal structures with bound osimertinib revelated a novel binding mode where the N-methylindole directly interacts with T790M through van der Waals interactions responsible for osimertinib mutant selectivity. 13We observe analogous intermolecular interactions in the case of the bound lazertinib phenyl showcasing that both inhibitors are made selective for T790M-containing EGFR through van der Waals interactions (Figure 4B).Additionally, versatile H-bonding methyleneamine moiety of lazertinib is distinctive potentially enabling the modest improvement in binding compared to osimertinib.5] By comparison, the lazertinib pyrazole substituents sterically block K745 potentially diminishing binding to WT EGFR (Figure 4C).The correlation of these differences in binding mode demonstrates how the pyrazole moiety of lazertinib affords distinct interactions with EGFR to enable T790M mutant selectivity.
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. 8,14 ir 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 4B) or methionine pi-stacking (LN2057, Figure 4C).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 4B). 15It 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. 8For completeness, we confirmed lazertinib forms a covalent bond at Cys-805 within the HER2 purified kinase domain with LC-MS/MS (Figure S5).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 5A). 8Uniquely, 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 5B).These findings are consistent with our biochemical activity assays (Table 1) indicating that lazertinib potently and selectively inhibits mutant EGFR(L858R/T790M) and WT EGFR while simultaneously affording limited activity against HER2.
In conclusion, we have determined the molecular basis of the novel EGFR TKI lazertinib bound to EGFR in X-ray co-crystal structures showcasing that the lazertinib pyrazole ring binds facilitates H-bonds and van der Waals interactions consistent with drug efficacy and T790M selectivity.Structural and functional correlation to osimertinib and LN2057 demonstrate the importance of productive intermolecular interactions with T790M.Additionally, we find that lazertinib does not H-bond with K745, which likely contributes to lower potency for WT EGFR.Another important feature to lazertinib is the lack of potency on HER2 that is often associated with dose-limiting adverse events, 17 as confirmed here in biochemical and cell-based studies.Our present structural analysis, however, does not reveal a discrete molecular origin for the preference of lazertinib for EGFR when compared to HER2.We speculate that differences in the sequence and dynamics of the HER2 kinase domain, as evident from reported crystal structures, 18 negatively impacts lazertinib reversible binding to HER2 and not osimertinib and LN2057 and motivates future structural studies to understand the structural basis for EGFR kinase specificity.Results from these studies define the binding mode of a novel third-generation mutant-selective EGFR TKI lazertinib with improved dose-limiting toxicity as well as ontarget potency and selectivity and serves as a noteworthy example for developing nextgeneration kinase inhibitors.
lazertinib is superior to osimertinib in several key respects including in vivo efficacy against H1975 (L858R/T790M) xenograft mouse models, brain penetrance, target specificity, and dose-limiting toxicity.8These improvements have motivated clinical evaluation of lazertinib in a variety of trials as front-line (NCT04248829)

Figure 2 .
Figure 2. Lazertinib bound to WT EGFR.Binding mode of lazertinib in complex with WT EGFR with the kinase domain in the active ɑC-helix in conformation.P-loop cartoon removed for clarity.(PDB ID 7UKV).

Figure 3 .
Figure 3. Lazertinib bound to inactive EGFR(T790M/V948R) with distinct conformations.A) Lazertinib bound with phenyl ring anchored within van der Waals distance to the T790M methionine (~3.8 Å) and methane amine involved in intramolecular H-bonding.B) A distinct conformation of lazertinib consisting of a "flipped" conformation with outward phenyl and methyleneamine donating a H-bond to D855.Other interactions seen in this structure include an intramolecular dipoledipole pyrazole to carbonyl (3.2 Å) as well as van der Waals methyleneamine methyl to thioether (3.8 Å).P-loop cartoon removed for clarity.(PDB ID 7UKW)

Table 1 . Biochemical activity assays (Homogenous time-resolved fluorescence HTRF) of recombinant EGFR and HER2 kinase domains
. IC50 values were16easured from a single experiment in triplicate.The ATP concentration was 100 µM.Errors are reported as ± the standard error.Data from ref.Wittlinger et al.,16 a