Acquired drug resistance in epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer is a persistent challenge in cancer therapy. Previous studies of trisubstituted imidazole inhibitors led to the serendipitous discovery of inhibitors that target the drug resistant EGFR(L858R/T790M/C797S) mutant with nanomolar potencies in a reversible binding mechanism. To dissect the molecular basis for their activity, we determined the binding modes of several trisubstituted imidazole inhibitors in complex with the EGFR kinase domain with X-ray crystallography. These structures reveal that the imidazole core acts as an H-bond acceptor for the catalytic lysine (K745) in the "αC-helix out" inactive state. Selective N-methylation of the H-bond accepting nitrogen ablates inhibitor potency, confirming the role of the K745 H-bond in potent, noncovalent inhibition of the C797S variant. Insights from these studies offer new strategies for developing next generation inhibitors targeting EGFR in non-small-cell lung cancer.

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 the nature of lazertinib inhibition, 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 EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The thirdgeneration agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximabindependent and mutant-selective cellular efficacies on the L858R (1.2 μM) and L858R/T790M (4.4 μM) variants.

No abstract

Specificity for a desired enzyme target is an essential property of small-molecule inhibitors. Molecules targeting oncogenic driver mutations in the epidermal growth factor receptor (EGFR) kinase domain have had a considerable clinical impact due to their selective binding to cancer-causing mutants compared to wild type. Despite the availability of clinically approved drugs for cancers driven by EGFR mutants, persistent challenges in drug resistance in the past decades have led to newer generations of drugs with divergent chemical structures. The present clinical complications are mainly due to acquired resistance to third-generation inhibitors by the acquisition of the C797S mutation. Diverse fourth-generation candidates and tool compounds with C797S selectivity have emerged and their structural characterization has allowed for understanding of the molecular factors that allow for EGFR mutant selective binding. Here, we have reviewed all known structurally-characterized EGFR TKIs targeting clinically-relevant mutations to identify consistent binding mode features that enable C797S inhibition. Newer generation EGFR inhibitors exhibit consistent and previously underutilized hydrogen bonding interactions with the conserved K745 and D855 residue side chains. We also consider binding modes and hydrogen bonding interactions of inhibitors targeting the classical ATP and the more unique allosteric sites.