Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation

Abstract

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Keywords: DFG-in/c-Helix-out; EGFR kinase; Ibrutinib; NSCLC; inactive conformation.

Figure 1. Ibrutinib irreversible binding mode exploration

(A) Ibrutinib and WZ4002 anti-proliferation effects against the H1975 cell line by removal of drug after 1 h, 4 h and 72 h treatment. (B) Ibrutinib and WZ4002 inhibitory effects on EGFRY1068 auto-phosphorylation in the H1975 cell line at different time points by removal of drug after 4 h pretreatment. (C) Ibrutinib and WZ4002 anti-proliferation effects against the HCC827 cell line by removal of drug after 1 h, 4 h and 72 h treatment. (D) Ibrutinib and WZ4002 inhibitory effects on EGFRY1068 auto-phosphorylation in the HCC827 cell line at different time points by removal of drug after 4 h pretreatment. (E) Micro-Scale Thermophoresis (MST) technology based binding Kd test of Ibrutinib and WZ4002 against EGFR T790M/L858R kinase.

Figure 2. Crystal structure of EGFR T790M+ibrutinib and the comparison with other EGFR T790M+inhibitor co-crystal structures

(A) Chemical structures of Ibrutinib, HKI-272, Lapatinib and WZ4002 shown in the orientation roughly indicating their binding mode to EGFR. “Hinge” indicates the hinge peptide of EGFR that connects N-lobe and C-lobe of the kinase and interacts with the inhibitors through hydrogen bonds (indicated by dashed lines). The green arrows indicate the extra methylene in HKI-272 and Lapatinib. The purple arrow indicates the cyano group of HKI-272. (B) Superimposition of the four molecules in the asymmetric unit of the EGFR T790M+ibrutinib co-crystal structure. The red arrows indicate the slight difference between the two binding modes of ibrutinib. (C) Superimposition of the EGFR T790M+ibrutinib structure (slate) and the V948R+AMP-PnP structure (drawn from PDB ID 2GS7, cyan). (D) Superimposition of the EGFR T790M+ibrutinib structure (slate) and the T790M+HKI-272 structure (yellow). The double-headed orange arrow indicates the hydrophobic interaction between the inhibitor and the Phe856 side-chain. (E) Superimposition of the EGFR T790M+ibrutinib structure (slate) and the T790M+WZ4002 structure (green).