Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors

Abstract

Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

Fig. 1. Structural characterization of osimertinib co-binding with different allosteric inhibitors.

a Chemical structures of ATP-competitive and allosteric inhibitors. b Crystal structure of EGFR(T790M/V948R) in complex with osimertinib and EAI045 (PDB 7JXM). The C-helix is colored red, Asp-Phe-Gly (DFG) motif in orange, ATP-site inhibitor in magenta, and allosteric inhibitor in green. c EGFR(L858R/V948R) in complex with osimertinib and JBJ-063 (PDB 7K1H). The P-loop and side chain of F723 from EGFR(L858R/V948R) in complex with AMP-PNP and JBJ-063 is shown in dark gray for comparison (PDB 7K1I). d EGFR(T790M/V948R) in complex with osimertinib and DDC4002 (PDB 6XL4).

Fig. 2. Evaluation of cooperative binding.

a Pulldown of EGFR protein from L858R/T790M/F723A Ba/F3 cells using biotinylated allosteric inhibitor following treatment with different irreversible tyrosine kinase inhibitors (TKIs) (n = 3 independent experiments). The dibenzodiazepinone allosteric inhibitor (b-DDC) failed to pulldown EGFR, whereas pulldown with b-JBJ-125 was enhanced or abolished depending on TKI pre-treatment. Biotinylated linker (b-linker) was used as a control. b Pulldown of EGFR protein from L858R/T790M/F723A Ba/F3 using biotinylated JBJ-125 (b-JBJ-125) following treatment with AZ5104 (n = 3 independent experiments). c Chemical structures of mavelertinib, naquotinib, and the osimertinib metabolite AZ5104. d Inhibition synergy evaluation between osimertinib (OSI) and mavelertinib (Mav) or naquotinib (Naq) in H3255GR cells (n = 3 independent experiments with 3 technical replicates per experiment). e Fluorescence polarization (FP) binding experiments using BODIPY-JBJ and purified EGFR kinase domain. Osimertinib, AZ5104, mavelertinib, and naquotinib enhanced binding of the allosteric inhibitor. The TKI neratinib, which extends into the allosteric pocket, was prevented binding of the allosteric inhibitor. Reported as mean (n = 2 independent experiments). Source data are provided as a Source Data file.

Fig. 3. Biochemical evaluation of inhibition synergy.

a Inhibition synergy analysis using purified EGFR kinase domain. Compound combinations were dispensed using a digital drug dispenser and IC₅₀s of one inhibitor were plotted as a function of the concentration of the other inhibitor. Similar to the FP binding experiments, osimertinib, AZ5104, and naquotinib enhanced the potency of JBJ-125, and vice versa. Mavelertinib was neither synergistic nor antagonistic with regard to JBJ-125 potency. Reported as mean ± SD (n = 3 independent experiments). b Labeling by osimertinib or mavelertinib in the presence or absence of allosteric inhibitor. Percent labeling was assessed via intact mass spectrometry. Reported as mean (n = 2 independent experiments). c Synergy evaluation with EGFR(L858R/C797S) and the osimertinib+JBJ−125 combination. The allosteric inhibitor enhances the potency of osimertinib despite the C797S mutation. Reported as mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 4. Structural characterization of additional inhibitor combinations.

a Structure of EGFR(T790M/V948R) in complex with mavelertinib and JBJ-125 (PDB 7JXK, light gray) and P-loop comparison with AMP-PNP+JBJ-125 (PDB 7JXP, gray) and mavelertinib (PDB 7JXI, black). b Crystal structure of EGFR(T790M/V948R) in complex with naquotinib and JBJ-063 (PDB 7LG8, light gray) and P-loop comparison with AMP-PNP+JBJ-063 (PDB 7K1I, gray) and naquotinib (PDB 5Y9T, black). c Crystal structure of EGFR(L858R/V948R) in complex with osimertinib and JBJ-063 (PDB 7K1I). Comparison of P-loop conformation in complex with osimertinib (PDB 4ZAU) in black, AMP−PNP+JBJ−063 in gray (PDB 7JXQ), and osimertinib+JBJ−063 in light gray. F723 contacts the allosteric inhibitor and occludes the putative entrance and exit tunnel to the allosteric site.

Fig. 5. Inhibitor features and role of F723 in cooperative binding and synergy.

a Pulldown of EGFR protein from HEK293T/17 cells transiently expressing EGFR(L858R/T790M) or EGFR(L858R/T790M/F723A) using b-JBJ-125 following treatment with different irreversible TKIs. F723A decreased the ability of select TKIs to enhance pulldown using b-JBJ-125 (n = 3 independent experiments). b Cooperative binding was not observed in the F723A variant in an FP binding assay using BODIPY-JBJ. Data are reported as mean (n = 2 independent experiments) EGFR(L858R/T790M) data are repeated from Fig. 2e for comparison. c Inhibition synergy resulted in a 20-fold increase in potency for JBJ-125 in the presence of osimertinib compared to approximately 2-fold in the L858R/T790M/F723A variant (top). Similarly, only a 3-fold increase in osimertinib potency was observed in the F723A variant compared to a 7.5-fold increase in L858R/T790M (bottom). Data are reported as mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 6. Proposed structural mechanism underlying cooperative binding and inhibition synergy.

When bound to nucleotide, the phosphates force the P-loop to be in an extended conformation with the side chain of F723 outward (grays, labeled 1 and 3). When bound to a TKI (osimertinib shown, magenta), F723 often folds under the P-loop (black, labeled 2) where the phosphates of ATP would be located. When in complex with both TKI and an allosteric inhibitor (green), the P-loop folds down into the space usually occupied by the phosphates and the side chain of F723 forms a π-stacking interaction with the inhibitor (light gray, labeled 4), closing the putative exit tunnel (see Fig. 4c). This more compact ternary complex conformation facilitates cooperative binding, which leads to inhibition synergy.