Fig. 2: Evaluation of cooperative binding.
From: Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors
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.
