We managed to study this question in SKMG3 cells harboring the EGFR A289D mutant, because we had previously shown that this mutant, unlike EGFRvIII, does not abrogate the power of EGFR to respond to EGF. Erlotinib, to the other hand, was stronger than lapatinib at curbing EGFR in lung cancer cell lines with the EGFR kinase domain mutants EGFR746 750 and EGFR L858R, consistent with previous studies. Dub inhibitors Akt and Erk, two well documented effector kinases of the analyzed EGFR kinase domain mutants, were also more potently inhibited by erlotinib in comparison with lapatinib in these lines. Curiously, inhibition of EGFR in SKMG3 GBM cells did not end up in Akt or Erk inhibition, suggesting that the A289D mutant utilizes other downstream effector pathways. We also examined the consequences of lapatinib and erlotinib on cell death. Lapatinib, however not erlotinib, induced cell death in most examined GBM cell lines with EGFR ectodomain mutants. In EGFR mutant lung cancer cell lines, erlotinib induced cell death at lower levels than lapatinib. 3. Type II EGFR inhibitors efficiently displace ATP from EGFR EC mutants Our results with four different EGFR kinase inhibitors proposed that the catalytic domain of substitution reaction EGFR ectodomain mutants may possibly favor an inactive like conformation that is more available to lapatinib or HKI 272 than to erlotinib or CI 1033. To further test this model, we developed an assay that measures the capacity of EGFR kinase inhibitors to compete entirely mobile lysates with ATP for binding to the ATP cleft of the EGFR kinase domain. Coincubation of whole cell lysates from A289D EGFR mutant SKMG3 cells with biotinylated ATP and erlotinib demonstrated reduced ATP presenting with increasing erlotinib concentrations. Coincubation of a sample of the same whole cell lysate with increasing concentrations of lapatinib blocked ATP binding at lower concentrations Lu AA21004 of lapatinib than erlotinib. As a specificity get a handle on, we established ATP binding to the kinase domain of SRC and found no displacement of ATP binding by both lapatinib or erlotinib. We also repeated these experiments with whole mobile lysates from H3255 lung cancer cells, and discovered that erlotinib blocked ATP binding to the EGFR kinase domain better than lapatinib. Since differences in off rates involving the reversible EGFR kinase inhibitors erlotinib and lapatinib might affect results of the ATP opposition assay, we conducted additional tests with the permanent EGFR kinase inhibitors CI 1033 and HKI 272. Entirely cell lysates from A289D EGFR SKMG3 cells, HKI 272 better blocked ATP binding to the EGFR kinase domain than CI 1033, consistent with our model. Last but not least, we investigated whether a forced change in receptor conformation, induced by ligand binding, may possibly alter the ability of EGFR inhibitors to achieve access to the kinase domain and block EGFR phosphorylation.