tained experienced patients, low levels of the Thr790Met mutation were observed in 40% of the treatment naïve patients . Although the resistance allele was detected in only a CAL-101 GS-1101 small number of cells, it remains possible that tyrosine kinase inhibitor therapy might select for those tumor cells harboring the pre existing Thr790Met mutation. It was originally believed that transformation of the threonine at the gatekeeper position to a bulkier methionine caused resistance to erlotinib and gefitinib through steric interference, analogous to how the ABL Thr315Ile mutation confers resistance to imatinib. However, this steric argument for EGFR resistance became tenuous upon the discovery that irreversible EGFR inhibitors can overcome the resistance caused by this mutation in cellular assays.
In order to further investigate this seemingly unique mechanism of resistance, Yun and co workers employed a direct binding assay to determine the affinities of gefitinib and AEE788 for wild type, Leu858Arg, Thr790Met and Leu858Arg/Thr790Met EGFR kinase . As expected, gefitinib has a low nanomolar affinity for the Leu858Arg mutant, which is a 15 fold increase AZD8931 in potency over the wild type enzyme. The Thr790Met gatekeeper single mutant of EGFR is also quite sensitive to gefitinib, with a Kd 4.6 nM. Surprisingly, the Thr790Met/Leu858Arg double mutant was found to have only a moderately lower binding affinity for gefitinib, which is only a 4 fold difference compared to the Leu858Arg single mutant.
Clearly, conversion of the threonine gatekeeper residue to a methionine does not create a large steric clash that prevents inhibitor binding. Furthermore, the modest difference in binding affinity to the double mutant cannot fully explain the drug resistance that is observed in cellular assays and clinically.In order to further study how EGFR can become resistant to small molecule inhibition, crystal structures of the Thr790Met mutant, in the apo form and bound to the inhibitors AEE788 and neratinib, were obtained. As described earlier, AEE788 has similar binding interactions with the pocket adjacent to the gatekeeper residue as gefitinib. Like gefitinib, the binding affinity of AEE788 for Thr790Met and Thr790Met/ Leu858Arg is very similar to wild type EGFR.
Consistent with conversion of the Thr gatekeeper to Met having only a minimal effect on binding affinity, the superimposed crystal structures of AEE788 bound to wild type and Thr790Met EGFR show that there is little difference in the binding mode of the inhibitor. The pyrrolopyrimidine scaffold of AEE788 is in an identical orientation when bound to wildtype and Thr790Met EGFR. Furthermore, there is no apparent steric clash between the bulkier methionine residue and phenethylamine substituent as it enters the hydrophobic pocket adjacent to the adenine site, the gatekeeper residue adopts a slightly different orientation that allows the phenethylamine access to the pocket. Presumably, the Krishnamurty and Maly Page 7 ACS Chem Biol. Author manuscript, available in PMC 2011 January 15. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript gatekeeper residue of Thr790Met EGFR undergoes a similar conformational change when bound to gefitinib or erlotinib. To gain a better understanding of how the Thr790Met mutation leads to drug resistance, kinetic characterizati