T is much more active than the wild Kinesin Spindle Protein type enzyme and also has a 5-h Here kcat / Km, the L858R mutant. Because TKIs such as gefitinib competes with ATP for binding to the kinase active site, should the increased Hte affinity t reduce the force ATP inhibitor. In this mutant L858R/T790M M effect Combined with the small difference in binding affinity t for gefitinib clearly explained by our results Observation Ren surprising that irreversible inhibitors anilinoquinazoline maintain efficacy against the mutant T790M resistance, and they are for fully understand the nature of important resistance and m Possible pathways for the development of more effective drugs. The fact that the substitution T790M resistance by erh Increase the affinity t for ATP, liked t, as Ren with inhibitor binding sterically st Means that T790M is a eneric Mutant of resistance, it is more likely to confer to resistance to an ATP-competitive inhibitor. To our knowledge, this mechanism of drug resistance, resistance transferred by a mutation, the affinity t for a physiological substrate increased competition Ht, was not previously documented in a clinical setting. Interestingly, a separate but related effect was recently described in a mutant of the mitotic kinesin KSP. The KSP mutant was discovered in a laboratory screen and confers resistance by an allosteric mechanism with an increased Hten affinity t for ATP. As a class, irreversible inhibitors can overcome T790M resistance through covalent bond, once by F Bound, covalent, they are no longer in a competitive environment, the reversible equilibrium with ATP. A number of these compounds are in clinical trials in oncology confinement Lich HKI 272, but none has yet again U approval. One concern with covalent inhibitors is the potential toxicity T caused by off-target effects.
At least 10 kinases, additionally Tzlich to a reactive cysteine residue of EGFR in the position corresponding to Cys 797 in EGFR, so it will be important to understand the activity T to have irreversible agents against these kinases, in particular, including normal Tec family kinases JAK3 and other kinases important for h matopoetische development etic and immune function. However, our results show that ben not irreversible binding for effective inhibition of the T790M mutant CONFIRMS. A reversible inhibitor that binds with sufficient affinity displace t to ATP Lengths should also work. Calculations Similar to those of FIG. 3A show that reversible inhibitors should be effective with an affinity t of 200 pM or tighter against the T790M mutant. Protein preparation and crystallization processes. Geb ude For 696 1022 Residues Walls of human EGFR and having the sequence WT or L858R and T790M mutations were expressed and purified using a baculovirus / insect cells as described. The crystals T790M mutant were obtained in 0.1 M Hepes buffer, 21% PEG6000, 0.3 M NaCl and 5 mM tris phosphine, w While the crystals T790M/HKI 272 weremadeby complex co-crystallization in Hepes 0, 1 M , 0.2 M Li2SO4, 28% PEG3350 and 5 mM TCEP. T790M/AEE788 complex crystals were obtained by soaking crystals of apo-T790M made 300 m AEE788 inhibitor overnight. Structure determination and refinement. Diffraction data were collected at Argonne.