olvent. AEE788 has been shown to be an extremely potent inhibitor of ErbB family kinases and VEGFR, with low nanomolar potency Several oncogenic mutations in EGFR have been identified that give rise to NSCLCs. These include exon 19 deletions and a point mutation in exon 21 that mutates Leu858 in the activation loop to an Arg, the latter accounting for approximately BMS-540215 Brivanib 40% of all mutations. A third point mutation that occurs less frequently is the conversion of Gly719 in the P loop to a Ser. Both Leu858Arg and Gly719Ser are gain of function mutations, and the success of gefitinib and erlotinib partially arises from their increased potency against these mutant kinases over the wild type enzyme . Several studies have been conducted to characterize the structure and activity of the Leu858Arg and Gly791Ser mutants of EGFR.
Crystal structures of the Leu858Arg and Gly791Ser mutants bound to the non hydrolyzable ATP analog AMP PNP show that these kinases exist in an active conformation, similar to that of the wild type kinase. To understand the mechanism of activation of the Leu858Arg mutant, crystal structures of wild type EGFR bound to lapatinib were studied. Lapatinib binds AZD8330 to an inactive form of the kinase domain, with the activation loop segment forming a helical turn that displaces the C helix from the regulatory site. Leu858 is one of several hydrophobic residues on the activation loop that helps to stabilize this inactive conformation. Upon substitution of leucine to arginine, the charged residue is no longer favorably accommodated in the hydrophobic pocket, effectively destabilizing the inactive form of the kinase.
Similar reasoning is applied to the Gly179Ser mutant, the serine residue destabilizes the inactive conformation of the P loop. These structural changes results in the Leu858Arg and Gly791Ser mutants of EGFR having a 50 and 10 fold increase in activity over wild type in the presence of excess ATP and peptide substrate, respectively. Further Krishnamurty and Maly Page 6 ACS Chem Biol. Author manuscript, available in PMC 2011 January 15. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript kinetic analysis demonstrated that these mutations result in a 10 to 20 fold increase in the kcat for ATP. However, this is compensated by a 5 to 10 fold higher Km for ATP.
Because cellular concentrations of ATP are much higher than EGFR,s Km for ATP, the increase in kcat is the most relevant parameter in a cellular context. Although patients with NSCLC that bear the Leu858Arg mutation respond well to gefitinib and erlotinib treatment, relapse due to drug resistance is common. Molecular analysis of tumor material obtained from patients with acquired resistance to gefitinib/erlotinib treatment has found that a single amino acid substitution in the catalytic domain of EGFR coincides with a majority of cases of drug resistance, conversion of the Thr790 gatekeeper residue to methionine. Significantly, the Thr790Met mutant occurs in the context of the Leu858Arg sensitizing mutation. Therefore, it appears that the gatekeeper mutation eliminates the drug sensitivity that Leu858Arg confers. This resistance mutation has been identified in almost 50% of cases of acquired resistance, making it a significant target of research towards more effective therapies. In a more recent study involving tumor cells ob