Based on the structure of Aurora bound to AMP PNP and the kinetic data for these mutants, these substitutions do not affect the binding of ATP. Despite the diverse chemical structures of ZM477439, VX 680 and Hesperadin, these inhibitors exploit similar contacts with the ATP binding CCT128930 pocket of Aurora, which leads to their uniform sensitivity to mutations in these region. In a related study, Scutt and co workers identified mutations in Aurora A that confer resistance to the inhibitor VX 680. Upon structural analysis of the binding mode of VX 680 in Aurora A kinase, the analogous glycine residue that confers resistance to Aurora B was identified. Mutation of this residue to a bulkier amino acid conferred resistance to VX 680 and ZM447439, with Gly216Leu showing the greatest loss in sensitivity compared to wild type Aurora A .
However, these substitutions in Aurora A greatly reduced the overall activity of this enzyme, which is in contrast to their effect on the catalytic activity of Aurora B. Notably, the Gly216Leu, Gly216Val and Gly216Glu mutants of Aurora A were found to have 6%, 1% and 12% of the activity of the wild type enzyme, respectively. Despite the overlapping inhibitor sensitivities Bergenin and structural similarities between Aurora A and B, resistance mutations do not affect these enzymes uniformly. Like the Aurora family, several studies have been conducted with other disease relevant protein kinases to anticipate potential mechanisms of resistance to their respective small molecule inhibitors.
Upregulation of the mitogen activated protein kinase pathway has been implicated in a number of human cancers. For example, a gain of function mutation in the MAPK kinase kinase B RAF is found in many melanomas. Thus, small molecule inhibitors that target proteins in the MAPK pathway, such as BRAF and its downstream kinase substrate MEK1, are promising drug candidates. Potent and selective inhibitors of the catalytic activity of MEK1 have been developed, with a series of non ATP competitive inhibitors showing potential in clinical trials. Garraway and coworkers conducted a study to identify mutations that may arise to confer resistance to the non ATP competitive inhibitors AZD6244 or CI 1040 . To do this, a random mutagenesis screen in melanoma cells harboring Val600Glu B RAF was performed in the presence of cytotoxic concentrations of these drugs.
Sequencing of resistant clones identified a set of MEK1 mutant alleles, a majority of which contained point mutations surrounding the Krishnamurty and Maly Page 10 ACS Chem Biol. Author manuscript, available in PMC 2011 January 15. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript site of inhibitor binding. It is likely that these mutants confer resistance through direct interference with inhibitor binding or by altering the conformation of the C helix. In addition, several mutations were identified in regions of the catalytic domain that are not close to the site of site of drug binding, a subset of which may cause resistance by upregulating the intrinsic catalytic activity of MEK1. Several drug resistant MEK1 mutants expressed in A375 melanoma cells showed increased AZD6244 GI50 values relative to wild type A375 cells. Analysis of cells expressing these resistant MEK1 mutants showed that phosphorylation of the downstream MAPK ERK was rescued in the prese