, giving rise to a total of five different aminoacid substitutions in the catalytic domain. Three of the seven cell lines contained two different Aurora B single mutants, His250Tyr with Gly160Val and His250Tyr with Gly160Glu. All of the Aurora mutants, with the exception of Leu308Pro, were ectopically expressed as Myc tagged fusions in DLD 1 CAY10505 cells and shown to localize correctly and maintain normal kinase function. In the presence of ZM447439, phosphorylation of the Aurora B substrate histone H3 was rescued in cells expressing drug resistant mutants of this kinase. Expression of similar levels of wild type Aurora B did not show a comparable effect. The most drug resistant mutant proved to be the Gly160Val of Aurora B, followed by Tyr156His and His250Tyr.
In vitro activity assays using histone H3 as a substrate showed that the Tyr156His mutant is 10 fold less sensitive to the drug than wild type kinase, while the Gly160Val and Gly160Glu mutants are completely BAY 73-4506 resistant to 500 M ZM447439. Most strikingly, these mutations were found to confer resistance to other known Aurora kinase inhibitors of unrelated structure to ZM447439. In the same in vitro activity assay, VX 680 was 20 fold less potent against the Tyr156His and Gly160Glu mutants of Aurora B than the wildtype kinase. Resistance mutations also diminished the ability of Hesperadin to block the catalytic activity of Aurora B. Consistent with the types of drug resistance mutations that have Krishnamurty and Maly Page 9 ACS Chem Biol. Author manuscript, available in PMC 2011 January 15.
NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript been identified in BCR ABL and EGFR, the Tyr156His, Gly160Val, Gly160Glu and His250Tyr mutants of Aurora B do not have compromised catalytic activity. In fact, an in vitro assay in the presence of 200 M ATP demonstrated that these mutants of Aurora B have higher catalytic activities than the wild type enzyme. Further analysis of the kinetic parameters of these Aurora B mutants was not performed. Structural studies were performed to characterize the specific mechanism of resistance. A crystal structure of the Xenopus laevis Aurora B:INCENP complex bound to ZM447439 shows that the inhibitor sits in the ATP binding pocket with the quinazoline core lying against the hinge region of the kinase, the benzamide directed towards the C helix and the morpholino substituent directed out of the pocket into solvent .
Mapping of the human Aurora mutations onto the Xenopus model places the Tyr156 residue at the hinge region of the kinase in close proximity to the aromatic quinazoline core of ZM447439. The authors hypothesize that mutation of the tyrosine to a histidine may weaken the van der Waals contacts that this hinge region amino acid makes with the smallmolecule inhibitor. The Gly160 residue maps to the hinge loop as well. In a similar fashion to the Thr315Ile gatekeeper mutation that renders ABL insensitive to imatinib, substitution of glycine for a larger residue most likely introduces a steric clash with the bound inhibitor. From a model of human Gly160Val bound to ZM447439, it is apparent that the morpholinyl propoxy moiety extends over the hinge loop and would be expected to collide with the valine or glutamate residue. A similar steric clash would be expected to occur with the pipe