Validating aurora b as an anticancer drug target
Microtubule inhibitor dosage can be significantly reduced when combined with a PLK1 inhibitor.The efficacy of these inhibitor combinations was validated by our experimental results.We demonstrate that a VX-680-resistant Aurora A mutant remains sensitive to the distinct anti-proliferative agent MLN8054 in human cells and that Aurora B is the critical target of VX-680 that promotes cell death in a cancer cell model.Furthermore, by analyzing a Plk1 mutant with decreased sensitivity to BI 2536, we establish that a mitotic phenotype arising from exposure to this drug is indeed due to Plk1 inhibition and that, during mitosis, Plk1 controls Aurora A phosphorylation at the critical activating residue Thr c DNA encoding full-length human Aurora A or the T210D Plk1 kinase domain mutant (encoding amino acids 1–364) was inserted into plasmid p ET30-Ek/LIC (Novagen) and subjected to PCR to generate the desired point mutants.Moreover, inhibitors of many distinct protein kinases have emerged as indispensable biological tools, in part through their rapid and often reversible mode of action, but also because of their widespread availability and utility in a range of research settings.
This makes interpretation of experiments in which Aurora A or Plk1 inhibitors are employed potentially confusing because phenotypes assigned to one inhibitor target might actually be due to indirect inhibition of the other kinase.
The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1.
In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks.
To begin to address these issues, we have investigated the cellular plasticity of kinase inhibition by both VX-680 and BI 2536.
By evaluating drug-resistant Aurora A and B proteins and exploiting these mutants in stable human cell lines, we demonstrate that drug-resistant forms of these kinases can be used to prove that phenotypes arising from VX-680 exposure are actually due to inhibition of the predicted mitotic targets.
In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants.