Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks

David H Williams,Matthew L.H Chu,Dominic A Sloane,Colin R Bignell,Paul J Scutt,Patrick A Eyers,Mike Cherry

doi:10.1074/jbc.m109.005694

Abstract

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. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. 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. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.

Highlights

Protein kinase inhibitors are prime examples of small molecules with the potential to revolutionize the treatment of chronic disease states such as inflammation and cancer (1, 2)
By evaluating drug-resistant Aurora A and B proteins in vitro and exploiting these mutants in stable human cell lines, we demonstrate that drugresistant forms of these kinases can be used to prove that phenotypes arising from VX-680 exposure are due to inhibition of the predicted mitotic targets
VX-680 Inhibits Human Aurora and ABL Kinases in Vitro— VX-680 is an equipotent Aurora inhibitor, with IC50 values of between 18 and 25 nM for human Aurora kinases when assessed in our standard radiometric assay (Table 1)

Summary

Introduction

Protein kinase inhibitors are prime examples of small molecules with the potential to revolutionize the treatment of chronic disease states such as inflammation and cancer (1, 2). Conclusions drawn in many thousands of peer-reviewed research papers every year rely upon experiments conducted with kinase inhibitors, but in only a handful of studies is the important question of inhibitor specificity explicitly addressed (4 –7) This is a vital issue because claims for specificity have rarely stood the test of time, yet a detailed knowledge of kinase inhibitor promiscuity would be beneficial in the clinic, where the simultaneous blockade of multiple signaling pathways can be exploited as an anticancer strategy (8). The key mitotic events of centrosome separation, bipolar spindle formation, and chromosome segregation are linked to the physical separation of the genomes during cytokinesis (11) These conserved signaling programs are controlled by dedicated mitotic protein kinases, which include two prominent human gene families, the Aurora kinases (comprising Aurora A, B, and C) and the Polo-like kinases (comprising Plk1– 4), whose overexpression in a spectrum of cancers make them outstanding drug candidates (12).

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