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

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.

TAK-960, a Novel, Orally Available, Selective Inhibitor of Polo-Like Kinase 1, Shows Broad-spectrum Preclinical Antitumor Activity in Multiple Dosing Regimens

“…Resistance to PLK1 inhibition might also be due to mutations in the ATP-binding site of PLK1. Scutt and colleagues (24) found that mutation of Arg136 to Gly in the PLK1 ATPbinding pocket could alter the sensitivity to PLK1 inhibitors in a compound-specific manner. Further efforts will be required to define potential patient stratification factors to help identify the tumors most vulnerable to TAK-960 and to determine whether combinatorial approaches may lead to durable tumor regression.…”

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

“…Flow cytometric assay showed that after incubation with LRD-22 at indicated concentrations for 24 h, the percentage of apoptotic HepG2 cells increased from 6.71% in the control group to 15.11% in the group treated with 8 mM LRD-22 (Fig. S2).…”

“…Activation of AURKA requires binding to specific cofactors including Ajuba, Bora and TPX2, leading to the autophosphorylation of a residue in its T-loop (Thr 288 ) [15]. Its kinase activity increases from late G2-phase onwards and peaks in prometaphase.…”

The structural mechanisms that underpin mitotic kinase activation

“…Immunofluorescence microscopy images show that centrosomal Aurora-A is strongly phosphorylated, whereas Aurora-A bound to TPX2 to spindle microtubules is weakly phosphorylated [16,17]. It would appear that, at the centrosome, Aurora-A is primarily activated by phosphorylation, whereas on the mitotic spindle the action of PP6 (protein phosphatase 6) (the cellular phosphatase for Aurora-A [18]) keeps Aurora-A in a mainly dephosphorylated state and ensures that activation is primarily via TPX2 binding.…”