Abstract
MASTL (microtubule-associated serine/threonine kinase-like), more commonly known as Greatwall (GWL), has been proposed as a novel cancer therapy target. GWL plays a crucial role in mitotic progression, via its known substrates ENSA/ARPP19, which when phosphorylated inactivate PP2A/B55 phosphatase. When over-expressed in breast cancer, GWL induces oncogenic properties such as transformation and invasiveness. Conversely, down-regulation of GWL selectively sensitises tumour cells to chemotherapy. Here we describe the first structure of the GWL minimal kinase domain and development of a small-molecule inhibitor GKI-1 (Greatwall Kinase Inhibitor-1). In vitro, GKI-1 inhibits full-length human GWL, and shows cellular efficacy. Treatment of HeLa cells with GKI-1 reduces ENSA/ARPP19 phosphorylation levels, such that they are comparable to those obtained by siRNA depletion of GWL; resulting in a decrease in mitotic events, mitotic arrest/cell death and cytokinesis failure. Furthermore, GKI-1 will be a useful starting point for the development of more potent and selective GWL inhibitors.
Highlights
The mitotic kinase MASTL - more commonly known as Greatwall kinase or GWL - belongs to the AGC family of serine/threonine protein kinases and has recently emerged as a potential target for cancer chemoprevention [1,2,3,4,5]
We noted that the amino acid sequence preceding the conserved ‘APE’ kinase motif of hGWL resembled that of the PKC (Protein Kinase C) family of kinases, in particular the alpha isozyme [19] (Figure 1A, Bottom)
To determine whether GKI-1 had any level of selectivity towards kinases within the AGC-family, we examined its effect on the activities of two commercially available proteins, ROCK1 and PKA; selected as they are known to be potently inhibited by the AGC kinase inhibitor AT13148, with experimentally determined IC50 values of ~6 and 11 nM respectively [36] (Table 2)
Summary
The mitotic kinase MASTL (microtubule-associated serine/threonine kinase-like) - more commonly known as Greatwall kinase or GWL - belongs to the AGC family of serine/threonine protein kinases and has recently emerged as a potential target for cancer chemoprevention [1,2,3,4,5]. In pursuit of developing human GWL as a target for drug discovery we have developed a minimal GWL kinase domain construct, in which the NCMR has been deleted and a conventional AGC-kinase activation segment inserted in its place This construct is soluble even when purified from E.coli, and displays specific kinase activity towards its bona fide substrate ENSA. We have determined the X-ray crystal structure of this construct and have developed a first generation inhibitor displaying in cellulo efficacy, based on a small-scale inhibitor screen and rational SAR-driven design This molecule, GKI-1, may find utility as a lead / tool compound to inform the ongoing development of potent and specific GWL inhibitors
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