A first generation inhibitor of human Greatwall kinase, enabled by structural and functional characterisation of a minimal kinase domain construct

// Cory A. Ocasio 1,3,* , Mohan B. Rajasekaran 2,* , Sarah Walker 3 , Darren Le Grand 3 , John Spencer 4 , Frances M.G. Pearl 4 , Simon E. Ward 3 , Velibor Savic 1,5 , Laurence H. Pearl 2 , Helfrid Hochegger 1 and Antony W. Oliver 2 1 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK 2 Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK 3 Sussex Drug Discovery Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK 4 School of Life Sciences, University of Sussex, Falmer, Brighton, UK 5 Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, UK * The authors wish it to be known, that in their opinion, the first 2 authors should be regarded as joint first authors Correspondence to: Cory A. Ocasio, email: // Helfrid Hochegger, email: // Antony W. Oliver, email: // Keywords : kinase, inhibitor, Greatwall, ENSA, cancer Received : July 20, 2016 Accepted : August 02, 2016 Published : August 22, 2016 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.