A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

Mathieu Unbekandt,Martin Drysdale,Simone Belshaw,Daniel R Croft,Mokdad Mezna,Michael F Olson,Justin Bower,Patricia Mcconnell,Duncan Mcarthur,Andrew Pannifer,Alexander W Schüttelkopf,Mairi Sime,Diane Crighton

doi:10.1186/s12964-014-0054-x

Abstract

BackgroundThe myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.ResultsScreening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.ConclusionsBDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

Highlights

The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis
Iterative rounds of structureactivity relationship (SAR) chemistry on representatives of the initial screening hit matter, in vitro profiling and X-ray crystallography resulted in the discovery of 4-chloro-1(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3carboxamide (BDP5290; Figure 1A) as an Myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) inhibitor with excellent potency and selectivity profiles
Inhibitor dose– response assays at 1 μM Adenosine triphosphate (ATP), which was close to the experimentally-determined Km ATP values for the test kinases under the conditions of the enzyme assay (MRCKα, 0.9 μM; MRCKβ, 0.4 μM; ROCK1, 0.6 μM; ROCK2, 0.6 μM), revealed IC50 values of 17 nM for MRCKβ, 230 nM for ROCK1 and 123 nM for ROCK2 (Figure 1B)

Summary

Introduction

The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. By subduing cancer cell invasion, in an adjuvant setting, molecularly-targeted inhibitors. Metastasis is a multi-step process powered by dynamic reorganization of the actin-myosin cytoskeleton and remodelling of the extracellular matrix, allowing cells to invade their local environment, cross tissue boundaries and spread via blood and lymphatic vessels to distal regions of the body [2]. Contraction of actin-myosin cytoskeletal structures generates the mechanical force required for cell motility and invasion [2]. A key element of the cytoskeletal contractile machinery is myosin II, which is regulated by phosphorylation of myosin II light chain proteins (MLC) at two key sites (Thr and Ser19) [3]

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