title: A novel small-molecule MRCK inhibitor blocks cancer cell invasion.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Screening 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.
  
  
  CONCLUSIONS
  BDP5290 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.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Unbekandt
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Croft
authors.forename: Diane
authors.initials: D
authors.lastname: Crighton
authors.forename: Mokdad
authors.initials: M
authors.lastname: Mezna
authors.forename: Duncan
authors.initials: D
authors.lastname: McArthur
authors.forename: Patricia
authors.initials: P
authors.lastname: McConnell
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Schüttelkopf
authors.forename: Simone
authors.initials: S
authors.lastname: Belshaw
authors.forename: Andrew
authors.initials: A
authors.lastname: Pannifer
authors.forename: Mairi
authors.initials: M
authors.lastname: Sime
authors.forename: Justin
authors.initials: J
authors.lastname: Bower
authors.forename: Martin
authors.initials: M
authors.lastname: Drysdale
authors.forename: Michael F
authors.initials: MF
authors.lastname: Olson
mesh_terms: D000577:Amides / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D006801:Humans; D065636:Myotonin-Protein Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011720:Pyrazoles / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C000602147:4-chloro-1-(4-piperidyl)-N-(5-(2-pyridyl)-1H-pyrazol-4-yl)pyrazole-3-carboxamide; D000577:Amides; D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; C108830:Y 27632; C105518:CDC42BPA protein, human; C118666:CDC42BPB protein, human; D065636:Myotonin-Protein Kinase; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases