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.

A water-mediated allosteric network governs activation of Aurora kinase A

“…However, there is another group of AGC kinases in which the polar residue linked to the water network is instead located at a third cavity position (104 in PKA numbering) (Figure 5f). The x-ray structure of MRCK, a representative member of this group, shows that this leads to a more AurA-like hexagonal water network (Figure 5f) 47 . Apparently there are several classes of water network in different kinases, distinguished by the position of the polar amino acid that participates in the hydrogen bonding.…”

Migrastatics—Anti-metastatic and Anti-invasion Drugs: Promises and Challenges

“…Moreover, the ability of human SCC12 squamous cell carcinoma cells to invade a 3D collagen matrix was strongly inhibited by 2-μM BDP5290 but not by the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation [120].…”

“…Nevertheless, it encouraged the development of second-generation ROCK and/or MRCK inhibitors such as RKI-18, BDP5290 or DJ4, which show substantially better specificity. Although these inhibitors are widely used in experimental conditions, no in vivo data are yet available for RKI-18 [119], BDP5290 [120] or DJ4 [122]. However, DJ4 was found to inhibit the invasiveness of human breast carcinoma MDA-MB-231 cells [122].…”

A Novel Small-Molecule Inhibitor of MRCK Prevents Radiation-Driven Invasion in Glioblastoma

“…This mode of invasion is characterized by an elongated cell body, actin-rich protrusions, and actinmyosin contractility at the rear of the migrating cells. Unbekandt and colleagues have recently demonstrated that MRCK can drive cancer cell migration, a process that can be inhibited by specific small-molecule inhibitors (15)(16)(17). Because CDC42 activity is upregulated in glioma (18,19) and may drive the mesenchymal mode of migration employed by infiltrating glioma cells, we investigated a potential role for MRCK-driven GBM cell invasion in the pathogenesis of GBM.…”