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Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.

ABSTRACT: The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

SUBMITTER: Montalvo-Ortiz BL

PROVIDER: S-EPMC3339933 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The myotonic dystrophy kinase-related CDC42-binding kinases MRCK? and MRCK? 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.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 MRCK? kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCK? 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 ?M BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.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.

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Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.

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