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

Unbekandt M, Croft DR, Crighton D, Mezna M, McArthur D, McConnell P, Schüttelkopf AW, Belshaw S, Pannifer A, Sime M, Bower J, Drysdale M, Olson MF - Cell Commun. Signal (2014)

Bottom Line: Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK.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.

View Article: PubMed Central - PubMed

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.

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Inhibition of kinase activity by BDP5290 in cells. (A) MDA-MB-231 breast cancer cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were established as indicated. Following treatment with doxycycline for 18 hours to induce expression, cell lysates were western blotted with antibodies as indicated. (B) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with BDP5290 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (C) Inhibition of MLC phosphorylation by BDP5290 for each induced kinase domain. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with Y27632 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (E) Inhibition of MLC phosphorylation by Y27632 for each induced kinase domain. All results are shown as mean ± standard error of n = 4 independent replicates.
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Fig4: Inhibition of kinase activity by BDP5290 in cells. (A) MDA-MB-231 breast cancer cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were established as indicated. Following treatment with doxycycline for 18 hours to induce expression, cell lysates were western blotted with antibodies as indicated. (B) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with BDP5290 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (C) Inhibition of MLC phosphorylation by BDP5290 for each induced kinase domain. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with Y27632 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (E) Inhibition of MLC phosphorylation by Y27632 for each induced kinase domain. All results are shown as mean ± standard error of n = 4 independent replicates.

Mentions: To determine whether the inhibitor BDP5290 would inhibit MRCK in cells, we established cell lines expressing doxycycline-inducible ROCK1, ROCK2 or MRCKβ kinases domains that led to increased pMLC following doxycycline treatment for 18 hours (Figure 4A). By treating cells in which ROCK1, ROCK2 or MRCKβ had been induced with doxycycline with varying concentrations of BDP5290 from 0 to 3 μM (Figure 4B), cell based EC50 values were determined to be 166 nM for MRCKβ, 501 nM for ROCK1 and 447 nM for ROCK2 (Figure 4C). Interestingly, 3 μM BDP5290 completely inhibited MLC phosphorylation induced by MRCKβ, but not by ROCK1 or ROCK2. For comparison, similar experiments were performed with the ROCK selective inhibitor Y27632 [20] with concentrations ranging from 0 to 30 μM (Figure 4D). Inhibition of MLC phosphorylation induced by ROCK1 had an EC50 value of 4.27 μM and for ROCK2 was 1.62 μM (Figure 4E). Although Y27632 had some effect on MRCKβ activity, inhibition was not greater than 50% at the highest 30 μM concentration. These results demonstrate that BDP5290 is a potent inhibitor of MLC phosphorylation in cells with selectivity for MRCK over ROCK1 or ROCK2.Figure 4


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

Unbekandt M, Croft DR, Crighton D, Mezna M, McArthur D, McConnell P, Schüttelkopf AW, Belshaw S, Pannifer A, Sime M, Bower J, Drysdale M, Olson MF - Cell Commun. Signal (2014)

Inhibition of kinase activity by BDP5290 in cells. (A) MDA-MB-231 breast cancer cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were established as indicated. Following treatment with doxycycline for 18 hours to induce expression, cell lysates were western blotted with antibodies as indicated. (B) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with BDP5290 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (C) Inhibition of MLC phosphorylation by BDP5290 for each induced kinase domain. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with Y27632 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (E) Inhibition of MLC phosphorylation by Y27632 for each induced kinase domain. All results are shown as mean ± standard error of n = 4 independent replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Inhibition of kinase activity by BDP5290 in cells. (A) MDA-MB-231 breast cancer cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were established as indicated. Following treatment with doxycycline for 18 hours to induce expression, cell lysates were western blotted with antibodies as indicated. (B) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with BDP5290 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (C) Inhibition of MLC phosphorylation by BDP5290 for each induced kinase domain. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with Y27632 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (E) Inhibition of MLC phosphorylation by Y27632 for each induced kinase domain. All results are shown as mean ± standard error of n = 4 independent replicates.
Mentions: To determine whether the inhibitor BDP5290 would inhibit MRCK in cells, we established cell lines expressing doxycycline-inducible ROCK1, ROCK2 or MRCKβ kinases domains that led to increased pMLC following doxycycline treatment for 18 hours (Figure 4A). By treating cells in which ROCK1, ROCK2 or MRCKβ had been induced with doxycycline with varying concentrations of BDP5290 from 0 to 3 μM (Figure 4B), cell based EC50 values were determined to be 166 nM for MRCKβ, 501 nM for ROCK1 and 447 nM for ROCK2 (Figure 4C). Interestingly, 3 μM BDP5290 completely inhibited MLC phosphorylation induced by MRCKβ, but not by ROCK1 or ROCK2. For comparison, similar experiments were performed with the ROCK selective inhibitor Y27632 [20] with concentrations ranging from 0 to 30 μM (Figure 4D). Inhibition of MLC phosphorylation induced by ROCK1 had an EC50 value of 4.27 μM and for ROCK2 was 1.62 μM (Figure 4E). Although Y27632 had some effect on MRCKβ activity, inhibition was not greater than 50% at the highest 30 μM concentration. These results demonstrate that BDP5290 is a potent inhibitor of MLC phosphorylation in cells with selectivity for MRCK over ROCK1 or ROCK2.Figure 4

Bottom Line: Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus