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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 myosin light chain phosphorylation in MDA-MB-231 breast cancer cells by BDP5290. (A) MDA-MB-231 human breast cancer cells were treated with indicated concentrations of BDP5290 or Y27632 for 30 minutes prior to cell lysis and quantitative western blotting with indicated antibodies. (B) Inhibition of MLC phosphorylation by BDP5290 and Y27632. All results are shown as mean ± standard error of n = 3 independent replicates. (C) Immunofluorescence imaging of MDA-MB-231 cells that were untreated or treated with 0.5 μM Y27632 or BDP5290 for 30 minutes. After fixation and permeabilization, cells were stained with phalloidin to visualize filamentous actin structures and pMLC (S19) primary antibody as indicated.
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Fig5: Inhibition of myosin light chain phosphorylation in MDA-MB-231 breast cancer cells by BDP5290. (A) MDA-MB-231 human breast cancer cells were treated with indicated concentrations of BDP5290 or Y27632 for 30 minutes prior to cell lysis and quantitative western blotting with indicated antibodies. (B) Inhibition of MLC phosphorylation by BDP5290 and Y27632. All results are shown as mean ± standard error of n = 3 independent replicates. (C) Immunofluorescence imaging of MDA-MB-231 cells that were untreated or treated with 0.5 μM Y27632 or BDP5290 for 30 minutes. After fixation and permeabilization, cells were stained with phalloidin to visualize filamentous actin structures and pMLC (S19) primary antibody as indicated.

Mentions: To determine how inhibition of endogenous MRCK and ROCK affected pMLC levels, we treated parental MDA-MB-231 breast cancer cells with BDP5290 or Y27632 at varying concentrations (Figure 5A). BDP5290 had an EC50 of 316 nM while Y27632 was slightly less potent with an EC50 of 407 nM (Figure 5B). At higher concentrations, BDP5290 reduced pMLC to undetectable levels while Y27632 was unable to completely inhibit pMLC on western blots. Immunofluorescence microscopy of MDA-MB-231 cells revealed both cytoplasmic stress-fibre associated and cortical pMLC (Figure 5C). Treatment for 30 minutes with inhibitors at concentrations near their EC50 on Western blots showed that 0.5 μM Y27632 effectively reduced stress-fibre associated pMLC staining but had little effect on cortical pMLC, in agreement with previous reports showing that ROCK principally phosphorylates cytoplasmic MLC [10]. In contrast, application of 0.5 μM BDP5290 lessened both cytoplasmic and cortical pMLC levels, which is consistent with previous reports showing that an important site of MRCK function is at cortical cytoskeletal structures proximal to the plasma membrane [21,22].Figure 5


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 myosin light chain phosphorylation in MDA-MB-231 breast cancer cells by BDP5290. (A) MDA-MB-231 human breast cancer cells were treated with indicated concentrations of BDP5290 or Y27632 for 30 minutes prior to cell lysis and quantitative western blotting with indicated antibodies. (B) Inhibition of MLC phosphorylation by BDP5290 and Y27632. All results are shown as mean ± standard error of n = 3 independent replicates. (C) Immunofluorescence imaging of MDA-MB-231 cells that were untreated or treated with 0.5 μM Y27632 or BDP5290 for 30 minutes. After fixation and permeabilization, cells were stained with phalloidin to visualize filamentous actin structures and pMLC (S19) primary antibody as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195943&req=5

Fig5: Inhibition of myosin light chain phosphorylation in MDA-MB-231 breast cancer cells by BDP5290. (A) MDA-MB-231 human breast cancer cells were treated with indicated concentrations of BDP5290 or Y27632 for 30 minutes prior to cell lysis and quantitative western blotting with indicated antibodies. (B) Inhibition of MLC phosphorylation by BDP5290 and Y27632. All results are shown as mean ± standard error of n = 3 independent replicates. (C) Immunofluorescence imaging of MDA-MB-231 cells that were untreated or treated with 0.5 μM Y27632 or BDP5290 for 30 minutes. After fixation and permeabilization, cells were stained with phalloidin to visualize filamentous actin structures and pMLC (S19) primary antibody as indicated.
Mentions: To determine how inhibition of endogenous MRCK and ROCK affected pMLC levels, we treated parental MDA-MB-231 breast cancer cells with BDP5290 or Y27632 at varying concentrations (Figure 5A). BDP5290 had an EC50 of 316 nM while Y27632 was slightly less potent with an EC50 of 407 nM (Figure 5B). At higher concentrations, BDP5290 reduced pMLC to undetectable levels while Y27632 was unable to completely inhibit pMLC on western blots. Immunofluorescence microscopy of MDA-MB-231 cells revealed both cytoplasmic stress-fibre associated and cortical pMLC (Figure 5C). Treatment for 30 minutes with inhibitors at concentrations near their EC50 on Western blots showed that 0.5 μM Y27632 effectively reduced stress-fibre associated pMLC staining but had little effect on cortical pMLC, in agreement with previous reports showing that ROCK principally phosphorylates cytoplasmic MLC [10]. In contrast, application of 0.5 μM BDP5290 lessened both cytoplasmic and cortical pMLC levels, which is consistent with previous reports showing that an important site of MRCK function is at cortical cytoskeletal structures proximal to the plasma membrane [21,22].Figure 5

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