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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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Matrigel invasion of MDA-MB-231 cells is inhibited by BDP5290. (A) MDA-MB-231 human breast cancer cells were plated in 96 well plates, then 24 hours later a ~800 μm scratch wound was created. Matrigel was overlayed for 1 hour, then images were acquired after 0 and 24 hours in the presence of DMSO vehicle or 3 μM BDP5290. (B) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated BDP5290 concentrations. (C) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated Y27632 concentrations. (D) Relative wound closure of MDA-MB-231 cells at 24 hours in the presence of indicated BDP5290 or Y27632 doses. All results are shown as mean ± standard error of n ≥ 4 independent replicates. (E) Alamar Blue metabolism of MDA MB 231 cells after 24 hours in the presence of DMSO vehicle, indicated concentrations of BDP5290 or 10 μM Y27632. Readings were compared to the Alamar Blue metabolism in DMSO treated cells that were used as the viability standard of 100% for each replicate experiment. All results are shown as mean ± standard error of 3 independent replicates.
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Fig6: Matrigel invasion of MDA-MB-231 cells is inhibited by BDP5290. (A) MDA-MB-231 human breast cancer cells were plated in 96 well plates, then 24 hours later a ~800 μm scratch wound was created. Matrigel was overlayed for 1 hour, then images were acquired after 0 and 24 hours in the presence of DMSO vehicle or 3 μM BDP5290. (B) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated BDP5290 concentrations. (C) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated Y27632 concentrations. (D) Relative wound closure of MDA-MB-231 cells at 24 hours in the presence of indicated BDP5290 or Y27632 doses. All results are shown as mean ± standard error of n ≥ 4 independent replicates. (E) Alamar Blue metabolism of MDA MB 231 cells after 24 hours in the presence of DMSO vehicle, indicated concentrations of BDP5290 or 10 μM Y27632. Readings were compared to the Alamar Blue metabolism in DMSO treated cells that were used as the viability standard of 100% for each replicate experiment. All results are shown as mean ± standard error of 3 independent replicates.

Mentions: We and others used siRNA to show that MRCK activity contributes to the ability of MDA-MB-231 human breast cancer cells to invade three dimensional Matrigel [6,11] and SCC12 human squamous cell carcinoma cells to invade collagen in an organotypic skin culture model of invasion [12]. Using a 96-well based Matrigel invasion assay and measurements determined with an Incucyte live content imaging instrument at time points up to 24 h (Figure 6A), we found that BDP5290 reduced MDA-MB-231 invasion at all tested concentrations starting from 0.1 μM, with virtually complete inhibition at 10 μM (Figure 6B). In contrast, Y27632 was dramatically less effective at inhibiting invasion at all concentrations, (Figure 6C). Comparing the dose–response relationship at 24 h after the start of the experiments, the EC50 for BDP5290 was 440 nM (Figure 6D), which was similar to the EC50 for inhibiting MLC phosphorylation (Figure 5B). However, Y27632 inhibition of invasion was not greater than 50% even at 30 μM (Figure 6D). To ensure that BDP5290 did not affect MDA MB 231 cell viability, a range of BDP5290 concentrations were tested for their effects on metabolic activity relative to DMSO vehicle using Alamar Blue [23]. After 24 hours in the presence of BDP5290 cell viability as measured by Alamar Blue metabolism was slightly reduced with an EC50 > 10 μM (Figure 6E). Wound closure was inhibited by > 60% at 1 μM BDP5290 (Figure 6D), a concentration that had no effect on cell viability (Figure 6E), indicating that the MRCK inhibitor can directly block cell motility independent of effects on cell proliferation. Treatment with 10 μM Y27632 had no effect on cell viability (Figure 6E) but inhibited wound closure by ~40%, indicating that ROCK inhibition also reduced cell motility directly. Given that both inhibitors had similar effects on total MLC phosphorylation (Figure 5A and B) but their effects on pMLC in different cellular compartments varied (Figure 5C), one possibility is that the phosphorylation of cortical MLC is an important contributor to cell motility and invasion. Previous studies found that polarized cell motility was dependent on recruitment of MRCK to the leading edge [22], where it promotes actin-myosin retrograde flow to generate tractive forces for cell movement [9]. One mechanism identified for this recruitment is the translocation of MRCKα associated with the PDK1 kinase that binds membrane phosphatidylinositol (3,4,5)-trisphosphate [24]. Blocking this translocation impairs the ability of MRCKα to promote lamellipodia retraction with consequent inhibition of directional migration. In addition, MRCK was found to be required for the assembly of matrix degrading complexes containing MT1-MMP [25] and promote cathepsin B expression [26] to permit cell invasion via matrix degradation. Therefore, BDP5290 may also affect matrix degradation as well as cell motility, resulting in significant inhibition of invasion.Figure 6


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)

Matrigel invasion of MDA-MB-231 cells is inhibited by BDP5290. (A) MDA-MB-231 human breast cancer cells were plated in 96 well plates, then 24 hours later a ~800 μm scratch wound was created. Matrigel was overlayed for 1 hour, then images were acquired after 0 and 24 hours in the presence of DMSO vehicle or 3 μM BDP5290. (B) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated BDP5290 concentrations. (C) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated Y27632 concentrations. (D) Relative wound closure of MDA-MB-231 cells at 24 hours in the presence of indicated BDP5290 or Y27632 doses. All results are shown as mean ± standard error of n ≥ 4 independent replicates. (E) Alamar Blue metabolism of MDA MB 231 cells after 24 hours in the presence of DMSO vehicle, indicated concentrations of BDP5290 or 10 μM Y27632. Readings were compared to the Alamar Blue metabolism in DMSO treated cells that were used as the viability standard of 100% for each replicate experiment. All results are shown as mean ± standard error of 3 independent replicates.
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Related In: Results  -  Collection

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Fig6: Matrigel invasion of MDA-MB-231 cells is inhibited by BDP5290. (A) MDA-MB-231 human breast cancer cells were plated in 96 well plates, then 24 hours later a ~800 μm scratch wound was created. Matrigel was overlayed for 1 hour, then images were acquired after 0 and 24 hours in the presence of DMSO vehicle or 3 μM BDP5290. (B) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated BDP5290 concentrations. (C) Relative wound closure of MDA-MB-231 cells imaged up to 24 hours at 3 hour intervals in the presence of indicated Y27632 concentrations. (D) Relative wound closure of MDA-MB-231 cells at 24 hours in the presence of indicated BDP5290 or Y27632 doses. All results are shown as mean ± standard error of n ≥ 4 independent replicates. (E) Alamar Blue metabolism of MDA MB 231 cells after 24 hours in the presence of DMSO vehicle, indicated concentrations of BDP5290 or 10 μM Y27632. Readings were compared to the Alamar Blue metabolism in DMSO treated cells that were used as the viability standard of 100% for each replicate experiment. All results are shown as mean ± standard error of 3 independent replicates.
Mentions: We and others used siRNA to show that MRCK activity contributes to the ability of MDA-MB-231 human breast cancer cells to invade three dimensional Matrigel [6,11] and SCC12 human squamous cell carcinoma cells to invade collagen in an organotypic skin culture model of invasion [12]. Using a 96-well based Matrigel invasion assay and measurements determined with an Incucyte live content imaging instrument at time points up to 24 h (Figure 6A), we found that BDP5290 reduced MDA-MB-231 invasion at all tested concentrations starting from 0.1 μM, with virtually complete inhibition at 10 μM (Figure 6B). In contrast, Y27632 was dramatically less effective at inhibiting invasion at all concentrations, (Figure 6C). Comparing the dose–response relationship at 24 h after the start of the experiments, the EC50 for BDP5290 was 440 nM (Figure 6D), which was similar to the EC50 for inhibiting MLC phosphorylation (Figure 5B). However, Y27632 inhibition of invasion was not greater than 50% even at 30 μM (Figure 6D). To ensure that BDP5290 did not affect MDA MB 231 cell viability, a range of BDP5290 concentrations were tested for their effects on metabolic activity relative to DMSO vehicle using Alamar Blue [23]. After 24 hours in the presence of BDP5290 cell viability as measured by Alamar Blue metabolism was slightly reduced with an EC50 > 10 μM (Figure 6E). Wound closure was inhibited by > 60% at 1 μM BDP5290 (Figure 6D), a concentration that had no effect on cell viability (Figure 6E), indicating that the MRCK inhibitor can directly block cell motility independent of effects on cell proliferation. Treatment with 10 μM Y27632 had no effect on cell viability (Figure 6E) but inhibited wound closure by ~40%, indicating that ROCK inhibition also reduced cell motility directly. Given that both inhibitors had similar effects on total MLC phosphorylation (Figure 5A and B) but their effects on pMLC in different cellular compartments varied (Figure 5C), one possibility is that the phosphorylation of cortical MLC is an important contributor to cell motility and invasion. Previous studies found that polarized cell motility was dependent on recruitment of MRCK to the leading edge [22], where it promotes actin-myosin retrograde flow to generate tractive forces for cell movement [9]. One mechanism identified for this recruitment is the translocation of MRCKα associated with the PDK1 kinase that binds membrane phosphatidylinositol (3,4,5)-trisphosphate [24]. Blocking this translocation impairs the ability of MRCKα to promote lamellipodia retraction with consequent inhibition of directional migration. In addition, MRCK was found to be required for the assembly of matrix degrading complexes containing MT1-MMP [25] and promote cathepsin B expression [26] to permit cell invasion via matrix degradation. Therefore, BDP5290 may also affect matrix degradation as well as cell motility, resulting in significant inhibition of invasion.Figure 6

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