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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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Selectivity profile of BDP5290. The percentage inhibition of 36 kinases by 10 μM BDP5290 was mapped onto the kinome phylogenetic tree. Kinases inhibited less than 25% have their names in green text. Kinases inhibited >25% and ≤50% have yellow circles, >50% and ≤75% have orange circles, and >75% have red circles. In each case, the size of the circle is proportional to the percentage inhibition. ROCK1 and MRCKα positions are indicated on the tree for orientation purposes. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
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Fig2: Selectivity profile of BDP5290. The percentage inhibition of 36 kinases by 10 μM BDP5290 was mapped onto the kinome phylogenetic tree. Kinases inhibited less than 25% have their names in green text. Kinases inhibited >25% and ≤50% have yellow circles, >50% and ≤75% have orange circles, and >75% have red circles. In each case, the size of the circle is proportional to the percentage inhibition. ROCK1 and MRCKα positions are indicated on the tree for orientation purposes. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Mentions: Kinase selectivity was determined by measuring the residual activity in the presence of 10 μM BDP5290 of a panel of 36 kinases. The results of this screening were mapped over the annotated human kinome phylogenetic tree [17] using Kinome Render [18] (Figure 2). Overall, the selectivity profile revealed that most kinases were unaffected by BDP5290 (green labels), with the AGC family kinases PRK2 and PKAα as well as the CAMK kinase PhKγ2 being the only ones inhibited >75% (red circles) at this concentration. These results show that BDP5290 is a potent MRCK inhibitor suitable as a chemical biology tool to characterize the contribution made by MRCK to cancer cell invasion.Figure 2


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)

Selectivity profile of BDP5290. The percentage inhibition of 36 kinases by 10 μM BDP5290 was mapped onto the kinome phylogenetic tree. Kinases inhibited less than 25% have their names in green text. Kinases inhibited >25% and ≤50% have yellow circles, >50% and ≤75% have orange circles, and >75% have red circles. In each case, the size of the circle is proportional to the percentage inhibition. ROCK1 and MRCKα positions are indicated on the tree for orientation purposes. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Selectivity profile of BDP5290. The percentage inhibition of 36 kinases by 10 μM BDP5290 was mapped onto the kinome phylogenetic tree. Kinases inhibited less than 25% have their names in green text. Kinases inhibited >25% and ≤50% have yellow circles, >50% and ≤75% have orange circles, and >75% have red circles. In each case, the size of the circle is proportional to the percentage inhibition. ROCK1 and MRCKα positions are indicated on the tree for orientation purposes. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
Mentions: Kinase selectivity was determined by measuring the residual activity in the presence of 10 μM BDP5290 of a panel of 36 kinases. The results of this screening were mapped over the annotated human kinome phylogenetic tree [17] using Kinome Render [18] (Figure 2). Overall, the selectivity profile revealed that most kinases were unaffected by BDP5290 (green labels), with the AGC family kinases PRK2 and PKAα as well as the CAMK kinase PhKγ2 being the only ones inhibited >75% (red circles) at this concentration. These results show that BDP5290 is a potent MRCK inhibitor suitable as a chemical biology tool to characterize the contribution made by MRCK to cancer cell invasion.Figure 2

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