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In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

Latham AM, Kankanala J, Fearnley GW, Gage MC, Kearney MT, Homer-Vanniasinkam S, Wheatcroft SB, Fishwick CW, Ponnambalam S - PLoS ONE (2014)

Bottom Line: However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro.In silico design is an attractive and innovative method to aid such drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Endothelial Cell Biology Unit, School of Molecular & Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT

Background: Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.

Methodology: We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.

Conclusions: We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

No MeSH data available.


Related in: MedlinePlus

JK-31 inhibits cell cycle progression in human breast cancer cells.Human breast cancer cells were treated with either (A) DMSO (ASYNCHR), (B) 200 nM nocodazole, (C) 1 µM JK-31, (D) 10 µM JK-31, (E) 50 µM JK-31, (F) 100 nM sunitinib or (G) 10 µM bohemine for 48 h, prior to staining with propidium iodide and assessment of DNA content using flow cytometry. (H) Quantification of cell cycle ratios after inhibitor treatment. Error bars represent ±SEM (n = 3).). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
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pone-0110997-g007: JK-31 inhibits cell cycle progression in human breast cancer cells.Human breast cancer cells were treated with either (A) DMSO (ASYNCHR), (B) 200 nM nocodazole, (C) 1 µM JK-31, (D) 10 µM JK-31, (E) 50 µM JK-31, (F) 100 nM sunitinib or (G) 10 µM bohemine for 48 h, prior to staining with propidium iodide and assessment of DNA content using flow cytometry. (H) Quantification of cell cycle ratios after inhibitor treatment. Error bars represent ±SEM (n = 3).). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Mentions: To investigate this further, human endothelial or breast cancer cells were treated with nocodazole, JK-31, sunitinib or bohemine for 48 h prior to fixation and analysis of cellular DNA status using flow cytometry. Cells containing n = 1 and n = 2 amount of genomic DNA were quantified (Figure 6A–7H; 7A–7H and Figure S10A–S10B), revealing that there was a significant decrease in the proportion of cells in the G2/M phase of the cell cycle in both endothelial and breast cancer cells treated with JK-31 at 10 or 50 µM, in comparison to asynchronous controls (Figure 6H and 7H). This correlated with a significant increase in the number of cells present in G1 (Figure 6H and 7H). Interestingly, there was not a significant change in the number of endothelial cells present in S phase upon JK-31 treatment (Figure 6H). Contrastingly, upon JK-31 treatment the number of breast cancer cells present in the S phase of the cell cycle was significantly decreased (Figure 7H). Treatment with nocodazole arrested both endothelial and human breast cancer cells at the G2/M phase boundary, promoting a significant shift in the cell cycle profile (Figure 6H and 7H and Figure S10A–S10B). However, sunitinib treatment had no effect on cell cycle progression vs. asynchronous controls in both cell types (Figure 6H and 7H). Upon treatment with the CDK1 inhibitor bohemine, there was a trend towards an increase in the number of endothelial and breast cancer cells present in both the S and G2/M phases of the cell cycle (Figure 6H and 7H). Interestingly, this suggested that JK-31 and bohemine inhibition of CDK1 have opposing effects on the cell cycle.


In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

Latham AM, Kankanala J, Fearnley GW, Gage MC, Kearney MT, Homer-Vanniasinkam S, Wheatcroft SB, Fishwick CW, Ponnambalam S - PLoS ONE (2014)

JK-31 inhibits cell cycle progression in human breast cancer cells.Human breast cancer cells were treated with either (A) DMSO (ASYNCHR), (B) 200 nM nocodazole, (C) 1 µM JK-31, (D) 10 µM JK-31, (E) 50 µM JK-31, (F) 100 nM sunitinib or (G) 10 µM bohemine for 48 h, prior to staining with propidium iodide and assessment of DNA content using flow cytometry. (H) Quantification of cell cycle ratios after inhibitor treatment. Error bars represent ±SEM (n = 3).). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230991&req=5

pone-0110997-g007: JK-31 inhibits cell cycle progression in human breast cancer cells.Human breast cancer cells were treated with either (A) DMSO (ASYNCHR), (B) 200 nM nocodazole, (C) 1 µM JK-31, (D) 10 µM JK-31, (E) 50 µM JK-31, (F) 100 nM sunitinib or (G) 10 µM bohemine for 48 h, prior to staining with propidium iodide and assessment of DNA content using flow cytometry. (H) Quantification of cell cycle ratios after inhibitor treatment. Error bars represent ±SEM (n = 3).). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Mentions: To investigate this further, human endothelial or breast cancer cells were treated with nocodazole, JK-31, sunitinib or bohemine for 48 h prior to fixation and analysis of cellular DNA status using flow cytometry. Cells containing n = 1 and n = 2 amount of genomic DNA were quantified (Figure 6A–7H; 7A–7H and Figure S10A–S10B), revealing that there was a significant decrease in the proportion of cells in the G2/M phase of the cell cycle in both endothelial and breast cancer cells treated with JK-31 at 10 or 50 µM, in comparison to asynchronous controls (Figure 6H and 7H). This correlated with a significant increase in the number of cells present in G1 (Figure 6H and 7H). Interestingly, there was not a significant change in the number of endothelial cells present in S phase upon JK-31 treatment (Figure 6H). Contrastingly, upon JK-31 treatment the number of breast cancer cells present in the S phase of the cell cycle was significantly decreased (Figure 7H). Treatment with nocodazole arrested both endothelial and human breast cancer cells at the G2/M phase boundary, promoting a significant shift in the cell cycle profile (Figure 6H and 7H and Figure S10A–S10B). However, sunitinib treatment had no effect on cell cycle progression vs. asynchronous controls in both cell types (Figure 6H and 7H). Upon treatment with the CDK1 inhibitor bohemine, there was a trend towards an increase in the number of endothelial and breast cancer cells present in both the S and G2/M phases of the cell cycle (Figure 6H and 7H). Interestingly, this suggested that JK-31 and bohemine inhibition of CDK1 have opposing effects on the cell cycle.

Bottom Line: However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro.In silico design is an attractive and innovative method to aid such drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Endothelial Cell Biology Unit, School of Molecular & Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT

Background: Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.

Methodology: We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.

Conclusions: We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

No MeSH data available.


Related in: MedlinePlus