Limits...
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 angiogenesis in an ex vivo murine model and in vitro.(A) Effects of JK-31 on ex vivo angiogenic sprouting from wild-type mouse aortic ring explants. Aortic rings were seeded onto Matrigel and treated with JK-31 (0, 1, 10 or 50 µM) for six days and imaged by phase-contrast microscopy. Freehand black lines delineate the outermost extent of sprouting. (B) Absolute quantification of angiogenic sprout length. For each aortic ring, the mean of three longest sprouts was calculated. Error bars represent ±SEM (n = 18; ***p<0.001). (C) JK-31 inhibits endothelial tube formation in vitro in response to exogenous VEGF-A. HUVECs were seeded onto a confluent layer of primary fibroblasts and treated with VEGF-A (10 ng/ml) for seven days in the presence of DMSO or JK-31 (1, 10 or 50 µM). Co-cultures were stained with CD31 antibody followed by HRP-conjugated secondary antibody and visualized by light microscopy using 1,1-diaminobenzidine (DAB)-staining (see Materials and Methods). (D) Fluorescence microscopy analysis of endothelial cell phenotypes and filopodia formation during tubulogenesis in the presence of VEGF-A and JK-31. White arrowheads indicate vestigial lamellipodia-like structures. (E) JK-31 inhibits endothelial tube formation in vitro in response to exogenous bFGF (20 ng/ml). Treatment and processing was carried out as described above. Quantification of (F) mean tubule branch length and (G) mean number of tubule branch points in response to JK-31 and exogenous growth factors. Light micrograph fields were chosen at random and quantification performed using Image J software. Error bars represent ± SEM (n = 10; *p<0.05; **p<0.01; ***p<0.001) for each respective growth factor.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230991&req=5

pone-0110997-g003: JK-31 inhibits angiogenesis in an ex vivo murine model and in vitro.(A) Effects of JK-31 on ex vivo angiogenic sprouting from wild-type mouse aortic ring explants. Aortic rings were seeded onto Matrigel and treated with JK-31 (0, 1, 10 or 50 µM) for six days and imaged by phase-contrast microscopy. Freehand black lines delineate the outermost extent of sprouting. (B) Absolute quantification of angiogenic sprout length. For each aortic ring, the mean of three longest sprouts was calculated. Error bars represent ±SEM (n = 18; ***p<0.001). (C) JK-31 inhibits endothelial tube formation in vitro in response to exogenous VEGF-A. HUVECs were seeded onto a confluent layer of primary fibroblasts and treated with VEGF-A (10 ng/ml) for seven days in the presence of DMSO or JK-31 (1, 10 or 50 µM). Co-cultures were stained with CD31 antibody followed by HRP-conjugated secondary antibody and visualized by light microscopy using 1,1-diaminobenzidine (DAB)-staining (see Materials and Methods). (D) Fluorescence microscopy analysis of endothelial cell phenotypes and filopodia formation during tubulogenesis in the presence of VEGF-A and JK-31. White arrowheads indicate vestigial lamellipodia-like structures. (E) JK-31 inhibits endothelial tube formation in vitro in response to exogenous bFGF (20 ng/ml). Treatment and processing was carried out as described above. Quantification of (F) mean tubule branch length and (G) mean number of tubule branch points in response to JK-31 and exogenous growth factors. Light micrograph fields were chosen at random and quantification performed using Image J software. Error bars represent ± SEM (n = 10; *p<0.05; **p<0.01; ***p<0.001) for each respective growth factor.

Mentions: Multiple lines of evidence thus far in this study have revealed JK-31 as an inhibitor of pro-angiogenic receptor tyrosine kinase-regulated signal transduction in endothelial cells (Table 2; Figures 1E and 1F; Figure 2). Hence we assessed whether JK-31 could indeed inhibit angiogenesis using both in vitro and ex vivo models (Figure 3). In an ex vivo assay, mouse aortic ring explants were seeded onto Matrigel in full growth medium and incubated with VEGF-A and a dose range of JK-31 for six days (Figure 3A). At concentrations of 10 µM and above, JK-31 elicited more than 40% inhibition of radial angiogenic sprout formation (Figure 3B).


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 angiogenesis in an ex vivo murine model and in vitro.(A) Effects of JK-31 on ex vivo angiogenic sprouting from wild-type mouse aortic ring explants. Aortic rings were seeded onto Matrigel and treated with JK-31 (0, 1, 10 or 50 µM) for six days and imaged by phase-contrast microscopy. Freehand black lines delineate the outermost extent of sprouting. (B) Absolute quantification of angiogenic sprout length. For each aortic ring, the mean of three longest sprouts was calculated. Error bars represent ±SEM (n = 18; ***p<0.001). (C) JK-31 inhibits endothelial tube formation in vitro in response to exogenous VEGF-A. HUVECs were seeded onto a confluent layer of primary fibroblasts and treated with VEGF-A (10 ng/ml) for seven days in the presence of DMSO or JK-31 (1, 10 or 50 µM). Co-cultures were stained with CD31 antibody followed by HRP-conjugated secondary antibody and visualized by light microscopy using 1,1-diaminobenzidine (DAB)-staining (see Materials and Methods). (D) Fluorescence microscopy analysis of endothelial cell phenotypes and filopodia formation during tubulogenesis in the presence of VEGF-A and JK-31. White arrowheads indicate vestigial lamellipodia-like structures. (E) JK-31 inhibits endothelial tube formation in vitro in response to exogenous bFGF (20 ng/ml). Treatment and processing was carried out as described above. Quantification of (F) mean tubule branch length and (G) mean number of tubule branch points in response to JK-31 and exogenous growth factors. Light micrograph fields were chosen at random and quantification performed using Image J software. Error bars represent ± SEM (n = 10; *p<0.05; **p<0.01; ***p<0.001) for each respective growth factor.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110997-g003: JK-31 inhibits angiogenesis in an ex vivo murine model and in vitro.(A) Effects of JK-31 on ex vivo angiogenic sprouting from wild-type mouse aortic ring explants. Aortic rings were seeded onto Matrigel and treated with JK-31 (0, 1, 10 or 50 µM) for six days and imaged by phase-contrast microscopy. Freehand black lines delineate the outermost extent of sprouting. (B) Absolute quantification of angiogenic sprout length. For each aortic ring, the mean of three longest sprouts was calculated. Error bars represent ±SEM (n = 18; ***p<0.001). (C) JK-31 inhibits endothelial tube formation in vitro in response to exogenous VEGF-A. HUVECs were seeded onto a confluent layer of primary fibroblasts and treated with VEGF-A (10 ng/ml) for seven days in the presence of DMSO or JK-31 (1, 10 or 50 µM). Co-cultures were stained with CD31 antibody followed by HRP-conjugated secondary antibody and visualized by light microscopy using 1,1-diaminobenzidine (DAB)-staining (see Materials and Methods). (D) Fluorescence microscopy analysis of endothelial cell phenotypes and filopodia formation during tubulogenesis in the presence of VEGF-A and JK-31. White arrowheads indicate vestigial lamellipodia-like structures. (E) JK-31 inhibits endothelial tube formation in vitro in response to exogenous bFGF (20 ng/ml). Treatment and processing was carried out as described above. Quantification of (F) mean tubule branch length and (G) mean number of tubule branch points in response to JK-31 and exogenous growth factors. Light micrograph fields were chosen at random and quantification performed using Image J software. Error bars represent ± SEM (n = 10; *p<0.05; **p<0.01; ***p<0.001) for each respective growth factor.
Mentions: Multiple lines of evidence thus far in this study have revealed JK-31 as an inhibitor of pro-angiogenic receptor tyrosine kinase-regulated signal transduction in endothelial cells (Table 2; Figures 1E and 1F; Figure 2). Hence we assessed whether JK-31 could indeed inhibit angiogenesis using both in vitro and ex vivo models (Figure 3). In an ex vivo assay, mouse aortic ring explants were seeded onto Matrigel in full growth medium and incubated with VEGF-A and a dose range of JK-31 for six days (Figure 3A). At concentrations of 10 µM and above, JK-31 elicited more than 40% inhibition of radial angiogenic sprout formation (Figure 3B).

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