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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 growth factor-stimulated receptor activation and downstream signal transduction.(A) HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 7.5 min stimulation with VEGF-A (25 ng/ml) in the presence of JK-31. Total cell lysates were prepared and processed for immunoblotting. Levels of phosphorylated VEGFR2, PLCγ1, Akt and ERK were analysed using phospho-specific antibodies. Membranes were stripped and re-probed for total protein levels and a loading control (α-tubulin). (B) Densitometric quantification of phosphorylated VEGFR2-pY1175 levels in response to VEGF-A and JK-31 treatment. Error bars represent ± SEM (n = 4; ***p<0.001). (C-D) JK-31 inhibits FGF-stimulated intracellular signaling in endothelial cells. HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 10 min stimulation with either (C) bFGF (50 ng/ml) or (D) aFGF (50 ng/ml) in the presence of JK-31. Cells were lysed and processed for immunoblotting. Representative immunoblots of three independent experiments are shown.
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pone-0110997-g002: JK-31 inhibits growth factor-stimulated receptor activation and downstream signal transduction.(A) HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 7.5 min stimulation with VEGF-A (25 ng/ml) in the presence of JK-31. Total cell lysates were prepared and processed for immunoblotting. Levels of phosphorylated VEGFR2, PLCγ1, Akt and ERK were analysed using phospho-specific antibodies. Membranes were stripped and re-probed for total protein levels and a loading control (α-tubulin). (B) Densitometric quantification of phosphorylated VEGFR2-pY1175 levels in response to VEGF-A and JK-31 treatment. Error bars represent ± SEM (n = 4; ***p<0.001). (C-D) JK-31 inhibits FGF-stimulated intracellular signaling in endothelial cells. HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 10 min stimulation with either (C) bFGF (50 ng/ml) or (D) aFGF (50 ng/ml) in the presence of JK-31. Cells were lysed and processed for immunoblotting. Representative immunoblots of three independent experiments are shown.

Mentions: A key feature of VEGFR kinase inhibitors is their ability to block VEGF-A-stimulated downstream signaling in endothelial cells [3], [6], [18], [27], [34]. Primary human umbilical vein endothelial cells (HUVECs) recapitulate many features of in vivo vascular function, including the expression of VEGFR2, Von Willebrand Factor (VWF) and PECAM-1 (Figure S1). We used immunoblot analysis of HUVECs treated with VEGF-A and JK-31 to examine the effects on VEGFR2 autophosphorylation and downstream signaling to PLCγ1, Akt and ERK1/2 (Figures 2A and B). At concentrations of 10 µM and above, JK-31 caused>60% inhibition of phosphorylation of residue Y1175 within the VEGFR2 cytoplasmic domain (Figure 2B). JK-31 also inhibited VEGF-A-stimulated signal transduction in a dose-dependent manner affecting multiple phosphorylation events such as production of PLCγ1-pY783, Akt-pS473 and ERK1/2-pT202/pY204 epitopes (Figure 2A). Quantification and statistical analysis clearly showed a dose-dependent inhibition of VEGF-A-stimulated signal transduction leading to altered phosphorylation status of key enzymes (Figure S5).


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 growth factor-stimulated receptor activation and downstream signal transduction.(A) HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 7.5 min stimulation with VEGF-A (25 ng/ml) in the presence of JK-31. Total cell lysates were prepared and processed for immunoblotting. Levels of phosphorylated VEGFR2, PLCγ1, Akt and ERK were analysed using phospho-specific antibodies. Membranes were stripped and re-probed for total protein levels and a loading control (α-tubulin). (B) Densitometric quantification of phosphorylated VEGFR2-pY1175 levels in response to VEGF-A and JK-31 treatment. Error bars represent ± SEM (n = 4; ***p<0.001). (C-D) JK-31 inhibits FGF-stimulated intracellular signaling in endothelial cells. HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 10 min stimulation with either (C) bFGF (50 ng/ml) or (D) aFGF (50 ng/ml) in the presence of JK-31. Cells were lysed and processed for immunoblotting. Representative immunoblots of three independent experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230991&req=5

pone-0110997-g002: JK-31 inhibits growth factor-stimulated receptor activation and downstream signal transduction.(A) HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 7.5 min stimulation with VEGF-A (25 ng/ml) in the presence of JK-31. Total cell lysates were prepared and processed for immunoblotting. Levels of phosphorylated VEGFR2, PLCγ1, Akt and ERK were analysed using phospho-specific antibodies. Membranes were stripped and re-probed for total protein levels and a loading control (α-tubulin). (B) Densitometric quantification of phosphorylated VEGFR2-pY1175 levels in response to VEGF-A and JK-31 treatment. Error bars represent ± SEM (n = 4; ***p<0.001). (C-D) JK-31 inhibits FGF-stimulated intracellular signaling in endothelial cells. HUVECs were pre-treated with JK-31 (0, 0.1, 1, 10 or 50 µM) for 30 min followed by 10 min stimulation with either (C) bFGF (50 ng/ml) or (D) aFGF (50 ng/ml) in the presence of JK-31. Cells were lysed and processed for immunoblotting. Representative immunoblots of three independent experiments are shown.
Mentions: A key feature of VEGFR kinase inhibitors is their ability to block VEGF-A-stimulated downstream signaling in endothelial cells [3], [6], [18], [27], [34]. Primary human umbilical vein endothelial cells (HUVECs) recapitulate many features of in vivo vascular function, including the expression of VEGFR2, Von Willebrand Factor (VWF) and PECAM-1 (Figure S1). We used immunoblot analysis of HUVECs treated with VEGF-A and JK-31 to examine the effects on VEGFR2 autophosphorylation and downstream signaling to PLCγ1, Akt and ERK1/2 (Figures 2A and B). At concentrations of 10 µM and above, JK-31 caused>60% inhibition of phosphorylation of residue Y1175 within the VEGFR2 cytoplasmic domain (Figure 2B). JK-31 also inhibited VEGF-A-stimulated signal transduction in a dose-dependent manner affecting multiple phosphorylation events such as production of PLCγ1-pY783, Akt-pS473 and ERK1/2-pT202/pY204 epitopes (Figure 2A). Quantification and statistical analysis clearly showed a dose-dependent inhibition of VEGF-A-stimulated signal transduction leading to altered phosphorylation status of key enzymes (Figure S5).

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