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

Effects of JK-31 on proliferation and cell cycle status of primary human endothelial cells and human breast cancer cells.(A) HUVECs and (B) MCF-7 cells were treated with DMSO, JK-31 (0.1, 1, 10, 50 µM), vatalanib (10 µM) or bohemine (10 µM) in full growth medium for 16 h followed by labeling with bromodeoxyuridine (BrdU; 10 µM) for 2 h and subsequent ELISA. Absorbance readings at OD450 were normalized and expressed relative to DMSO control. Error bars represent ± SEM (n = 4; ***p<0.001). (C) HUVECs and (D) MCF-7 cells were treated with nocodazole (200 nM), bohemine (10 µM), sunitinib (100 nM) or JK-31 (0, 0.1, 1, 10, 50 µM) for 48 h. Total cell lysates were prepared and immunoblotting carried out as described in Experimental Section. Membranes were probed for phosphorylated and total CDK1 in addition to cyclin A, B and D1 and a loading control (α-tubulin). Representative immunoblots of three independent experiments shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110997-g005: Effects of JK-31 on proliferation and cell cycle status of primary human endothelial cells and human breast cancer cells.(A) HUVECs and (B) MCF-7 cells were treated with DMSO, JK-31 (0.1, 1, 10, 50 µM), vatalanib (10 µM) or bohemine (10 µM) in full growth medium for 16 h followed by labeling with bromodeoxyuridine (BrdU; 10 µM) for 2 h and subsequent ELISA. Absorbance readings at OD450 were normalized and expressed relative to DMSO control. Error bars represent ± SEM (n = 4; ***p<0.001). (C) HUVECs and (D) MCF-7 cells were treated with nocodazole (200 nM), bohemine (10 µM), sunitinib (100 nM) or JK-31 (0, 0.1, 1, 10, 50 µM) for 48 h. Total cell lysates were prepared and immunoblotting carried out as described in Experimental Section. Membranes were probed for phosphorylated and total CDK1 in addition to cyclin A, B and D1 and a loading control (α-tubulin). Representative immunoblots of three independent experiments shown.

Mentions: JK-31 inhibits both a key pro-angiogenic receptor tyrosine kinase, VEGFR2, and a well-described regulator of the cell cycle, CDK1. Are such effects evident in different cellular responses involving VEGFR2 and/or CDK1 that could contribute to growth of a neovascularized tumor? To address such a scenario, we first examined the effects of JK-31 on the proliferation of primary endothelial cells (Figure 5A) and the human breast cancer cell line MCF-7 (Figure 5B). Immunoblot analysis showed that both cell types express CDK1 but only endothelial cells express VEGFR2 (Figure S7A). Using a bromodeoxyuridine (BrdU) incorporation assay to monitor new DNA synthesis, we found that JK-31 inhibited proliferation of both human cell types, albeit at a higher dose range in MCF-7 breast epithelial cells (Figure 5B). A control CDK1-selective inhibitor, bohemine, showed pronounced inhibition of proliferation of both cell types; however, the VEGFR-specific inhibitor vatalanib did not significantly inhibit cell proliferation in endothelial cells (Figure 5A) or breast cancer cells (Figure 5B). A cell viability assay showed that JK-31 had no significant cytotoxic effects on endothelial cells when treated for two days at concentrations of up to 50 µM (Figure S7B).


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)

Effects of JK-31 on proliferation and cell cycle status of primary human endothelial cells and human breast cancer cells.(A) HUVECs and (B) MCF-7 cells were treated with DMSO, JK-31 (0.1, 1, 10, 50 µM), vatalanib (10 µM) or bohemine (10 µM) in full growth medium for 16 h followed by labeling with bromodeoxyuridine (BrdU; 10 µM) for 2 h and subsequent ELISA. Absorbance readings at OD450 were normalized and expressed relative to DMSO control. Error bars represent ± SEM (n = 4; ***p<0.001). (C) HUVECs and (D) MCF-7 cells were treated with nocodazole (200 nM), bohemine (10 µM), sunitinib (100 nM) or JK-31 (0, 0.1, 1, 10, 50 µM) for 48 h. Total cell lysates were prepared and immunoblotting carried out as described in Experimental Section. Membranes were probed for phosphorylated and total CDK1 in addition to cyclin A, B and D1 and a loading control (α-tubulin). Representative immunoblots of three independent experiments shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110997-g005: Effects of JK-31 on proliferation and cell cycle status of primary human endothelial cells and human breast cancer cells.(A) HUVECs and (B) MCF-7 cells were treated with DMSO, JK-31 (0.1, 1, 10, 50 µM), vatalanib (10 µM) or bohemine (10 µM) in full growth medium for 16 h followed by labeling with bromodeoxyuridine (BrdU; 10 µM) for 2 h and subsequent ELISA. Absorbance readings at OD450 were normalized and expressed relative to DMSO control. Error bars represent ± SEM (n = 4; ***p<0.001). (C) HUVECs and (D) MCF-7 cells were treated with nocodazole (200 nM), bohemine (10 µM), sunitinib (100 nM) or JK-31 (0, 0.1, 1, 10, 50 µM) for 48 h. Total cell lysates were prepared and immunoblotting carried out as described in Experimental Section. Membranes were probed for phosphorylated and total CDK1 in addition to cyclin A, B and D1 and a loading control (α-tubulin). Representative immunoblots of three independent experiments shown.
Mentions: JK-31 inhibits both a key pro-angiogenic receptor tyrosine kinase, VEGFR2, and a well-described regulator of the cell cycle, CDK1. Are such effects evident in different cellular responses involving VEGFR2 and/or CDK1 that could contribute to growth of a neovascularized tumor? To address such a scenario, we first examined the effects of JK-31 on the proliferation of primary endothelial cells (Figure 5A) and the human breast cancer cell line MCF-7 (Figure 5B). Immunoblot analysis showed that both cell types express CDK1 but only endothelial cells express VEGFR2 (Figure S7A). Using a bromodeoxyuridine (BrdU) incorporation assay to monitor new DNA synthesis, we found that JK-31 inhibited proliferation of both human cell types, albeit at a higher dose range in MCF-7 breast epithelial cells (Figure 5B). A control CDK1-selective inhibitor, bohemine, showed pronounced inhibition of proliferation of both cell types; however, the VEGFR-specific inhibitor vatalanib did not significantly inhibit cell proliferation in endothelial cells (Figure 5A) or breast cancer cells (Figure 5B). A cell viability assay showed that JK-31 had no significant cytotoxic effects on endothelial cells when treated for two days at concentrations of up to 50 µM (Figure S7B).

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