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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

Dual targeting of VEGFR2 and CDK1.(A) Chemical structure of the aminotriazole-based compound JK-31. A full description of the chemical synthesis can be found in the Materials and Methods section. (B) In silico molecular modeling of JK-31 (magenta structure) in the VEGFR2 and CDK1 kinase domains. A homology model of CDK1 was created using a structurally-related family member (CDK2 PDB code: 3s2p). The CDK1 and VEGFR2 kinase domain sequences were aligned and overlapped. JK-31 was docked into the overlapped crystal structure using Glide program and important amino acid residues identified. VEGFR2 crystal structure is shown in yellow and residues annotated in bold; CDK1 crystal structure is shown in blue/grey and residues annotated in italics. (C) JK-31 was docked into a VEGFR2 crystal structure alone (PDB code: 3cjg) and predicted hydrogen bond contacts identified. (D) JK-31 was docked into the CDK1 crystal structure homolog alone and predicted hydrogen bond contacts identified. Dotted lines denote predicted hydrogen bond contacts. Green structures denote important kinase domain residues. Further details of these models can be found in Figure S2. (E–F) JK-31 inhibits the intrinsic kinase activity of both CDK1 and VEGFR2. IC50 curves were generated by incubating recombinant protein kinases with a peptide substrate, radiolabeled [γ-33P]-ATP and either JK-31 or bohemine (5 nM to 100 µM). Effects of JK-31 and bohemine on phosphate transfer from a (E) recombinant human VEGFR2 to a peptide substrate, poly[Glu∶Tyr] (4∶1) and (F) recombinant human CDK1/cyclin B complex to histone H1 substrate in vitro. Line graphs of three independent replicate experiments are shown.
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pone-0110997-g001: Dual targeting of VEGFR2 and CDK1.(A) Chemical structure of the aminotriazole-based compound JK-31. A full description of the chemical synthesis can be found in the Materials and Methods section. (B) In silico molecular modeling of JK-31 (magenta structure) in the VEGFR2 and CDK1 kinase domains. A homology model of CDK1 was created using a structurally-related family member (CDK2 PDB code: 3s2p). The CDK1 and VEGFR2 kinase domain sequences were aligned and overlapped. JK-31 was docked into the overlapped crystal structure using Glide program and important amino acid residues identified. VEGFR2 crystal structure is shown in yellow and residues annotated in bold; CDK1 crystal structure is shown in blue/grey and residues annotated in italics. (C) JK-31 was docked into a VEGFR2 crystal structure alone (PDB code: 3cjg) and predicted hydrogen bond contacts identified. (D) JK-31 was docked into the CDK1 crystal structure homolog alone and predicted hydrogen bond contacts identified. Dotted lines denote predicted hydrogen bond contacts. Green structures denote important kinase domain residues. Further details of these models can be found in Figure S2. (E–F) JK-31 inhibits the intrinsic kinase activity of both CDK1 and VEGFR2. IC50 curves were generated by incubating recombinant protein kinases with a peptide substrate, radiolabeled [γ-33P]-ATP and either JK-31 or bohemine (5 nM to 100 µM). Effects of JK-31 and bohemine on phosphate transfer from a (E) recombinant human VEGFR2 to a peptide substrate, poly[Glu∶Tyr] (4∶1) and (F) recombinant human CDK1/cyclin B complex to histone H1 substrate in vitro. Line graphs of three independent replicate experiments are shown.

Mentions: Due to the high sequence homology between CDK1 and CDK2 (66% homology in kinase domain), an available crystal structure of CDK2 (PDB code: 3s2p) [19] was used to build a homology model of CDK1. The sequence of CDK1 was imported into the program Prime (Schrödinger Inc.) [20] and the model of CDK1 was built using 3s2p as a template, which was further subjected to protein preparation using Maestro and PrimeX (Schrödinger Inc.) (Figure 1B, Figure S2) [21]. Use of the programs SPROUT (SimBioSys Inc.) [22], [23] and Glide (Schrödinger Inc.) [24]–[26] for molecular modelling have been described previously [27]. Briefly, JK-31 was docked into both the protein kinase domains of either CDK1 and VEGFR2 (PDB code: 3cjg) [28] using both programs. SPROUT identified a target region where JK-31 would interact most strongly and was scored to give an estimated pKi (Figure 1B, Table 1). Glide was also used to predict the binding affinity of JK-31 (presented as a Glide score where a lower score represents lower energy and thus greater affinity; Table 1). Images from Glide software are used in this publication (Figures 1B-1D, S2A and S2B). The binding mode of JK-31 within the VEGFR2 and CDK1 kinase domains (with respect to hydrogen bonding) were confirmed to be similar to oneanother (Figures 1B–1D). A full description of the structure-activity relationship of JK-31 and other compounds of the same class is currently ongoing.


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)

Dual targeting of VEGFR2 and CDK1.(A) Chemical structure of the aminotriazole-based compound JK-31. A full description of the chemical synthesis can be found in the Materials and Methods section. (B) In silico molecular modeling of JK-31 (magenta structure) in the VEGFR2 and CDK1 kinase domains. A homology model of CDK1 was created using a structurally-related family member (CDK2 PDB code: 3s2p). The CDK1 and VEGFR2 kinase domain sequences were aligned and overlapped. JK-31 was docked into the overlapped crystal structure using Glide program and important amino acid residues identified. VEGFR2 crystal structure is shown in yellow and residues annotated in bold; CDK1 crystal structure is shown in blue/grey and residues annotated in italics. (C) JK-31 was docked into a VEGFR2 crystal structure alone (PDB code: 3cjg) and predicted hydrogen bond contacts identified. (D) JK-31 was docked into the CDK1 crystal structure homolog alone and predicted hydrogen bond contacts identified. Dotted lines denote predicted hydrogen bond contacts. Green structures denote important kinase domain residues. Further details of these models can be found in Figure S2. (E–F) JK-31 inhibits the intrinsic kinase activity of both CDK1 and VEGFR2. IC50 curves were generated by incubating recombinant protein kinases with a peptide substrate, radiolabeled [γ-33P]-ATP and either JK-31 or bohemine (5 nM to 100 µM). Effects of JK-31 and bohemine on phosphate transfer from a (E) recombinant human VEGFR2 to a peptide substrate, poly[Glu∶Tyr] (4∶1) and (F) recombinant human CDK1/cyclin B complex to histone H1 substrate in vitro. Line graphs of three independent replicate experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110997-g001: Dual targeting of VEGFR2 and CDK1.(A) Chemical structure of the aminotriazole-based compound JK-31. A full description of the chemical synthesis can be found in the Materials and Methods section. (B) In silico molecular modeling of JK-31 (magenta structure) in the VEGFR2 and CDK1 kinase domains. A homology model of CDK1 was created using a structurally-related family member (CDK2 PDB code: 3s2p). The CDK1 and VEGFR2 kinase domain sequences were aligned and overlapped. JK-31 was docked into the overlapped crystal structure using Glide program and important amino acid residues identified. VEGFR2 crystal structure is shown in yellow and residues annotated in bold; CDK1 crystal structure is shown in blue/grey and residues annotated in italics. (C) JK-31 was docked into a VEGFR2 crystal structure alone (PDB code: 3cjg) and predicted hydrogen bond contacts identified. (D) JK-31 was docked into the CDK1 crystal structure homolog alone and predicted hydrogen bond contacts identified. Dotted lines denote predicted hydrogen bond contacts. Green structures denote important kinase domain residues. Further details of these models can be found in Figure S2. (E–F) JK-31 inhibits the intrinsic kinase activity of both CDK1 and VEGFR2. IC50 curves were generated by incubating recombinant protein kinases with a peptide substrate, radiolabeled [γ-33P]-ATP and either JK-31 or bohemine (5 nM to 100 µM). Effects of JK-31 and bohemine on phosphate transfer from a (E) recombinant human VEGFR2 to a peptide substrate, poly[Glu∶Tyr] (4∶1) and (F) recombinant human CDK1/cyclin B complex to histone H1 substrate in vitro. Line graphs of three independent replicate experiments are shown.
Mentions: Due to the high sequence homology between CDK1 and CDK2 (66% homology in kinase domain), an available crystal structure of CDK2 (PDB code: 3s2p) [19] was used to build a homology model of CDK1. The sequence of CDK1 was imported into the program Prime (Schrödinger Inc.) [20] and the model of CDK1 was built using 3s2p as a template, which was further subjected to protein preparation using Maestro and PrimeX (Schrödinger Inc.) (Figure 1B, Figure S2) [21]. Use of the programs SPROUT (SimBioSys Inc.) [22], [23] and Glide (Schrödinger Inc.) [24]–[26] for molecular modelling have been described previously [27]. Briefly, JK-31 was docked into both the protein kinase domains of either CDK1 and VEGFR2 (PDB code: 3cjg) [28] using both programs. SPROUT identified a target region where JK-31 would interact most strongly and was scored to give an estimated pKi (Figure 1B, Table 1). Glide was also used to predict the binding affinity of JK-31 (presented as a Glide score where a lower score represents lower energy and thus greater affinity; Table 1). Images from Glide software are used in this publication (Figures 1B-1D, S2A and S2B). The binding mode of JK-31 within the VEGFR2 and CDK1 kinase domains (with respect to hydrogen bonding) were confirmed to be similar to oneanother (Figures 1B–1D). A full description of the structure-activity relationship of JK-31 and other compounds of the same class is currently ongoing.

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