Roscovitine(CYC202)

• Catalog No.: S1153
• CAS Number: 186692-46-6
• M. F.: C19H26N6O
• M. W.: 354.44934
• Storage: -20°C

SYNONYMS

• IUPAC name: (2R)-2-{[6-(benzylamino)-9-(propan-2-yl)-9H-purin-2-yl]amino}butan-1-ol
• IUPAC Traditional name: seliciclib
• Synonyms: Seliciclib; R-roscovitine; CYC202

DATABASE IDS

• CAS Number: 186692-46-6

PROPERTIES

• Target: CDK
• Salt Data: Free Base
• Solubility: DMSO
• Storage Condition: -20°C

DETAILS

Description (English)

Research Area

• Description: Cancer

Biological Activity

• Description: Roscovitine (Seliciclib, CYC202, R-roscovitine) is a potent and selective CDK inhibitor for Cdc2/cyclin B, CDK2/cyclin A, CDK2/cyclin E and CDK5/p53 with IC50 of 0.65 μM, 0.7 μM, 0.7 μM and 0.16 μM, respectively.
• Targets: Cdc2/cyclin B; CDK2/cyclin A; CDK2/cyclin E; CDK5/p35
• IC50: 0.65 μM; 0.7 μM; 0.7 μM; 0.16 μM ^[1]
• In Vitro: Roscovitine displays high efficiency and high selectivity towards some cyclin-dependent kinases with IC50 of 0.65, 0.7, 0.7 and 0.16 μM for cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p53, respectively. ^[1] Roscovitine reversibly inhibits the prophaselmetaphase transition in the micromolar range of starfish oocytes and sea urchin embryos, inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts, and suppresses the proliferation of mammalian cell lines with an average IC50 of 16 μM. ^[1] In mesangial cells, Roscovitine results in a dose-dependent reduction of CDK2 activity that at concentrations of 7.5, 12.5 and 25 mM, Roscovitine causes a 25, 50% and 100% decrease in CDK2 activity, respectively. ^[2] A recent study shows that Roscovitine inhibits cdk5 kinase activity, cell proliferation, multicellular development, and cdk5 nuclear translocation in Dictyostelium discoideum, without affecting the expression of cdk5 protein during axenic growth. ^[3]
• In Vivo: Roscovitine, at a dose of 50 mg/kg, significantly inhibits growth of The Ewing's sarcoma family of tumors (ESFT) xenografts. ^[4] Roscovitine enhances the antitumor effect of doxorubicin without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis in nude mice bearing established MCF7 xenografts. ^[5]
• Clinical Trials: Roscovitine is currently in Phase I clinical trial in patients with advanced solid tumors.

Protocol

• Protocol: Kinase Assay ^[1]
• Enzymes: Kinases activities are assayed at 30 °C in buffer C. Blank values are subtracted from the data and activities calculated as molar amount of phosphate incorporated in protein acceptor during a 10-minute incubation. Controls are performed with appropriate dilutions of DMSO. In a few cases, phosphorylation of the substrate is assessed by autoradiography after SDS/PAGE. p34^cdc2/cyclin B is purified from M-phase starfish (M. glacialis) oocytes by affinity chromatography. It is assayed with 1 mg histone Hl/mL, in the presence of 15 μM [γ-^32P]ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL. After a 10-minute incubation at 30 °C, 25-μL aliquots of supernatant are spotted onto pieces of Whatman P81 phosphocellulose paper, and, after 20 seconds, the filters are washed five times (for at least 5 minutes each time) in a solution of 10mL phosphoric acid/L water. The wet filters are transferred into 6-mL plastic scintillation vials, 5 mL ACS scintillation fluid is added and the radioactivity measured in a Packard counter. The kinase activity is expressed as molar amount of phosphate incorporated in histone H1 during a 10-minutes incubation or as a percentage of maximal activity. p33^cdk2/cyclin A and p33^cdk2/cyclinE are reconstituted from extracts of sf9 insect cells infected with various baculoviruses. Cyclins A and E are fusion proteins with glutathione S-transferase and the complexes are purified on glutathione-agarose beads. Kinase activities are assayed with 1 mg/mL histone H1, in the presence of 15 μM [γ-^32P]ATP, during 10 minutes, in a final volume of 30 μL, as described for the p34^cdc2/cyclin B kinase. p33^cdk5/p35 is purified from bovine brain, excluding the Mono S-chromatographic step. The active fractions from the Superose 12 column are pooled and concentrated to a final concentration of approximately 25 μg enzyme/mL. The kinase is assayed with 1 mg/mL histone HI in the presence of 15 μM [γ-^32P]ATP, during 10 minutes in a final volume of 30 μL, as described for the p34^cdc2/cyclin B kinase. p33^cdk5/cyclin D1 is obtained from insect cell lysates. Cdk4 is a fusion protein with glutathione-S-transferase and the active complex is purified on glutathione-agarose beads. Its kinase activity is assayed with purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 μM [γ-^32P]ATP, in a final volume of 30 μL. After a 15-minute incubation, 30 μL Laemmli sample buffer is added. The phosphorylated substrate is resolved by 10 % SDS/PAGE and analysed by autoradiography by overnight exposure to Hyperfilm MP and densitometry. p33^cdk4/cyclinD 2 is obtained from insect cell lysates. It is assayed with purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 μM [γ-^32P]ATP in a final volume of 30 μL. After a 30-minute incubation, 30 μL Laemmli sample buffer is added. The phosphorylated substrate is resolved by 10% SDS/PAGE and analysed by autoradiography by overnight exposure to Hyperfilm MP and densitometry. MAP kinase erkl (tagged with glutathione-S-transferase), is expressed in bacteria, purified on glutathione-agarose beads and assayed with 1 mg myelin basic protein/ml in the presence of 15 μM [γ-^32P]ATP as described above for the p34^cdc2cyclin B kinase. His-tagged erkl and erk2 are activated in vitro by mitogen-activated protein kinase kinase, purified (Ni-affinity and Mono Q) and assayed as described above during 10 minutes in a final volume of 30 μL. Protein kinase C isoforms are purified from baculovirus infected sf9 insect cells and assayed with 1 mg/mL protamine sulfate in the presence of 15 μM [γ-^32P]ATP, during 10 minutes at 30 °C, in a final volume of 30 μL. Phosphorylated protamine sulfate is recovered on Whatman P81 phosphocellulose paper as described for the cdc2 kinase. The catalytic subunit of cAMP-dependent protein kinase, purified from bovine heart, is assayed with 1 mg histone Hl/ml, in the presence of 15 μM [γ-^32P]ATP as described for the p34^cdc2/cyclin B kinase. cGMP-dependent protein kinase, purified to homogeneity from bovine tracheal smooth muscle, is assayed with 1 mg histone Hl/mL, in the presence of 15 μM [γ-^32P]ATP as described for the p34^cdc2/cyclin B kinase. Casein kinase 2 is isolated from rat liver cytosol and assayed with 1 mg casein/mL and 15 μM [γ-^32P]ATP. The substrate is spotted on Whatmann 3MM filters and washed with 10% (mass/vol.) trichloroacetic acid. Myosin light chain kinase, purified from chicken gizzard is assayed in the presence of 100 nM calmodulin, 100 μM CaCl₂, 50 mM Hepes, 5 mM MgCI,, 1 mM dithiothreitol and 0.1 mg BSA/ml at pH 7.5 using a synthetic peptide based on the smooth-muscle myosin light-chain phosphorylation site and in the presence of 15 μM [γ-^32P]ATP, in a final volume of 50 μL. Incorporation of radioactive phosphate is monitored on phosphocellulose filters as described above. ASK-γ, a plant homologue of GSK-3, is expressed as a glutathione-S-transferase fusion protein in Escherichia coli and purified on glutathione-agarose. ASK-γ kinase is assayed, for 10 minutes at 30 °C, with 5 μg myelin basic protein, in the presence of 15 μM [γ-^32P]ATP in a final volume of 30 μL. The phosphorylated myelin basic protein is recovered on Whatman P81 phosphocellulose paper as described for the p34^cdc2/cyclin B kinase. Insulin receptor tyrosine kinase domain (CIRK-41) is overexpressed in a baculovirus system and purified to homogeneity. Its kinase activity is assayed, for 10 minutes at 30 °C, with 5 μg Raytide, in the presence of 15 μM [γ-^32P]ATP, in a final volume of 30 μL. The phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper as described for the p34^cdc2/cyclin B kinase. c-src kinase is purified from infected Sf9 cells. The v-abl kinase is expressed in E. coli and affinity purified on IgG Affigel 10. Both kinases are assayed for 10 minutes at 30 °C, with 5 μg Raytide, in the presence of 15 μM [γ-^32P]ATP, in a final volume of 30 μL. The phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper as described for the p34^cdc2/cyclin B kinase.
• Protocol: Cell Assay ^[1]
• Cell Lines: Leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer
• Concentrations: 0.01 - 100 μM
• Incubation Time: 48 hours
• Methods: 60 human tumour cell lines comprising nine tumor types are cultured for 24 hours prior to a 48-hour continuous exposure to 0.01-100 μM roscovitine. A sulforhodaminine B protein assay is used to estimate the cytotoxicity.
• Protocol: Animal Study ^[4]
• Animal Models: A4573 cells are injected s.c. into the right posterior flank of CD1 nu/nu mice.
• Formulation: Roscovitine is dissolved in either absolute methanol or DMSO and then diluted in 10% Tween 80, 20% N-N-dimethylacetamide, and 70% polyethylene glycol 400.
• Doses: ≤50 mg/kg
• Administration: Administered via i.p.

References

• References: [1] Meijer L, et al. Eur J Biochem, 1997, 243(1-2), 527-536.
• References: [2] Pippin JW, et al. J Clin Invest, 1997, 100(10), 2512-2520.
• References: [3] Huber RJ, et al. J Cell Biochem, 2012, 113(3), 868-876.
• References: [4] Tirado OM, et al. Cancer Res, 2005, 65(20), 9320-9327.
• References: [5] Appleyard MV, et al. Int J Cancer, 2009, 124(2), 465-472.

REFERENCES

• Meijer L, et al. Eur J Biochem, 1997, 243(1-2), 527-536.
• Pippin JW, et al. J Clin Invest, 1997, 100(10), 2512-2520.
• Huber RJ, et al. J Cell Biochem, 2012, 113(3), 868-876.
• Tirado OM, et al. Cancer Res, 2005, 65(20), 9320-9327.
• Appleyard MV, et al. Int J Cancer, 2009, 124(2), 465-472.