Seliciclib (Roscovitine)

cdc2/cyclin B (IC50 = 0.65 μM); cdk2/cyclin A (IC50 = 0.7 μM); Cdk2/cyclin E2 (IC50 = 0.7 μM); CDK5/p35 (IC50 = 0.16 μM); GST-erk1 (IC50 = 30 μM); erk1 (IC50 = 34 μM); erk2 (IC50 = 14 μM); IR tyrosine kinase (IC50 = 70 μM)

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] In vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts are inhibited by Roscovitine, which also reversibly inhibits the prophaselmetaphase transition in the micromolar range of starfish oocytes and sea urchin embryos. As an average IC50 of 16 μM, Roscovitine also suppresses molecular line proliferation. (Source: ) At doses of 7.5, 12.5, and 25 mM, roscovitine causes a 25, 50%, and 100% decrease in CDK2 activity in mesangial cells, respectively. This reduction in CDK2 activity is dose-dependent.[2] In Dictyostelium discoideum, a recent study demonstrates that roscovitine inhibits cdk5 kinase activity, cell proliferation, multicellular development, and cdk5 nuclear translocation without influencing the expression of cdk5 protein during axenic growth.[3]

Roscovitine significantly inhibits the growth of xenografts of The Ewing's Sarcoma Family of Tumors (ESFT) at a dose of 50 mg/kg.[4] In nude mice with established MCF7 xenografts, roscovitine increases the antitumor effect of doxorubicin without increasing toxicity through a mechanism involving cell cycle arrest rather than apoptosis.[5]

The kinase activities in buffer C are measured at 30 °C. The data are stripped of blank values, and activities are computed as the molar amount of phosphate incorporated in the protein acceptor over the course of a 10-minute incubation. The proper DMSO dilutions are used for the controls. After SDS/PAGE, autoradiography is sometimes used to evaluate the substrate's phosphorylation. By using affinity chromatography, p34^cdc2/cyclin B is isolated from M-phase starfish (M. glacialis) oocytes. In a final volume of 30 μL, 1 mg histone Hl/mL is used in the assay along with 15 μM [γ-³²P]ATP (3000 Ci/mmol; 1 mCi/mL). 25-μL aliquots of supernatant are spotted onto Whatman P81 phosphocellulose paper after a 10-minute incubation period at 30 °C. The filters are then washed five times (for a minimum of five minutes each time) in a solution of 10mL phosphoric acid/L water after 20 seconds. After transferring the wet filters into 6-mL plastic scintillation vials, 5 mL of ACS scintillation fluid is added, and a Packard counter is used to measure the radioactivity. The kinase activity is reported as a percentage of maximal activity or as the molar amount of phosphate incorporated in histone H1 after 10 minutes of incubation. Reconstituted p33^cdk2/cyclin A and p33^cdk2/cyclin E are made from extracts of baculovirus-infected sf9 insect cells. Glutathione S-transferase fusion proteins, cyclins A and E, are purified on glutathione-agarose beads. As with p34^cdk2/cyclin B kinase, kinase activities are measured using 1 mg/mL histone H1 and 15 μM [γ-³²P]ATP over the course of 10 minutes in a final volume of 30 μL. Bovine brain is used to purify p33^cdk5/p35; the Mono S-chromatographic step is not included. The Superose 12 column's active fractions are combined and concentrated until they reach a final concentration of about 25 μg enzyme/mL. As with the p34^cdk2/cyclin B kinase, the kinase is assayed using 1 mg/mL histone HI in the presence of 15 μM [γ-³²P]ATP, over the course of 10 minutes in a final volume of 30 μL. The source of p33^cdk5/cyclin D1 is insect cell lysates. Glutathione-S-transferase and Cdk4 form a fusion protein, and the active complex is purified using glutathione-agarose beads. In a final volume of 30 μL, its kinase activity is measured using purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 μM [γ-³²P]ATP. After the incubation period of 15 minutes, 30 μL of Laemmli sample buffer is added. The substrate that has been phosphorylated is separated using 10% SDS/PAGE and examined using autoradiography, densitometry, and an overnight exposure to Hyperfilm MP. The source of p33^cdk4/cyclinD 2 is insect cell lysates. In a final volume of 30 μL, it is tested using purified retinoblastoma protein (complexed with glutathione-S-transferase) and 15 μM [γ-³²P]ATP. After the incubation period of 30 minutes, 30 μL of Laemmli sample buffer is added. The phosphorylated substrate is separated using 10% SDS/PAGE and examined using densitometry and autoradiography after being exposed to Hyperfilm MP for an entire night. Purified on glutathione-agarose beads and assayed with 1 mg myelin basic protein/ml in the presence of 15 μM [γ-³²P]ATP, MAP kinase erkl (tagged with glutathione-S-transferase) is produced in bacteria, as previously mentioned for p34^cdc2cyclin B kinase. In vitro, mitogen-activated protein kinase kinase activates His-tagged erkl and erk2, which are then purified using Ni-affinity and Mono Q. The assay is conducted over ten minutes in a final volume of thirty microliters, following the previously mentioned protocol. Infected sf9 insect cells are used to isolate protein kinase C isoforms, which are then tested for 10 minutes at 30 °C in a final volume of 30 μL using 1 mg/mL protamine sulfate and 15 μM [γ-³²P]ATP. The Whatman P81 phosphocellulose paper is used to recover phosphorylated protamine sulfate, just like it is for CDC2 kinase. Purified from the heart of cows, the catalytic subunit of cAMP-dependent protein kinase is measured using 1 mg of histone Hl/ml and 15 μM [γ-³²P]ATP, just like p34^cdc2/cyclin B kinase. After being homogenized and purified from cow tracheal smooth muscle, cGMP-dependent protein kinase is measured using 1 mg of histone Hl/mL and 15 μM [γ-³²P]ATP, just like p34^cdc2/cyclin B kinase. Rat liver cytosol is used to isolate casein kinase 2, which is then tested using 1 mg casein/mL and 15 μM [γ-³²P]ATP. After being spotted on Whatmann 3MM filters, the substrate is cleaned with 10% (mass/vol.) trichloroacetic acid. A synthetic peptide based on the smooth-muscle myosin light-chain phosphorylation site is used to assay myosin light chain kinase that has been purified from chicken gizzard. The final volume of the assay is 50 μL, and the conditions include 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. As previously mentioned, radioactive phosphate incorporation is tracked on phosphocellulose filters. Plant homolog of GSK-3, ASK-γ, is purified on glutathione-agarose after being expressed in Escherichia coli as a glutathione-S-transferase fusion protein. For 10 minutes at 30°C, 5 μg of myelin basic protein is added to a final volume of 30 μL of 15 μM [γ-³²P]ATP to test ASK-γ kinase. On Whatman P81 phosphocellulose paper, the phosphorylated myelin basic protein is recovered in the same manner as the p34^cdc2/cyclin B kinase. In a baculovirus system, the insulin receptor tyrosine kinase domain (CIRK-41) is overexpressed and homogeneously purified. Its kinase activity is measured in a final volume of 30 μL, for 10 minutes at 30 °C, using 5 μg of Raytide and 15 μM [γ-³²P]ATP. As stated for the p34^cdc2/cyclin B kinase, the phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper. From Sf9 cells that are infected, c-src kinase is isolated. After being expressed in E. Coli, the v-abl kinase is affinity purified using IgG Affigel 10. The assay is conducted for 10 minutes at 30°C, using 5 μg of Raytide, 15 μM [γ-³²P]ATP, and a final volume of 30 μL. As stated for the p34^cdc2/cyclin B kinase, the phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper.

The cells used are rat kidney tubular epithelial cells (NRK52E). Treatment for NRK52E cells involves the use of CDK5 inhibitor (R)-Roscovitine (Seliciclib) (Ros.; 10 μM) and activator p35 (15 μM), PPARγ agonist BRL 49653 (Rosi.; 50 nM), and ERK1/2 inhibitor U0126 (50 nM). Following a 72-hour treatment period, cells are extracted from each group for additional analysis.

Rats: Male Sprague Dawley rats (6–8 weeks old) receive a single intraperitoneal injection of either citrate buffer (non-diabetic) or 0.1 M citrate buffer pH 4.5 (diabetic) diluted with streptozotocin (65 mg/kg). Three days following the injection, the glucose oxidase method is used on a glucose analyzer to measure plasma glucose concentrations. The study includes rats that are classified as diabetics if their blood glucose level is greater than 16.7 mM. The level of plasma glucose is measured once a week. Seliciclib (25 mg/kg) is injected intraperitoneally into diabetic rats once a day until they are sacrificed in order to study the impact of CDK5 inhibition on renal tubulointerstitial fibrosis. As controls, DMSO is used.
Mice: Subcutaneous injections of exponentially growing UMSCC47 cells are made into the sacral region of female NUDE mice. Each mouse is inoculated with 2×10⁵ cells in 50% matrigel and 50% PBS at a volume of 100 μL. The mice receive intraperitoneal injections of either vehicle or Seliciclib at a dose of 16.5 mg/kg once the tumors have grown to a detectable size. General behavior, tumor growth, and body weight are tracked. Every three days, tumor volumes are measured. Once the tumor grows larger than 0.5 cm³, the mice are killed.

[1]. Eur J Biochem . 1997 Jan 15;243(1-2):527-36.

[2]. J Clin Invest . 1997 Nov 15;100(10):2512-20.

[3]. J Cell Biochem . 2012 Mar;113(3):868-76.

[4]. Cancer Res . 2005 Oct 15;65(20):9320-7.

[5]. Int J Cancer . 2009 Jan 15;124(2):465-72.

C19H26N6O

354.45

354.22

C, 64.38; H, 7.39; N, 23.71; O, 4.51

186692-46-6

Solid powder

CC[C@H](CO)NC1=NC(=C2C(=N1)N(C=N2)C(C)C)NCC3=CC=CC=C3

BTIHMVBBUGXLCJ-OAHLLOKOSA-N

InChI=1S/C19H26N6O/c1-4-15(11-26)22-19-23-17(20-10-14-8-6-5-7-9-14)16-18(24-19)25(12-21-16)13(2)3/h5-9,12-13,15,26H,4,10-11H2,1-3H3,(H2,20,22,23,24)/t15-/m1/s1

(2R)-2-[[6-(benzylamino)-9-propan-2-ylpurin-2-yl]amino]butan-1-ol

Seliciclib; R-Roscovitine; CYC202; CYC202; Roscovitin; Roscovitine; CYC202; CYC 202

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)