生物活性

Roscovitine is a potent and selective inhibitor of cyclin-dependent kinases which suppresses the proliferation of mammalian cell lines. Selective for Cdk1/cyclin B kinase (IC50=450nM), Cdk2 (IC50=700nM) and Cdk5/p35 (IC50=160nM). It inhibits M phase promoting factor kinase activity and affects calcium channel gating. Roscovitine reversibly inhibits the transition from prophase to metaphase at micromolar concentrations in starfish oocytes and sea urchin embryos. It also inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. Also selective over a wide range of related kinases including ERK1 and ERK2. Arrests L1210 cells in G1 phase. Inhibits phosphorylation of vimentin in vivo. Antimitotic. 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. Roscovitine is an inhibitor of Cdk7, cyclin B, cyclin E, cyclin A, cyclin H, cdc2 and p53. R-roscovitine (Roscovitine, Seliciclib, CYC202) is an ATP-competitiveselective second-generation CDKs inhibitor, which has variousanti-inflammatory, -proliferative, -apoptotic, and neuroprotective effects, incell types ranging from cancer cell lines to keratinocytes and fibroblasts.

Enzymeactivities of roscovitine[1][2]

Kinaseactivities of Roscovitine (R/S isomer)

Growthinhibition of (R)-roscovitine

Functionalrestoration of F508del-CFTR activity by roscovitine[3]

Cell cycle inhibition of roscovitine[1]

体外研究

体内研究

1% DMSO+30% polyethylene glycol+1% Tween 80

激酶实验

Kinases activity assay[1]

Kinases activity was assayed at 30°Cin buffer C (60mM glycerol 2-phosphate, 15mM p-nitrophenyl phosphate, 25mM MopspH 7.2, 5mM EGTA, 15mM MgCl₂, 1mM dithiothreitol, 1mM sodium vanadate,1mM phenyl phosphate, 10μg leupeptin/ml, 10μg soybeantrypsin inhibitor/ml and 100μM benzamidine). Blank values were substracted from the data andactivities calculated as molar amount of phosphate incorporated in proteinacceptor during a 10-min incubation. Controls were performed with appropriatedilutions of Me₂SO. In a few cases, phosphorylation of the substratewas assessed by autoradiography after SDS/PAGE.

p34^cdc2/cyclin B was purifiedfrom M-phase starfish oocytes by affinity chromatography on p9^CKShs1-Sepharosebeads, from which it was eluted by free p9^CKShs1. It was assayedwith 1mg histone H1/ml, in the presence of 15μM [y-³³P]ATP (3000Ci/mmol; 1mCi/ml) in a final volume of 30μl. After a10-min incubation at 30^oC, 25-μl aliquots ofsupernatant were spotted onto pieces (2.5 X3cm) of Whatman P81 phosphocellulosepaper, and, after 20s, the filters were washed five times (for at least 5 mineach time) in a solution of 10ml phosphoric acid/1 water. The wet filters weretransferred into 6-ml plastic scintillation vials, 5ml ACS scintillation fluidwas added and the radioactivity measured in a Packard counter. The kinaseactivity was expressed as molar amount of phosphate incorporated in histone H1during a 10-min incubation or as a percentage of maximal activity.

细胞实验

PC12 Cell Culture, R-roscovitine Treatment andNeurite Outgrowth Analysis[8]

PC12 cells were seeded at 1.5x10⁵ and 4.5 x10⁵ (for western blot analysis) into 35mm and 60mm tissue culturedishes, respectively, in high serum-containing RPMI (10% horse serum, 5% fetal bovineserum) with penicillin-streptomycin. After 24h, cells were subjected to serum starvationin RPMI containing 0.2% horse serum and penicillin-streptomycin for18h. Cells werethen treated with 10ng/ml nerve growth factor (NGF), and/or graded concentrations(0.2, 2, 5, 10, 20 and 40mM) of R-roscovitine for 24h. Neurite outgrowth was examinedby light microscopy and images were analyzed using the Image-ProExpress software.The length of neuritis was analyzed using the Image-J software. Three optical fieldsand 50cells/field were evaluated for each condition. Cellviability was assessedby trypan blue assay. Measurements were taken from 50cells in each treatment (n=3).

动物实验

In vivo experiments[7]

Exponentially growing UMSCC47 cells wereinjected subcutaneously into the sacral area of female NUDE mice. Each mousewas inoculated with 2 × 10⁵ cells in 50% matrigel and 50% PBS at avolume of 100μL. Body weight, tumor growth, and general behavior weremonitored. Tumor volumes were measured every 3 days. Mice were sacrificed when thetumor exceeded a size of 0.5cm³.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Meijer L BA, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem. 1997;243(1-2):527-536.
[2] Cicenas J, Kalyan K, Sorokinas A, et al. Roscovitine in cancer and other diseases. Ann Transl Med. 2015;3(10):135.
[3] Norez C, Vandebrouck C, Bertrand J, et al. Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism. Br J Pharmacol. 2014;171(21):4831-4849.
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体内溶解度
约29 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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