生物活性

GSK461364 isa potent, selective, reversible, ATP-competitive Plk1 inhibitor that inhibitsthe proliferation of multiple human cancer cell lines by promoting G2/M cellcycle arrest at low concentrations. GSK461364 inhibits purified Plk1 with Ki of 2.2 nM in a cell-free assay. It is more than 1000-fold selective against Plk2/3. 

Enzyme activities of GSK461364

体外研究

体内研究

激酶实验

Enzyme assays[1]

Kinase reactions were performed in a finalassay volume of 10μL using the Z’-Lyte Assay kit (Ser/Thr peptide 16). Briefly, reactionscontained 50mmol/L HEPES (pH 7.5), 10mmol/L MgCl₂, 1mmol/L EGTA,1mmol/L DTT, 0.01% Brij 35, 0.01mg/mL casein, 200μmol/L ATP, 200μmol/LPolo Box peptide (NH2-MAGPMQS[pT]PLNGAKK-OH), and 6nmol/L recombinantPlk1(H6-tev-PLK 1-603). Plk1was preincubated for 60 min in the presence or absenceof 0 to 1,000nmol/L GSK461364A. Reactions were then initiated by the additionof 2μmol/L peptide. After 15 min at 23 ℃, reactions werequenched and processed according the Z’-Lyte protocol and read on an EnVisionplate reader. Raw fluorescence values were converted to concentration ofproduct formed using substrate and product standards. IC50 values weredetermined using two-parameter fit (Hill coefficient and IC50) using GraFitsoftware. Because the potency of inhibition for GSK461364A was observed to varyas a function of the ATP concentration in a manner consistent with anATP-competitive mode of inhibition, an upper limit for the Ki*app for GSK461364Awas determined by applying the Cheng-Prusoff relationship for a competitiveinhibitor (ATP Km*app = 16μmol/L) to the IC50 value obtained with 60min preincubation ofGSK461364A.

细胞实验

Cell lines[3]

Three OS cell lines, U2OS, MG63 and SJSAwere selected for in vitro study and maintained in either Dulbecco's modifiedEagle's medium (DMEM), Iscove's modified Dulbecco's medium, or DMEM/F12 basemedia with 10% fetal bovine serum.

Cellviability assay

TACS™3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cellproliferation assay was used in the cell viability assay. Approximately 2,000-20,000cells/100μl/wellwere seeded in 96-well plates overnight. Subsequently, reagents at differentconcentrations desired time at 37˚C, pulsed with 10μl MTT reagent, and incubatedfor an additional 4h at 37 ˚C. Furthermore, detergent reagent of 100μl/well wasadded and mixed thoroughly to dissolve the blue crystals. Absorbance of theconverted dye was measured using a Vmax microplate reader at 570 nm (test) and650nm wavelengths. Cell survival was calculated using the following equation: %survival = (mean experimental absorbance/mean control absorbance) x 100. Thepossible synergistic effect of GSK461364 and chemotherapy was measured usingthe combination index (CI) calculated using CalcuSyn software. CI >1 wasdefined as antagonism, CI = 1 as additivity, and CI <1 as synergy; theexperiment was performed in triplicate.

动物实验

Xenograft model[4]

SK-N-AS and IMR32 neuroblastoma cells were culturedto 80% confluency, harvested and suspended in 200μL Matrigel™ for subcutaneousinoculation of 2 × 10⁷ cells into the left flank of 8-week-oldfemale athymic nu/ nu mice (n = 12 mice). Mice were randomly assigned (6 miceper group) to either GSK461364 or vehicle (0.1% DMSO, 5% glucose) controlgroups after tumors reached 200 mm³. Vehicle control or 50mg GSK461364per kg body weight was intraperitoneally injected every second day until allcontrol mice reached the endpoint (tumor volume of 2,500mm³) but notlonger than 40 days. Tumor growth was monitored using a caliper, and tumorvolume was calculated using the formula, (breadth × length ×height)/2. Micewere euthanized by cervical dislocation when tumor size exceeded 2,500mm³.In mice, whose xenograft tumors were to be immunohistologically examined forapoptosis and proliferation, 2 doses of 100 mg GSK461364 per kg body weightwere administered every 12 hours over a 3-day course (n = 3 mice in bothGSK461364 and vehicle control groups). Mice were euthanized 4 hours after thelast treatment injection, and xenograft tumors were excised, formalinfixed and paraffin-embedded.

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] Gilmartin AG, Bleam MR, Richter MC, et al. Distinct concentration-dependent effects of the polo-like kinase 1-specific inhibitor GSK461364A, including differential effect on apoptosis. Cancer Res. 2009;69(17):6969-6977.
[2] Ferrarotto R, Goonatilake R, Young Yoo S, et al. Epithelial-Mesenchymal Transition Predicts Polo-Like Kinase 1 Inhibitor-Mediated Apoptosis in Non-Small Cell Lung Cancer. Clin Cancer Res. 2016;22(7):1674-1686.
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体内溶解度

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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