生物活性

GW788388 is a potent inhibitor of transforming growth factor-β type I receptor (ALK5) (IC50 values are 18 and 93 nM for ALK5 binding and for TGF-β cellular assay respectively). Inhibits esophageal squamous cell carcinoma (ESCC)-induced neoangiogenesis.

Kinaseand cellular potency of GW788388[1]

体外研究

体内研究

30% PEG400+0.5% Tween80+5% propylene glycol

激酶实验

ALK5Fluorescence Polarization Binding Assay[1]

Compound binding to ALK5 was tested onpurified recombinant GST-ALK5 (residues 198-503). Displacement of rhodaminegreen fluorescently labeled ATP competitive inhibitor by differentconcentrations of test compounds was used to calculate a binding pIC50.GST-ALK5 was added to a buffer containing 62.5mM N-(2-hydroxyethyl)- piperazine-N’-2-ethanesulfonicacid (Hepes), pH 7.5, 1mM dithiothreitol (DTT), 12.5mM MgCl2, 1.25mM3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonic acid (CHAPS), and1nM rhodamine green-labeled ligand so that the final ALK5 concentration is 10nMbased on active-site titration of the enzyme. The enzyme/ligand reagent (40μL)was added to 384-well assay plates containingμL ofdifferent concentrations of test compound. The plates are read immediately on aLJL Acquest fluorescence reader (Molecular Devices) with excitation, emission,and dichroic filters of 485, 530, and 505nm, respectively. The fluorescencepolarization for each well is calculated by the Acquest and is then importedinto curve-fitting software for construction of concentration-response curves.

细胞实验

Cellculture and reagents[2]

The human breast carcinoma MDA-MB 231 andT47D, the human osteosarcoma U2OS, and the monkey kidney COS cell lines were maintainedin Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100U/mlpenicillin, and 50 mg/ml streptomycin. The human renal carcinoma cell line(RCC4) stably expressing the VHL protein under neomycin selection. The murineepithelial breast cells NMuMG were maintained in Dulbecco’s modified Eagle’s mediumas above with 10 mg/ ml insulin. Cell lines were cultured at 37oC in5% CO2. Compounds were dissolved in dimethyl sulfoxide (DMSO). Weused 5 ng/ ml TGF-β3, 100 ng/ ml BMP6, and 50 ng/ ml, activin A.

Cellviability assay

NMuMG cells were treated with dilutions ofGW788388 and SB431542 for 72 h. Viability was measured with a modified MTSassay, measuring metabolically active cells.

动物实验

Electrocardiography[3]

All mice (129/Sv genetic background) weregenotyped by polymerase chain reaction. Mice were anaesthetized for ECGrecording with etomidate (25 mg/kg i.p.). Body temperature was maintained at 37oCwith a heating pad. Sixlead ECG was recorded with 25-gauge subcutaneouselectrodes on a computer through an analog-digital converter for monitoring andlater analysis.

Cardiac fibrosisremodeling investigations

Age-related cardiac fibrosis remodeling inWT and Scn5a+/- mice was investigated at the ages of 10, 30, 45 and 60 weeks.Cardiac fibrosis was also investigated at the age of 60 weeks in WT mice treatedchronically with flecainide or placebo and in Scn5a+/-mice treated withGW788388 or placebo. Mice were euthanized by cervical dislocation. Afterexcision, hearts were rinsed in saline solution, fixed by immersion in 4%paraformaldehyde, embedded in paraffin, and transverse sectioned at 5-µmintervals. Hearts from placebo- and flecainide-treated WT mice were snap-frozenin liquid nitrogen and sectioned at 6-µm intervals. Slices were hydrated inTissue-Clear®, decreased alcohol bath and deionized water. Then, they werestained during 1 hour in 0.1% picrosirius red solution (20 g of picric acid + 1g of Sirius Red in 1 liter of deionized water, pH 2.0). Then, slices weredecolorized for precisely 2 minutes in 0.01 N HCl and dehydrated in increasedalcohol bath and Tissue-Clear®. Sections were mounted in QPath Coverquick3000  and examined with a classic lightmicroscope (Nikon Eclipse E-600 microscope with NIS-Elements BR v4.10 Software,Nikon, Japan). Semi quantitative assessment of fibrosis was determined basedupon the extent of interstitial fibrosis. For each heart, 3 regions (apex,middle and base) were observed and at least 20 pictures of each region weretaken. Automatic analysis was performed using ImageJ software 1.45b.

In vivo treatments with flecainide and GW788388         

For flecainide, 129/Sv WT mice (male andfemale) were chronically treated orally (drinking water) with flecainide at 50mg/kg/day from the age of 6 weeks to the age of 20, 30, and 60 weeks.Flecainide was dissolved in water.

For GW788388, 45-week-old Scn5aþ/- micewere treated orally chronically until the age of 60 weeks at a dose of5mg/kg/day. GW788388 was dissolved in dimethyl sulfoxide (DMSO). The maximum concentrationof DMSO in drinking water was 0.2% in both GW788388- and placebo-treatedgroups.

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] Gellibert F dGA, Woolven J, Mathews N, Nguyen VL, Bertho-Ruault C, Patikis A, Grygielko ET, Laping NJ, Huet S. Discovery of 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H- pyran-4-yl)benzamide (GW788388): a potent, selective, and orally active transforming growth factor-beta type I receptor inhibitor. J Med Chem. . 2006 49(7):2210-2221.
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体内溶解度
约28 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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