生物活性

A66 is a potent and selective PI 3-kinase p110α inhibitor (IC50 = 32 nM) which exhibits >100-fold selectivity for p110α over other PI 3-kinase isoforms. Inhibits Akt signaling and tumor growth in SK-OV-3 xenografts in mice. Among the class-II PI3Ks, class-III PI3Ks, and PI4Ks, A66 only exhibits limited cross-reactivity with the class-II PI3K PI3K-C2β and the PI4Kβ isoform of PI4K with IC50 of 462 nM and 236 nM, respectively. A66 exhibits no inhibitory activity against other lipid kinases or the related kinases DNA-PK and mTOR. A66 treatment at 0.7 μM induces a 75-80% reduction in focus formation by the highly transforming p85α iSH2 mutants KS459delN, DKRMN-S560del, and K379E, and reduces the phosphorylation of Akt on T308 by all p85 mutants.

IC50 against different PI3K isoforms and PI3K-related kinases[1]

PI3Ks activities[2]

The anti- proliferation ability[3]

Cytokine secretion by CD4+ T cells: sensitivity to A66[4]

The IC50 against the protein kinase and the lipid kinase activities of wild-type and mutant p110α[5]

体外研究

体内研究

激酶实验

Protein Kinase Assays[5]

Protein kinaseassays were carried out in a buffer containing 50 mM NaCl, 20 mM Tris/Cl (pH 7.4), 0.1 mM Na-orthovanadate, 12 mM ATP, 5 mM DTT, 2 mCiγ33P-ATP, and either 5 mM MgCl2,or 5 mM MnCl2or both (as stated); Each reaction tube contained 0.5 mg kinase, 0.5 mg bic and inhibitors at stated concentrations. Unless otherwise stated, incubations were allowed to proceed for 20 minutes at 32oC and terminated by the addition of 5x electrophoresis sample buffer before complete denaturation at 99oC for 5 min. Components were separated by SDS PAGE, Coomassie-stained, dried and analysed by autoradiography (Molecular Dynamics Storm 680 PhosphorImager and quantified using ImageQuantTL software).

Lipid Kinase Assays

To verify the impact of phosphorylation on lipid kinase activity, kinases were either pretreated with ATP (phosphorylated) or PP2A (unphosphorylated) before determining lipid kinase activity using phosphoinositol (PI) as a substrate. More specifically, kinases were either treated according to the Protein Kinase Assay (for 1 hour at 37oC withoutγ33P-ATP) or PP2A Treatment methods outlined above before the addition of EDTA to a final concentration of 2 mM EDTA; 10μL of kinase (equivalent to 0.5 mg) was mixed with 90μL buffer containing 40 mM Tris/Cl, 200 mM NaCl, 1 mM EDTA (pH 7.4). Each reaction point consisted of 20μL of this kinase mixed with 10μL of 1 mg/mL PI (Lipid Products, Surrey UK) in 10 mM Tris/Cl, 1 mM EDTA (pH 7.4), and 30μL of ATP mix (10 mM MgCl2, 200μM ATP, 1μCiγ33P-ATP). The reaction was allowed to proceed for 1 hour at room temperature and stopped with 100μL of 1 M HCl, before chloroform lipid extraction with minor alterations described here. Specifically the re-extraction buffer consisted of 50:50 methanol/1 M HCl and the dried lipid was resuspended in 30 mL of chloroform: methanol (4:1 v/v). TLC plates were pre-treated with a solution containing 8 mM oxalic acid and 1 mM EDTA (pH 8) in MQ H2O/ethanol (3:1 v/v), and allowed to dry at room temperature overnight. Lipids were separated on the TLC plates using propan-1-ol/glacial acetic acid/MQ H2O (65:4:31 v/v). Assay results were analysed by autoradiography (Molecular Dynamics Storm 680 PhosphorImager and quantified using ImageQuantTL software).

Lipid Kinase IC50 Determination

IC50 values were determined using the PI3K (human) HTRF Assay. All PI 3-K isoforms were made in-house and used in the range of their EC65–80 titration (18 ng/Ml for H1047R, 6.5 ng/mL for E545K, 50 ng/mL for p110α, 400 ng/mL for p110β, 65 ng/ml for p110δand 400 ng/mL for p110γ). Drugs were dissolved in DMSO and serially diluted in the same. Final DMSO concentration in assay was 2.5%.

细胞实验

Cell lines and culture[3]

The human gallbladder cancer cell lines GBC-SD cells were grown in high-glucose DMEM supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin. The NOZ cells were grown in William’s medium supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin. All cells lines were maintained at 37 °C in a humidified atmosphere with 5 % CO2.

Cell proliferation assay

The proliferations of the GBC-SD and NOZ cells lines treated with siRNA, negative control (NC) siRNA (random null siRNA sequence), CON (only Lipofectamine 2000), WT (PIK3CA-WT plasmid), E545K (PIK3CA-E545K plasmid) and vector (empty vector) were measured using a Cell Counting Kit-8 assay according to the manufacturer’s instructions. The GBC-SD (8×104) and NOZ (5 × 104) cells were seeded into 6-well plates and incubated overnight. The cells were then transfected with siRNA, NC, CON, WT, E545K or vector. The cells were maintained for 12 h and then seeded into 96-well plates (500 cells in each well). The cells were maintained for 6, 24, 48, 72, 96 and 120 h, and CCK-8 was then added to each well, and cells were incubated at 37 °C for 3 h. The optical densities (ODs) were measured at a wavelength of 450 nm with a microplate reader. The results are represented as averages of five different wells. Cell proliferation was measured according to the following the formula: cell proliferation = (ODDay n/ODDay1)*100 %.

动物实验

In vivo subcutaneous xenograft analysis[3]

Nude mice were randomly divided into control, WT and E545K mutation groups that received subcutaneous injection inoculations with 1×106NOZ, NOZ-WT and NOZ-E545K cells, respectively.

The tumor volumes were measured using Vernier calipers once every three days, and the tumor volumes were calculated according to the following formula: tumor volume (mm3) = L×W2(where L and W represent the length and width of the tumor, respectively). On day 21, the mice were anesthetized and photographed. Afterwards, the mice in the control group were sacrificed. The WT group and the E545K mutation group were randomly divided into A66 group and normal saline groups. The mice in the A66 group were injected with 1 mg A66 once every three days through the tail vein for three weeks, and the mice in normal saline group were injected with same volume of normal saline. The tumor volumes were measured once every three days as previously described. On day 42, the mice were sacrificed, and the tumor tissues were removed and weighed.

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] Jamieson S, Flanagan JU, Kolekar S, et al. A drug targeting only p110alpha can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types. Biochem J. 2011;438(1):53-62.
[2] Fairhurst RA, Imbach-Weese P, Gerspacher M, et al. Identification and optimisation of a 4',5-bisthiazole series of selective phosphatidylinositol-3 kinase alpha inhibitors. Bioorg Med Chem Lett. 2015;25(17):3569-3574.
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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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