Combretastatin A4 (CA4) is a microtubule-targeting agent that binds β-tubulin (Kd: 0.4 μM).

β-tubulin:0.4 μM(Kd)

在NT2和MDA-MB-231乳腺肿瘤模型中,Combretastatin A4（100 mg/kg,i.p.）诱导脂质R1明显减少,并通过电子顺磁共振血氧定量法测得pO2下降. Combretastatin A4（100 mg/kg,i.p.）明显减少雄性NMRI小鼠的Ktrans.

Combretastatin A4（1 μM）抑制35%微管蛋白聚合,10 μM几乎完全可阻断。Combretastatin A4抑制MDA-MB-231,Hela,A549,HL-60,SF295,HCT-8,MDA-MB435,OVCAR-8,PC3M,NCI-H358M和淋巴细胞的生长,IC50分别为2.8,0.9,3.8,2.1,6.2,5.3,7.9,0.37,4.7,8和3.2 nM。

Competitive binding assay using LC-MS/MS: Colchicine (1.2 μ M) is incubated with tubulin (1.3 mg/mL) in the incubation bu?er (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH 6.9) at 37℃ for 1 h. Varying concentrations (0.1 ? 125 μ M) of Combretastatin A4 are used to compete with colchicine originally bound to tubulin. After incubation, the ?ltrate is obtained. The ability of the analogue to inhibit the binding of colchicine is expressed as a percentage of control binding in the absence of any competitor.

MDA-MB-231, A549, and HeLa cells are grown in DMEM medium (115 units/mL of penicillin G, 115 μg/mL of streptomycin, and 10% fetal bovine serum). Cells are seeded in 96-well plates (5000 cells/well) containing 50 μL of growth medium for 24 h. After medium removal, 100 μL of fresh medium containing individual analogue compounds at di?erent concentrations is added to each well and incubated at 37 ℃ for 72 h. After 24 h of culture, the cells are supplemented with 50 μL of analogue compounds dissolved in DMSO (less than 0.25% in each preparation). After 72 h of incubation, 20 μL of resazurin is added for 2 h before recording ?uorescence at 560 nm (excitation) and 590 nm (emission) using a Victor microtiter plate ?uorimeter. The IC50 is de?ned as the compound concentration required to inhibit cell proliferation by 50% in comparison with cells treated with the maximum amount of DMSO (0.25%) and considered as 100% viability.(Only for Reference)