| 名稱 | Tivantinib |
| 描述 | Tivantinib (ARQ 197) is an orally bioavailable small molecule inhibitor of c-Met with potential antineoplastic activity. |
| 細胞實驗 | HT29, MKN-45, and MDA-MB-231 cells are seeded in black 96-well plates at 5 × 103 cells per well overnight in a medium with 10% FBS. The next day, cells are treated with increasing concentrations of ARQ-197 (0.03-10 μM) for 24, 32, and 48 hours at 37 °C. After ARQ-197 treatment, the drug-containing medium is removed and cells are incubated for at least 10 minutes in a labeling solution (10 mM HEPES, 140 mM NaCl, and 6 mM CaCl2) containing 2 μg/mL Hoescht 33342 (blue channel), 500-times diluted Annexin V-FITC (green channel), and 1 μg/mL propidium iodide (red channel). High-content image acquisition and analysis are carried out. The program is set to take four images per well. The exposure time is set at 16.7 ms/10% gain, 500 ms/35% gain, and 300 ms/30% gain for the 4,6-diamidino-2-phenylindole, FITC, and rhodamine channels, respectively. Images are processed and the numbers of positive cells for each channel and each condition are determined. In addition, HT29 cells are treated with increasing concentrations of ARQ-197 for 32 hours in the absence or the presence of 25, 50, and 100 μM ZvAD-FMK (irreversible general caspase inhibitor), and the same procedures are undertaken. All experiments are done in triplicate. To determine whether the apoptotic effect is due to c-Met inhibition, the effect of ARQ-197 when glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and c-Met are knocked down using siRNA is investigated. HT29, MKN-45, and MDA-MB-231 cells are transfected with a nontargeted control siRNA, a gapgh-targeted control siRNA, or a met-targeted siRNA. After 3 days, c-Met, GAPDH, and β-actin expression levels are determined using specific antibodies. To determine if the effect is caspase dependent, HT29, MKN-45, and MDA-MB-231 cells are transfected with a met-targeted siRNA for 2 days and incubated in the absence or the presence of increasing concentrations of ZvAD-FMK for 1 additional day.A nontargeted siRNA and a gapgh-targeted siRNA (siRNA GAPDH) are also transfected in parallel, as controls. Cells are then stained with Annexin V-FITC and propidium iodide, and the percentage of apoptotic cells is determined.(Only for Reference) |
| 激酶實驗 | c-Met SDS-PAGE in vitro kinase assay: Recombinant c-Met protein (100 ng) is preincubated with increasing concentrations of ARQ-197 for 30 minutes at room temperature. Following preincubation, 100 μM of poly-Glu-Tyr substrate and various concentrations of ATP containing 5 μCi of [γ-32P]ATP are added to the reaction mixture. The reaction is incubated for 5 minutes at room temperature and then stopped by the addition of 5 μL of SDS-polyacrylamide gel, reducing sample buffer. The samples are then loaded onto a 7.5% acrylamide gel and SDS-PAGE is performed. The phosphorylated poly-Glu-Tyr substrates are ultimately visualized by autoradiography. c-Met activity is quantified by densitometry. |
| 體外活性 | 用ARQ-197處理的所有三種異種移植物模型顯示腫瘤生長減少：HT29模型中66％,MKN-45模型中45％,MDA-MB-231模型中79％.口服給藥200 mg/kg ARQ-197處理HT29,MKN-45和MDA-MB-231三種移植瘤模型,沒有顯著的體重變化.藥效學方面,ARQ-197強效抑制人類結(jié)腸移植瘤HT29中c-Met的磷酸化,單獨口服200 mg/kg ARQ-19724小時后,c-Met自磷酸化強烈下降.總之,ARQ-197抑制ARQ-197抑制c-Met依賴性異種移植人類腫瘤的生長. |
| 體內(nèi)活性 | ARQ-197具有抗腫瘤活性,抑制A549,DBTRG和NCI-H441細胞增殖,IC50分別為0.38,0.45,0.29 μM。用ARQ-197處理,導致MAPK信號級聯(lián)反應(yīng)的磷酸化減少及預(yù)防侵入和遷移。此外,在沒有內(nèi)源性c-Met表達的細胞系NCI-H661中c-Met的異位表達導致其獲得侵襲性表型,也被ARQ-197抑制。ARQ-197抑制人類重組c-Met,具有恒定的Ki值,為355 nM。ARQ-197抑制c-Met磷酸化,且阻斷下游c-Met信號通路。盡管增加濃度的ARQ-197不會顯著影響ATP的Km,但c-Met暴露于0.5 μMARQ-197會使c-Met的Vmax降低約3倍。ARQ-197降低Vmax而不影響ATP的Km值說明ARQ-197抑制c-Met是非ATP競爭抑制,也說明ARQ-197具有高度激酶選擇性。ARQ-197抑制組成型和配體介導的c-Met自磷酸化,并且進而抑制c-Met活性,繼而導致抑制下游c-Met效應(yīng)物。在表達c-Met的人類癌細胞（包括HT29,MKN-45和MDA-MB-231細胞）中ARQ-197誘導胱天蛋白酶依賴性細胞凋亡增加。 |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 68 mg/mL (184.1 mM)
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
H2O : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | c-Met/HGFR | Tivantinib | inhibit | Apoptosis | ARQ-197 | ARQ197 | Inhibitor |
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