| 名稱 | PNU-120596 |
| 描述 | PNU-120596 (NSC-216666) is a positive allosteric modulator of α7 nAChR with EC50 of 216 nM. |
| 細(xì)胞實(shí)驗(yàn) | SH-SY5Y-α7 cells are plated on 96-well plates at a density of 15,000 cells per well (100 μL of 1.5 × 105 cells per mL) in complete growth medium and placed into a 37 °C incubator for 20 to 24 hours. Complete growth medium then is replaced with experimental medium alone ("PNU-120596 free") or containing appropriate concentrations of PNU-120596 and returns to the 37 °C ncubator for 20 to 24 hours. The medium is then replaced with fresh experimental medium and 20 μL per well MTS solution and returned to the 37 °C incubator for 3 hours, after which the plate is read on a microplate spectrophotometer at an absorbance of 490 nm. For all data analysis, data are normalized to untreated compound-free wells (100% cell viability) and a background absorbance taken from wells containing experimental medium and MTS solution.(Only for Reference) |
| 激酶實(shí)驗(yàn) | Ca2+ Fluorescence Assay: SH-EP1 human epithelial cells expressing a variant of theα7 nAChR (α7*) are grown in minimal essential medium (MEM) containing nonessential amino acids supplemented with 10% fetal bovine serum, L-glutamine, 100 U/ml penicillin/streptomycin, 250 ng/mL fungizone, 400 μg/mL hygromycin B, and 800 μg/mL geneticin. α7* is a variant of the human α7 nAChR, with two point mutations in the first transmembrane domain (T230P and C241S) that allow for high functional expression in SH-EP1 cells [Groppi VE, Wolfe ML, Berkenpas MB (2003) U.S. Patent 6,693,172 B1]. Cells are grown in a 37 °C incubator with 6% CO2. Cells are trypsinized and plated in 96-well plates with dark side walls and clear bottoms at a density of 2 × 104 cells/well 2 days before analysis. Cells are loaded with a mixture of Calcium reen-1AM in anhydrous dimethylsulfoxide and 20% pluronic F-127. This reagent is added directly to the growth medium of each well to achieve a final concentration of 2 μM Calcium Green-1 AM. Cells are then incubated in the dye for 1 hour at 37 °C and then washed four times with Mark's modified Earle's balanced salt solution (MMEBSS) composed of the following (inmM): 4 CaCl2, 0.8 MgSO4, 20 NaCl, 5.3 KCl, 5.6 D-glucose, 20 Tris-HEPES, and 120 N-methyl-D-glucamine, pH 7.4. After the fourth cycle, the cells are allowed to incubate at 37 °C for at least 10 minutes. The final volume of MMEBSS in each well is 100 μL and atropine is added to all wells for a final concentration of 1 μM. A fluorometric imaging plate reader (FLIPR; Molecular Devices, Union City, CA) is set up to excite Calcium Green at 488 nm using 500 mW of power and reading fluorescence emission of >525 nm. A 0.5 seconds exposure is used to illuminate each well. Fluorescence is detected using an F-stop set of either 2.0 or 1.2. After 30 seconds of baseline recording, test compounds are added to each well of a 96-well plate in 50 μL of a 3 × stock.In each experiment, four wells are used as vehicle (0.2% DMSO) controls. |
| 動(dòng)物實(shí)驗(yàn) | Animal Models: male Sprague Dawley rats (weighing 250–300 g)Formulation: PNU-120596 is dissolved in 5% DMSO and 5% Solutol in PBS.Dosages: 1 mg/kgAdministration: PNU-120596 is intravenously administrated 5 minutes before auditory gating measurements. |
| 體外活性 | PNU-120596通過增強(qiáng)由人類α7 nAChR工程變體介導(dǎo)的乙酰膽堿(Ach)激發(fā)的鈣通量，以及通過野生型受體介導(dǎo)的乙酰膽堿和膽堿激發(fā)的電流，并在擬南芥卵母細(xì)胞中的激動(dòng)劑持續(xù)存在時(shí)顯著延長激發(fā)反應(yīng)。PNU-120596能增加α7 nAChRs的通道平均開啟時(shí)間，但對(duì)離子選擇性無影響，對(duì)單位電導(dǎo)率的影響極小。當(dāng)應(yīng)用于急性海馬切片時(shí)，PNU-120596增加了在錐體神經(jīng)元中測量的ACh激發(fā)的GABAergic突觸后電流的頻率；該效果被TTX抑制，表明PNU-120596調(diào)節(jié)位于海馬區(qū)星形細(xì)胞樹突膜上的α7 nAChRs的功能。除了對(duì)α7 nAChR的正向調(diào)節(jié)外，PNU-120596還顯著延緩了脫敏動(dòng)力學(xué)，增加了通過過度刺激α7 nAChR引起的Ca2+誘導(dǎo)的毒性的潛力。PNU-120596還改變了與α7 nAChR結(jié)合時(shí)內(nèi)部β折葉、轉(zhuǎn)換區(qū)和激動(dòng)劑結(jié)合位點(diǎn)的半胱氨酸可達(dá)性。PNU-120596的結(jié)合位點(diǎn)不在激動(dòng)劑結(jié)合位點(diǎn)，并通過引起與由Ach促進(jìn)的門控構(gòu)象相似但非完全相同的構(gòu)象效應(yīng)來增強(qiáng)煙堿型受體激動(dòng)劑激發(fā)的門控。 |
| 體內(nèi)活性 | 系統(tǒng)性給藥PNU-120596（1 mg/kg）給大鼠可改善由安非他明引起的聽覺閘門缺陷，這一模型被提出用來反映與精神分裂癥相關(guān)的電路級(jí)干擾。[1] 在卡拉膠之前給予PNU-1230596，30 mg/kg顯著減輕了機(jī)械性痛覺過敏和承重缺陷，效果持續(xù)達(dá)4小時(shí)。PNU-120596減弱了由卡拉膠引起的TNF-α和IL-6在后爪水腫中的增加水平，而雙氯芬酸僅減輕了IL-6水平。通過卡拉膠或CFA引起的已建立的機(jī)械性痛覺過敏也被PNU-120596部分逆轉(zhuǎn)。[4] |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 31.2 mg/mL (100 mM)
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| 關(guān)鍵字 | pain | anti-hyperalgesia | inhibit | inflammatory | Nicotinic acetylcholine receptors | TNF-α | Inhibitor | α7nAChR | disorders | PNU 120596 | neurological | NSC216666 | IL-6 | nAChR | PNU120596 | PNU-120596 | NSC-216666 |
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