| 名稱 | PNU-120596 |
| 描述 | PNU-120596 (NSC-216666) is a positive allosteric modulator of α7 nAChR with EC50 of 216 nM. |
| 細胞實驗 | 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) |
| 激酶實驗 | 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. |
| 動物實驗 | 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通過增強由人類α7 nAChR工程變體介導的乙酰膽堿(Ach)激發(fā)的鈣通量，以及通過野生型受體介導的乙酰膽堿和膽堿激發(fā)的電流，并在擬南芥卵母細胞中的激動劑持續(xù)存在時顯著延長激發(fā)反應。PNU-120596能增加α7 nAChRs的通道平均開啟時間，但對離子選擇性無影響，對單位電導率的影響極小。當應用于急性海馬切片時，PNU-120596增加了在錐體神經元中測量的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調節(jié)位于海馬區(qū)星形細胞樹突膜上的α7 nAChRs的功能。除了對α7 nAChR的正向調節(jié)外，PNU-120596還顯著延緩了脫敏動力學，增加了通過過度刺激α7 nAChR引起的Ca2+誘導的毒性的潛力。PNU-120596還改變了與α7 nAChR結合時內部β折葉、轉換區(qū)和激動劑結合位點的半胱氨酸可達性。PNU-120596的結合位點不在激動劑結合位點，并通過引起與由Ach促進的門控構象相似但非完全相同的構象效應來增強煙堿型受體激動劑激發(fā)的門控。 |
| 體內活性 | 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的電路級干擾。[1] 在卡拉膠之前給予PNU-1230596，30 mg/kg顯著減輕了機械性痛覺過敏和承重缺陷，效果持續(xù)達4小時。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引起的已建立的機械性痛覺過敏也被PNU-120596部分逆轉。[4] |
| 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 : 31.2 mg/mL (100 mM)
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| 關鍵字 | 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 |
| 相關產品 | Pilocarpine Hydrochloride | Amitriptyline hydrochloride | Choline chloride | Arecoline hydrobromide | Pilocarpine nitrate | CLOZAPINE N-OXIDE | Adenine | Forskolin | Propoxur | Adiphenine hydrochloride | Ribavirin | Nanofin |
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