- DBeQ
- Xanthohumol
| NMS-873VCP/p97 inhibitor,selective and allosteric |
Sample solution is provided at 25 µL, 10mM.
Quality Control & MSDS
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- Purity = 98.00%
- COA (Certificate Of Analysis)
- MSDS (Material Safety Data Sheet)
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Chemical structure
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| Description | NMS-873 is an allosteric and specific inhibitor of p97 with an IC50 value of 30 nM. | |||||
| Targets | p97 | |||||
| IC50 | 30 nM | |||||
| Kinase experiment [1]: | |
Biochemical assay development and HTS | The ATPase activity and the kinetic parameters of recombinant wild-type VCP and its mutants were evaluated by monitoring ADP formation in the reaction, using a modified NADH-coupled assay. As ADP and NADH are ATP-competitive inhibitors of VCP ATPase activity, the standard protocol for the NADH-coupled assay was modified into a two-step procedure. In the first part, an ATP-regenerating system (40 U/ml pyruvate kinase and 3 mM phosphoenolpyruvate) recycles the ADP produced by VCP activity, keeps the substrate concentration constant (thus preventing product inhibition) and accumulates a stoichiometric amount of pyruvate. In the second part, the VCP enzymatic reaction is quenched with 30 mM EDTA and 250 μM NADH and stoichiometrically oxidized by 40 U/ml lactic dehydrogenase to reduce accumulated pyruvate. The decrease of NADH concentration was measured at 340 nm using a Tecan Safire 2 reader plate. The assay was performed in 96- or 384-well UV plates in a reaction buffer with 50 mM Hepes, pH 7.5, 0.2 mg/ml BSA, 10 mM MgCl2 and 2 mM DTT. Experimental data were fitted with a cooperative equation obtaining a Ks* of about 60 μM and a Hill coefficient (n) of 2.0 ± 0.1.The HTS campaign was performed against a 1-million-compound library using a miniaturized assay in 1,536-well format and a more sensitive ADP detection system, Transcreener ADP FP. A 20-min preincubation of 10 nM VCP and 10 μM inhibitor was performed, after which 10 μM ATP was added to the reaction, which was allowed to proceed for 90 min before quenching. The average Z′ of the screening was 0.58, and the hit rate using 3× s.d. (38% inhibition) as cutoff was 1.7%. Primary hits with >60% inhibition at 10-μM concentration were pruned using physicochemical and structural filters to leave 7,516 compounds. At the end, reconfirmation was performed in duplicate on 3,988 primary hits, and 500 compounds were selected for a dose-response evaluation using the previously described NADH-modified coupled assay.The potency of the most interesting HTS hits was measured against both wild-type VCP and the C522T mutant. ATP concentrations that yielded the half-maximal velocity (Ks*) for each enzyme, corresponding to 60 μM and 130 μM for the wild type and C522T mutant, respectively, were used in the assay. To explore the dependency of reversible inhibitors from substrate concentration, their potency was evaluated also at saturating ATP concentration (1 mM) and compared to the potency of a standard ATP competitive inhibitor (AMP-PNP). |
| Cell experiment [1]: | |
Cell lines | HCT116, HeLa cells |
Preparation method | The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months. |
Reacting condition | 0.5, 2.5 μM for 6h |
Applications | NMS-873 activated the unfolded protein response, interfered with autophagy and induced cancer cell death. NMS-873 mediated stabilization of Mcl-1 in live cells. NMS-873 inhibited the proliferative activity in HCT116 and HeLa cells with IC50 values of 0.4 and 0.7 μM, respectively. |
References: 1Magnaghi, P., D"Alessio, R., Valsasina, B., Avanzi, N., Rizzi, S., Asa, D., Gasparri, F., Cozzi, L., Cucchi, U., Orrenius, C., Polucci, P., Ballinari, D., Perrera, C., Leone, A., Cervi, G., Casale, E., Xiao, Y., Wong, C., Anderson, D. J., Galvani, A., Donati, D., O"Brien, T., Jackson, P. K. and Isacchi, A. (2013) Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol. 9, 548-556 | |
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| Cas No. | 1418013-75-8 | SDF | Download SDF |
| Synonyms | N/A | ||
| Chemical Name | 3-[3-cyclopentylsulfanyl-5-[[3-methyl-4-(4-methylsulfonylphenyl)phenoxy]methyl]-1,2,4-triazol-4-yl]pyridine | ||
| Canonical SMILES | CC1=C(C=CC(=C1)OCC2=NN=C(N2C3=CN=CC=C3)SC4CCCC4)C5=CC=C(C=C5)S(=O)(=O)C | ||
| Formula | C27H28N4O3S2 | M.Wt | 520.67 |
| Solubility | ≥17.1mg/mL in DMSO | Storage | Store at -20°C |
| Physical Appearance | A solid | Shipping Condition | Evaluation sample solution : ship with blue ice.All other available size:ship with RT , or blue ice upon request |
| General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months. | ||
NMS-873 is a selective inhibitor of VCP with IC50 value of 30nM [1].
NMS-873 is the most potent and specific VCP inhibitor. It shows similar potency against both wild-type VCP and VCPC522T without affecting the oligomeric state of VCP. NMS-873 is selective against all of the AAA ATPases, HSP90 or some other kinases. The inhibition effect of NMS-873 is potent and somehow selective in a panel of tumor cell lines when its concentration ranges from 0.08μM to 2μM. NMS-873 suppresses cell proliferation in HCT-116 cell line through inducing accumulation of poly-Ub proteins and stabilization of cyclin E and Mcl-1 dose-dependently. Additionally, NMS-873 causes distribution of HCT116 cells, leading a dose and time-dependent increase in the G2 population and in the sub-G1 fraction. Further, NMS-873 shows cell killing activity in hematological tumors as well as a wide variety of solid tumors with IC50 range between 0.08μM and 2μM [1].
References:[1] Magnaghi P, D"Alessio R, Valsasina B, Avanzi N, Rizzi S, Asa D, Gasparri F, Cozzi L, Cucchi U, Orrenius C, Polucci P, Ballinari D, Perrera C, Leone A, Cervi G, Casale E, Xiao Y, Wong C, Anderson DJ, Galvani A, Donati D, O"Brien T, Jackson PK, Isacchi A. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol. 2013 Sep;9(9):548-56. doi: 10.1038/nchembio.1313. Epub 2013 Jul 28.
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先提取RNA,反转录成cDNA,然后根据目的基因设计PCR引物,通过半定量RT-PCR确定目的基因表达.
正因为检测的是mRNA,所以要先反转录成cDNA才能PCR.
1.将外源基因插入慢病毒载体
2.将构建完成的载体与慢病毒包装质粒混合，共转染靶细胞
3.收集病毒液
4.用病毒液感染靶细胞
5.用载体上带的抗生素进行筛选，如果没有，可以用无限稀释法
6.获得稳转株
一种是脂质体转染后,单克隆筛选稳定细胞株.另外一种是应用逆转录病毒,慢病毒转染,筛选稳定细胞株.
脂质体转染：在转染24小时后,消化细胞并计数.将细胞种到96孔板,保证每个孔2-3个细胞,这样才能得到单克隆.待细胞贴壁后,加入抗生素筛选.筛选时间和浓度视细胞而定.一般G418一个星期作用,嘌呤霉素2-3天.
脂质体法筛单克隆时间较长,且效率低,大概只有1%.
病毒转染：先要用包装细胞,一般为293细胞,包装出病毒,再用病毒转染目的细胞.包装病毒视不同类型的病毒而定,一般要3-5天的时间.包装好的病毒要测滴度,根据滴度决定转染目的细胞的病毒量.转染目的细胞1-2天后加抗生素筛选得到稳定细胞株.
病毒转染得到稳定细胞株的效率高,只是步骤繁琐.
细胞转染是指将外源分子如DNA，RNA等导入真核细胞的技术。随着分子生物学和细胞生物学研究的不断发展，转染已经成为研究和控制真核细胞基因功能的常规工具。在研究基因功能、调控基因表达、突变分析和蛋白质生产等生物学试验中，其应用越来越广泛。
方法
脂质体转染法
阳离子脂质体表面带正电荷，能与核酸的磷酸根通过静电作用，将DNA分子包裹入内，形成DNA脂复合物，也能被表面带负电的细胞膜吸附，再通过融合或细胞内吞进入细胞。脂质体转染适用于把DNA转染入悬浮或贴壁培养细胞中，是目前实验室最方便的转染方法之一，其转染率较高，优于磷酸钙法。由于脂质体对细胞有一定的毒性，所以转染时间一般不超过24小时。常用细胞类型：cos-7 、BHK、NIH3T3 、Hela等。
电穿孔转染法
电流能够可逆地击穿细胞膜形成瞬时的水通路或膜上小孔促使DNA分子进入胞内，这种方法就是电穿孔。当遇到某些脂质体转染效率很低或儿乎无法转入时建议用电穿孔法转染。一般情况下，高电场强度会杀死50%-70% 的细胞。现在针对细胞死亡开发出了一种电转保护剂，可以大大的降低细胞的死亡率，同时提高电穿孔转染效率。
病毒感染
对于脂质体转染与电穿孔转染都无法成功转染的细胞系建议用病毒感染，此法可以快速100%感染，检测成功率高。
常用步骤
1. 转染试剂的准备
① 将400ul去核酸酶水加入管中，震荡10秒钟，溶解脂状物。
② 震荡后将试剂放在－20摄氏度保存，使用前还需震荡。
2. 选择合适的混合比例(1：1－1：2/脂质体[1]体积：DNA质量)来转染细胞。在一个转染管中加入合适体积的无血清培养基。加入合适质量的MyoD或者EGFP的DNA，震荡后在加入合适体积的转染试剂，再次震荡。
3. 将混合液在室温放置10―15分钟。
4. 吸去培养板中的培养基，用PBS或者无血清培养基清洗一次。
5. 加入混合液，将细胞放回培养箱中培养一个小时。
6. 到时后，根据细胞种类决定是否移除混合液，之后加入完全培养基继续培养24－48小时。
建立稳定细胞株,一般是根据不同基因载体中所含有的抗性标志选用相应的药物对靶...专注整体实验·服务生命科学已开展了数千个实验外包项目
由于质粒的不兼容性,拥有同种复制子的质粒不能在同一细胞内稳定共存,经过几代的复制,会质粒丢失,所以并不是任何两个或两个以上质粒都可以在同一细胞内稳定存在,但是可以同时进入.
稳定转染的细胞株，就是转染后质粒可以稳定整合到基因组上不会因细胞分裂而丢失。区别于质粒瞬时转染不能长时间保留质粒在细胞里。
