loadingProduct Data?SheetAloxistatinCat. No.:CAS No.:分?式:分?量:作?靶点:作?通路:储存?式:HY-10022988321-09-9C??H??N?O?342.43Cathepsin; SARS-CoVMetabolic Enzyme/Protease; Anti-infectionPowderIn solvent-20°C4°C-80°C-20°C3 years2 years6 months1 monthInhibitors?Agonists?Screening Libraries溶解性数据体外实验Ethanol : ≥ 33.33 mg/mL (97.33 mM)DMSO : ≥ 23 mg/mL (67.17 mM)* \Mass1 mg5 mg10 mgSolventConcentration制备储备液1 mM5 mM10 mM2.9203 mL0.5841 mL0.2920 mL14.6015 mL2.9203 mL1.4602 mL29.2030 mL5.8406 mL2.9203 mL请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；?旦配成溶液，请分装保存，避免反复冻融造成的产品失效。储备液的保存?式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个?内使?，-20°C 储存时，请在 1 个?内使?。体内实验请根据您的实验动物和给药?式选择适当的溶解?案。以下溶解?案都请先按照 In Vitro ?式配制澄清的储备液，再依次添加助溶剂： 为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的?作液，建议您现?现配，当天使?； 以下溶剂前显?的百分?是指该溶剂在您配制终溶液中的体积占?；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的?式助溶1. 请依序添加每种溶剂：?10% DMSO ?? 40% PEG300 ?? 5% Tween-80 ?? 45% salineSolubility: ≥ 2.5 mg/mL (7.30 mM); Suspended solution此?案可获得 ≥ 2.5 mg/mL (7.30 mM，饱和度未知) 的均匀悬浊液，悬浊液可?于?服和腹腔注射。 以 1 mL ?作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加? 50 μL Tween-80，混合均匀；然后继续加? 450 μL ?理盐?定容? 1 mL。2. 请依序添加每种溶剂：?10% DMSO ?? 90% corn oilSolubility: ≥ 2.5 mg/mL (7.30 mM); Clear solutionPage 1 of 2 www.MedChemExpress.cn
此?案可获得 ≥ 2.5 mg/mL (7.30 mM，饱和度未知) 的澄清溶液，此?案不适?于实验周期在半个?以上的实验。 以 1 mL ?作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL ??油中，混合均匀。
3. 请依序添加每种溶剂：?10% EtOH ?? 40% PEG300 ?? 5% Tween-80 ?? 45% salineSolubility: ≥ 2.5 mg/mL (7.30 mM); Clear solution
此?案可获得 ≥ 2.5 mg/mL (7.30 mM，饱和度未知) 的澄清溶液。
以 1 mL ?作液为例，取 100 μL 25.0 mg/mL 的澄清 EtOH 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加? 50 μL Tween-80，混合均匀；然后继续加? 450 μL ?理盐?定容? 1 mL。
4. 请依序添加每种溶剂：?10% EtOH ?? 90% (20% SBE-β-CD in saline)Solubility: ≥ 2.5 mg/mL (7.30 mM); Clear solution
此?案可获得 ≥ 2.5 mg/mL (7.30 mM，饱和度未知) 的澄清溶液。
以 1 mL ?作液为例，取 100 μL 25.0 mg/mL 的澄清 EtOH 储备液加到 900 μL 20% 的 SBE-β-CD ?理盐??溶液中，混合均匀。
5. 请依序添加每种溶剂：?10% EtOH ?? 90% corn oilSolubility: ≥ 2.5 mg/mL (7.30 mM); Clear solution
此?案可获得 ≥ 2.5 mg/mL (7.30 mM，饱和度未知) 的澄清溶液，此?案不适?于实验周期在半个?以上的实验。 以 1 mL ?作液为例，取 100 μL 25.0 mg/mL 的澄清 EtOH 储备液加到 900 μL ??油中，混合均匀。
BIOLOGICAL ACTIVITY
?物活性
Aloxistatin (E64d) 是可渗透细胞,不可逆的?谱半胱氨酸蛋?酶 (cysteine protease) 抑制剂。Aloxistatin (E64d) 可抑制 MERS-CoV。
IC?? & Target体外研究
Cysteine protease[1]
Inhibition of protease-resistant prion protein (PrP-res) accumulation in ScNB cells by cysteine protease inhibitor Aloxistatin (E64d) with IC50 of 0.5±0.11 μM. For the cell surface PrP-sen detection, PrP-sen is immunoprecipitated from media treated with phosphatidylinositol-specific phospholipase C (PIPLC) to release pulse-35S-labeled PrP-sen from the cell surface. Aloxistatin is maintained at 15 μM, respectively, in the labeling media of all but the control cells
[1]. Aloxistatin (E64d) (which specifically blocks cysteine proteases, but not serine proteases such as granzymes) is able
to completely block turnover of the CatL substrate Z-Phe-Arg-aminomethylcoumarin, when pre-incubated with NK-92 or YT 5 cells[2]. Aloxistatin (E64d) is a broad-spectrum cell-permeable inhibitor of cysteine proteases[3].
体内研究
Oral administration of Aloxistatin (E64d) to guinea pigs results in a dose-dependent reduction in brain, CSF and plasma Aβ(40) and Aβ(42). Aloxistatin also causes a biphasic dose-dependent reduction in brain CTFβ. Aloxistatin causes a dose-dependent increase in brain sAβPPα. The mean sAβPPα levels are significantly higher than the no dose group for Aloxistatin doses of 5 mg/kg/day or greater with the highest Aloxistatin dose resulting in the maximum increase in sAβPPα of about 54% more than the control group. Similar to the Aβ effect, oral Aloxistatin administration produces a biphasic dose-dependent reduction in brain cathepsin B activity. The minimum effective dose is about 1 mg/kg/day with the highest Aloxistatin dose causing the maximum reduction in brain cathepsin B activity of about 91% lower than that of the control group. Thus, Aloxistatin reduces guinea pig brain cathepsin B activity in a manner which is consistent with the compound inhibiting cathepsin B β-secretase activity[4]. Aloxistatin (E64d) inhibits the increases in the expression of AT1AR and ACE genes in rats. Administration of Olmesartan or Aloxistatin reduces the increase in the superoxide production of the intramyocardial coronary arteries in HF rats[5].
PROTOCOL
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Kinase Assay [2]The CTLs and NK cells (0.8×106/mL) are treated with the inhibitors L1 (10-20 μM) or Aloxistatin (20-30 μM) for 24 hr at 37°C in 24-well plates. Cells are then used in 51Cr-release assays or are lysed to examine perforin in Western blots. The inhibitor is also added at the same concentration during the 4 hr reactions in some 51Cr-release assays, as indicated. Cell lysates are prepared using NP-40 lysis buffer (25 mM HEPES, 250 mM NaCl, 2.5 mM
ethylenediaminetetraacetic acid, 0.1% volume/volume Nonidet P-40) and total protein concentration is determined using the Bradford assay. Equal amounts of protein are loaded and resolved on 8% SDS-PAGE gels. Human or mouse perforin is detected using the appropriate antibodies as indicated. Anti-actin antibody is used as a loading control[2].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Cell Assay [3]
Cell proliferation and apoptosis are assessed by staining for a proliferation marker, Ki67, or an apoptotic marker, cleaved caspase 3, following the protocol described above for the polarity markers. MCF10 variants are grown in 3D rBM overlay cultures for 4 days and are treated with 0.1 % DMSO, 5 μM CA074Me or 5 μM Aloxistatin. The percentage of structures that are positive for Ki67 or cleaved caspase 3 is determined by counting a total of 100 structures on two separate coverslips with a Zeiss Axiophot epifluorescent microscope. Structures are considered Ki67 positive if they contained at least one cell staining for Ki67. Structures are considered to be caspase 3 positive if they contained at least one cell that is positive for cleaved caspase 3 and the positive cell(s) is not localized in the center of a developing lumen[3].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Animal
Administration [4][5]
Mice and Pigs[4]
Guinea Pigs (male, Hartley strain, average weight 400 g corresponding to animals about 6 weeks old) are used. Male transgenic mice expressing human AβPP containing the wt β-secretase site and the London mutant β-secretase site sequences are used. Delivering a drug by gavage offers the advantage of accurate dosing but is traumatic and thus only suitable for relatively short dosing periods (up to about a week). Delivery by gavage is used for the guinea pig studies. Aloxistatin is suspended in Me2SO at the indicated concentrations (0.1, 1.0, 5, and 10 mg/kg) and administered by gavage daily using a feeding tube. Vehicle control animals are treated by gavage of Me2SO alone. Rats[5]
Male inbred DS rats are used. Weaned rats are fed laboratory chow containing 0.3% NaCl until 7 weeks of age. DS rats fed an 8% NaCl diet after 7 weeks manifest compensated concentric left ventricular (LV) hypertrophy
secondary to hypertension at 12 weeks and a distinct stage of fatal LV failure with lung congestion at 19 weeks. DS rats are therefore fed an 8% NaCl diet from 7 weeks of age and are randomized to an HF group, an Aloxistatin group (10 mg per kg of body mass per day, administered intraperitoneally every other day), or an olmesartan group (3 mg/kg per day in chow) from 12 to 19 weeks of age (n=10 for each group). The doses of olmesartan (an ARB) and Aloxistatin are determined in preliminary experiments and previous studies. DS rats maintained on the 0.3% NaCl diet served as age-matched controls (control group, n=10). At 19 weeks of age, all of the rats are euthanized by an intraperitoneal overdose of sodium pentobarbital (50 mg/kg), and the hearts are removed for biological and histological analyses. Arterial blood is collected from the abdominal aorta for the measurement of renin activity. Systolic blood pressure and heart rate are measured in conscious rats from 7 weeks of age, every week, using a noninvasive tail-cuff method. In separate experiments, 12-week-old DS rats, fed a low-salt diet from 7 weeks of age, are given vehicle, olmesartan, or Aloxistatin in the same manner as in the above experiments (n=5 for each group), and the LV tissues for measuring targeting mRNAs and protein levels are immediately placed in liquid nitrogen and stored at -80°C.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
客户使?本产品发表的科研?献
????Nat Commun. 2020; 11: 1620.
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????Environ Sci Technol. 2017 Dec 5;51(23):13938-13948.????Vaccines (Basel).?2020 Jan 13;8(1). pii: E22.?????Toxicol Appl Pharmacol. 2018 Oct 1;356:159-171.????PLoS One.?2019 Dec 30;14(12):e0227278.
See more customer validations on www.MedChemExpress.cnREFERENCES
[1].?Doh-Ura K, et al. Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol. 2000 May;74(10):4894-7.
[2].?Konjar S, et al. Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L. Immunology. 2010 Oct;131(2):257-67.
[3].?Mullins SR, et al. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins. Biol Chem. 2012 Dec;393(12):1405-16.
[4].?Hook G, et al. The cysteine protease inhibitor, E64d, reduces brain amyloid-β and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B, but not BACE1, β-secretase activity. J Alzheimers Dis. 2011;26(2):387-408.
[5].?Cheng XW, et al. Superoxide-dependent cathepsin activation is associated with hypertensive myocardial remodeling and represents a target for angiotensin II type 1 receptor blocker treatment. Am J Pathol. 2008 Aug;173(2):358-69.
[6].?Ji Yeun Kim, et al. Safe, High-Throughput Screening of Natural Compounds of MERS-CoV Entry Inhibitors Using a Pseudovirus Expressing MERS-CoV Spike Protein. Int J Antimicrob Agents. 2018 Nov;52(5):730-732.
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