Aloxistatin (Loxistatin; E64d, NSC-694281)   中文名称: 阿洛司他丁

Cysteine protease

体外活性：Aloxistatin（也称为 E-64d、E-64c 乙酯；Loxistatin；NSC 694281）是一种有效的、选择性的、不可逆的、广谱的、细胞膜可渗透的半胱氨酸蛋白酶抑制剂。 Aloxistatin 可防止体外雨蛙蛋白诱导的胰蛋白酶原激活。阿洛司他汀可以进入完整细胞并抑制钙蛋白酶。 E-64d 已被证明对于治疗人类阿尔茨海默病是安全的。 Aloxistatin 通过调节异常的锌信号转导以及通过海马中的 ApoE/clusterin 途径调节脂质代谢改变，可潜在用于治疗发育性癫痫诱发的脑损伤。阿洛司他汀可以进入完整的血小板，通过抑制钙蛋白酶来抑制蛋白水解。 Aloxistatin 在体外可抑制甲状旁腺激素 (PTH) 诱导的细胞增殖并抑制成骨细胞的分化。激酶测定：在 24 孔板中，用抑制剂 L1 (10-20 μM) 或阿洛司他丁 (20-30 μM) 在 37°C 下处理 CTL 和 NK 细胞 (0.8×106/mL) 24 小时。然后将细胞用于 51 Cr 释放测定或裂解以在蛋白质印迹中检查穿孔素。如所示，在一些 51 Cr 释放测定中，在 4 小时反应期间也以相同浓度添加抑制剂。使用 NP-40 裂解缓冲液（25 mM HEPES、250 mM NaCl、2.5 mM 乙二胺四乙酸、0.1% 体积/体积 Nonidet P-40）制备细胞裂解物，并使用 Bradford 测定法测定总蛋白浓度。等量的蛋白质上样并在 8% SDS-PAGE 凝胶上解析。使用指定的适当抗体检测人或小鼠穿孔素。抗肌动蛋白抗体用作上样对照。细胞测定：用 A23187 加钙激活血小板，已知这种情况会导致钙蛋白酶催化 ABP 和 talin 蛋白水解。在没有抑制剂的情况下，A23187 导致 ABP 和talin 完全降解。 E 64d，渗透抑制剂，确实抑制细胞内蛋白水解。在最低测试浓度 20 μg/ml 下观察到了一些抑制作用，而在 50 μg/ml 下则获得了基本上完全的抑制作用。

E-64d 治疗后，用青霉素治疗小鼠以诱导复发性癫痫发作。结果表明，E-64d显着减少了齿状回颗粒上区和海马CA3亚区的异常苔藓纤维萌芽。在未经E-64d治疗的大鼠中，在齿状回和CA3亚区的锥体层中存在明显的苔藓纤维末端聚集。在用 E-64d 治疗的大鼠中，齿状回颗粒上区域和 CA3 亚区的苔藓纤维末端聚集显着减少。

将抑制剂 L1 (10–20 μM) 或 Aloxistatin (20–30 μM) 应用于 CTL 和 NK 细胞 (0.8×106/mL)，在 37°C 下在 24 孔板中持续 24 小时。之后，将细胞用于 51 Cr 释放实验或裂解细胞以在蛋白质印迹分析中观察穿孔素。一些 51Cr 释放测定还在 4 小时反应期间添加相同浓度的抑制剂，如图所示。使用 NP-40 裂解缓冲液（25 mM HEPES、250 mM NaCl、2.5 mM 乙二胺四乙酸、0.1% 体积/体积 Nonidet P-40）制备细胞裂解液，并使用 Bradford 法测定总蛋白浓度。将等量的蛋白质上样并解析到 8% SDS-PAGE 凝胶上。按照指示使用正确的抗体来检测人或小鼠穿孔素。使用抗肌动蛋白抗体作为上样对照。

增殖标记物 Ki67 或凋亡标记物 cleaved caspase 3 染色可用于评估细胞增殖和凋亡。极性标记的过程与之前相同。四天后，MCF10 变体在 3D rBM 覆盖培养物中生长，并用 5 μM CA074Me、5 μM Aloxistatin 或 0.1% DMSO 处理。通过使用 Zeiss Axiophot 落射荧光显微镜对两个不同盖玻片上总共 100 个结构进行计数，可以确定 Ki67 或裂解的 caspase 3 呈阳性的结构的百分比。如果一个结构至少有一个 Ki67 染色细胞，则被认为是 Ki67 阳性。当某个结构具有一个或多个裂解 caspase 3 阳性的细胞，并且这些细胞不位于发育管腔的中心时，该结构被称为 caspase 3 阳性[3]。

Mice and Pigs: Male Hartley strain guinea pigs, weighing an average of 400 g, or approximately six weeks old, are used. The London mutant β-secretase site sequences and the wt β-secretase site-containing human AβPP are expressed in male transgenic mice. Although accurate dosage can be achieved by gavage delivery, this method is traumatic and should only be used for brief dosage intervals (up to approximately one week). In the studies using guinea pigs, gavage delivery is utilized. The recommended dosages of aloxistatin (0.1, 1.0, 5, and 10 mg/kg) are suspended in Me2SO and given orally once a day through a feeding tube. Me2SO alone is administered by gavage to vehicle control animals.
Rats: The rats are inbred male DS rats. Up until the age of seven weeks, weaned rats are given laboratory chow containing 0.3% NaCl. DS rats given an 8% NaCl diet for seven weeks show signs of compensating for concentric left ventricular (LV) hypertrophy, which is related to hypertension at twelve weeks. At nineteen weeks, the rats show signs of a distinct stage of fatal LV failure, accompanied by lung congestion. To that end, DS rats are started on an 8% NaCl diet at 7 weeks of age. From 12 to 19 weeks of age, they are randomized into three groups: HF, Aloxistatin (10 mg per kg of body mass per day, administered intraperitoneally every other day), and RNH-6270 (3 mg/kg per day in chow) (n=10 for each group). Preliminary experiments and prior research determine the doses of aloxistatin and RNH-6270, an ARB. Age-matched controls (control group, n = 10) were DS rats fed a diet containing 0.3% NaCl. All of the rats are killed at 19 weeks of age by injecting an excess of 50 mg/kg of NSC 10816 intraperitoneally, and their hearts are taken out for histological and biological examinations. To measure renin activity, arterial blood is drawn from the abdominal aorta. Every week starting at 7 weeks of age, conscious rats have their heart rate and systolic blood pressure measured using a noninvasive tail-cuff technique. In independent studies, n = 5 per group of 12-week-old DS rats fed a low-salt diet starting at 7 weeks of age are given vehicle, RNH-6270, or Aloxistatin in the same way as in the previous studies. The LV tissues used to measure targeting mRNAs and protein levels are then promptly frozen in liquid nitrogen and kept at -80°C.

[1]. J Pharmacobiodyn . 1986 Aug;9(8):672-7.

[2]. Biochem Biophys Res Commun . 1989 Jan 31;158(2):432-5.

[3]. Metabolism . 1997 Sep;46(9):1090-4.

[4]. Ann N Y Acad Sci . 2001 Jun:939:436-49.

[5]. J Alzheimers Dis . 2011;26(2):387-408.

[6]. Nat Commun . 2022 Aug 16;13(1):4804.

Aloxistatin is an L-leucine derivative that is the amide obtained by formal condensation of the carboxy group of (2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid with the amino group of N-(3-methylbutyl)-L-leucinamide. It has a role as a cathepsin B inhibitor and an anticoronaviral agent. It is a L-leucine derivative, a monocarboxylic acid amide, an epoxide and an ethyl ester.
Aloxistatin is an inhibitor of cysteine protease with blood platelet aggregation inhibiting activity. Aloxistatin is an irreversible, membrane-permeable inhibitor of lysosomal and cytosolic cysteine proteases with the ability to inhibit calpain activity in intact platelets.

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E-64 isolated from a culture of Aspergillus japonicus is a specific inhibitor of cysteine proteinases. E-64-c, a synthetic analog of E-64, was effective in model animals of muscular dystrophy only when it was given intraperitoneally and by means of osmotic minipump. It showed no effects due to its low absorbability from intestine when it was administered orally. EST, the ethyl ester of E-64-c, was expected to be readily absorbed through intestinal membrane, since it is more lipophilic than E-64-c. Both EST and E-64-c have a high specificity to cysteine proteinase similar to E-64 but E-64-c was 100 to 1000 times stronger than EST in in vitro cathepsin inhibition. However, EST was stronger than E-64-c in cathepsin inhibition when given orally. The cathepsin B&L activities (whole activities of cathepsins B and L) in the skeletal muscle, heart and liver of hamsters were strongly inhibited soon after oral administration of 100 mg/kg body weight of EST. The inhibition continued for at least 3 h and then disappeared gradually. E-64-c was found in plasma of hamster treated with EST, but unchanged EST was not found. These results suggested that EST was converted to E-64-c, a more active form, during the permeation through intestinal membrane. The conversion of EST to E-64-c was also indicated by the absorption experiment using in situ loop method. EST was thus shown to be useful as an oral drug and expected to be effective in therapeutic trials using model animals.[1]

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E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.[2]

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Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.[3]

C17H30N2O5

342.43

342.215

C, 59.63; H, 8.83; N, 8.18; O, 23.36

88321-09-9

65663

White to off-white solid powder

1.2±0.1 g/cm3

470.5±55.0 °C at 760 mmHg

126.2°C

238.4±31.5 °C

0.0±2.6 mmHg at 25°C

1.530

3.64

97.03

2

5

11

24

450

3

C([C@H]1O[C@@H]1C(=O)OCC)(=O)N[C@@H](CC(C)C)C(=O)NCCC(C)C

SRVFFFJZQVENJC-IHRRRGAJSA-N

InChI=1S/C17H30N2O5/c1-6-23-17(22)14-13(24-14)16(21)19-12(9-11(4)5)15(20)18-8-7-10(2)3/h10-14H,6-9H2,1-5H3,(H,18,20)(H,19,21)/t12-,13-,14-/m0/s1

ethyl (2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.30 mM) (饱和度未知) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 EtOH 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (7.30 mM) (饱和度未知) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清乙醇储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (7.30 mM) (饱和度未知) in 10% EtOH + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清乙醇储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2.08 mg/mL (6.07 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清的DMSO储备液加入400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.08 mg/mL (6.07 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 6 中的溶解度: 2% DMSO+corn oil: 5mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。