E-64

Papain (IC50 = 9 nM)

体外活性：E-64快速抑制半胱氨酸蛋白酶组织蛋白酶B、H和L以及木瓜蛋白酶的活性，而对丝氨酸蛋白酶或金属蛋白酶没有影响。 E-64作为半胱氨酸蛋白酶的特异性抑制剂，在体外有效阻断卵巢癌细胞的侵袭。此外，E-64 还在体外表现出针对兰氏贾第鞭毛虫排泄的抗寄生虫活性。 E-64改善了牛体细胞核移植胚胎的着床前发育，这表明了E-64的另一个重要意义。激酶测定：使用 Z-Arg-Arg-4mβNA 作为底物并稍加修改来测定组织蛋白酶 B 活性。将粗提取物在含有 1 mM EDTA 和 5 mM DTT 的 50 mM 乙酸钠缓冲液（pH 5.0）中于 37°C 预孵育 5 分钟。添加底物（最终浓度，100 μM）以使最终测定体积为 1 mL。将反应混合物在 37°C 下孵育 30 分钟。通过添加等体积的含有 Fast Garnet GBC 盐 (1 mg/mL)、10 mM pHMB 和 50 mM EDTA（pH 6.0）的终止试剂来终止反应。用正丁醇萃取产物β-萘胺(β-NA)。完全层分离后，测量正丁醇层的吸光度，并使用 520 nm 处 31.5 M/cm/秒的 β-萘胺溶液摩尔消光系数计算活性。 1个酶活力单位定义为37℃下每分钟释放1μmol βNA的酶量。通过绘制不同浓度的 E-64 和组织蛋白酶 B 活性抑制百分比之间的图表来计算半数最大抑制浓度 (IC50)。这里，IC50表示将寄生组织蛋白酶B活性抑制一半所需的E-64浓度。细胞检测：E-64以剂量依赖性方式抑制H-59侵袭，在10μg/ml浓度时最大抑制率为97%，且无毒。用涂有 7.5 μg/过滤器 IV 型胶原蛋白的过滤器测量的细胞迁移仅减少了 25%，这表明在没有基底膜屏障的情况下，半胱氨酸蛋白酶在细胞迁移中发挥较小的作用。另一方面，即使浓度高达 100 μg/ml，E-64 处理也不会显着影响 M-27 侵袭。

E-64可显着减少M5076荷瘤小鼠的自发转移数量，但对实验性转移的形成没有影响。 E-64 还对感染小鼠中的贾第鞭毛虫排泄表现出抗寄生虫活性。

Z-Arg-Arg-4mβNA 经过一些修改，用作测量组织蛋白酶 B 活性的底物。50 mM 乙酸钠缓冲液，pH 5.0，含有 1 mM EDTA 和 5 mM DTT，与粗提物在 37°C 下反应 5 分钟。通过添加底物（最终浓度，100 μM）将测定体积增加至 1 mL。将反应混合物在 37°C 下孵育三十分钟。添加等体积的含有 50 mM EDTA、pH 6.0 和 10 mM pHMB、Fast Garnet GBC 盐 (1 mg/mL) 的终止试剂以终止反应。萃取过程中使用正丁醇产生产物β-萘胺（β-NA）。各层完全分离后，测量正丁醇层的吸光度，并使用 β-萘胺溶液的摩尔消光系数计算活性，该系数在 520 nm 处为 31.5 M/cm 每秒。酶的活性以 37°C 下每分钟释放 1 μmol βNA 为单位进行测量。绘制不同 E-64 浓度和组织蛋白酶 B 活性抑制百分比之间的图表，得出半数最大抑制浓度或 IC50。这里，IC50 表示将寄生组织蛋白酶 B 活性降低一半所需的 E-64 浓度[2]。

E-64对H-59入侵表现出剂量依赖性抑制作用，在无毒浓度10μg/ml时达到97%的最大抑制率。当使用涂有 7.5 μg/滤膜 IV 型胶原蛋白的滤膜测量细胞迁移时，减少幅度仅为 25%，表明在没有基底膜屏障的情况下，半胱氨酸蛋白酶对细胞迁移的影响相对不显着。然而，即使浓度高达 100 μg/ml，E-64 处理也不会显着改变 M-27 侵袭。

Mice: The effects of treatment with anti-serpin B13 monoclonal antibody (mAb) are investigated in NOD/LtJ and BDC2.5 T cell receptor (TCR) transgenic mice. 100 μg of anti-serpin B13 mAb is injected intravenously four times over a ten-day period into four-week-old female NOD/LtJ mice. Furthermore, during the same time frame, a few animals receive intraperitoneal injections of the protease inhibitor E64 at a daily dose of 10 mg/kg for a few days. Diluent, a sterile PBS solution containing 10% DMSO, is administered to control mice along with control IgG. Before being used, the solutions containing DMSO or E64 are quickly made. When the mice are killed 24 hours after the last injection, cells from their pancreatic islets and lymphoid organs are subjected to FACS analysis.
Rats: Male Dahl Salt Sensitive rats (SS/JrHsdMcwi) aged seven weeks are utilized. In summary, the left femoral artery and vein of eight-week-old anesthetized SS rats are catheterized. The arterial line is connected to a heparinized saline infusion pump that is in line with a blood pressure transducer, and the venous line is connected to a saline infusion pump. Both catheters are fixed and exteriorized from the back of the neck. Animals can move 360 degrees thanks to a tether-swivel system. Rats that were conscious and able to move around were prepared to receive a chronic venous infusion and have their arterial blood pressure measured. Four days are needed to establish a stable baseline blood pressure before introducing E-64 (1 mg/day; 280 mM stock in DMSO) or the vehicle (DMSO in saline) control at the same time to the venous catheter in both groups. The daily MAP is computed by taking an average of the MAP measurements made every minute during the first three hours of the rat sleep cycle.

[1]. Structural basis of inhibition of cysteine proteases by E-64 and its derivatives. Biopolymers. 1999;51(1):99-107.

[2]. Inhibition of cathepsin B by E-64 induces oxidative stress and apoptosis in filarial parasite. PLoS One. 2014 Mar 25;9(3):e93161.

[3]. Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes. J Biol Chem. 2013 Jan 18;288(3):1612-9.

[4]. Chronic cathepsin inhibition by E-64 in Dahl salt-sensitive rats. Physiol Rep. 2016 Sep;4(17). pii: e12950.

C15H27N5O5

357.41

357.20

C, 50.41; H, 7.61; N, 19.60; O, 22.38

66701-25-5

White to off-white solid powder

-1.46

169.930

CC(C)C[C@@H](C(=O)NCCCCN=C(N)N)NC(=O)[C@@H]1[C@H](O1)C(=O)O

LTLYEAJONXGNFG-DCAQKATOSA-N

InChI=1S/C15H27N5O5/c1-8(2)7-9(20-13(22)10-11(25-10)14(23)24)12(21)18-5-3-4-6-19-15(16)17/h8-11H,3-7H2,1-2H3,(H,18,21)(H,20,22)(H,23,24)(H4,16,17,19)/t9-,10-,11-/m0/s1

(2S,3S)-3-[[(2S)-1-[4-(diaminomethylideneamino)butylamino]-4-methyl-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic acid

E 64; E64; E-64

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.99 mM) (saturation unknown) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 50 mg/mL (139.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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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。