E-64
(Synonyms: N-(反式-环氧丁二酰基)-L-亮氨酸-4-胍基丁基酰胺,Proteinase inhibitor E 64) 目录号 : GC13418
E-64是一种有效的不可逆的半胱氨酸蛋白酶抑制剂，对木瓜蛋白酶的IC50为9nM。
Cas No.:66701-25-5
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
Cell lines | MDA-MB-231 cells |
Preparation Method | Confluent MDA-MB-231 cells were treated with E-64 concentrations ranging from 0 to 50μM for 24h. Cells were lysed and subjected to multiplex cathepsin zymography to detect changes in the amount of active material. |
Reaction Conditions | 0-50μM; 24h |
Applications | E-64 treatment significantly increased the amount of active cathepsin S, while significantly decreased the amount of cathepsin L in cell lysates. |
| Animal experiment [2]: | |
Animal models | Dahl Salt Sensitive rats (SS/JrHsdMcwi) |
Preparation Method | Dahl Salt Sensitive rats (SS/JrHsdMcwi) had their left femoral artery and vein catheterized. Both catheters were fixed and exteriorized from the back of the neck and the arterial line was connected to a heparinized saline infusion pump that was in line with a blood pressure transducer, and the venous line was connected to a saline infusion pump. Animals were allowed 360° movement using a tether-swivel system. A stable baseline blood pressure was obtained for 4 days prior to switching both groups to an 8.0% NaCl diet and the simultaneous addition of N-[N-(L-3-trans-carboxyox-irane-2-carbonyl)-L-leucyl]-agmatine (E-64, 1mg/day; 280mM stock in DMSO) or the vehicle control to the venous catheter. Daily mean arterial pressure (MAP) was calculated by averaging MAP taken every min over the beginning 3 h period of the rat sleep cycle. |
Dosage form | 1mg/day; i.v. |
Applications | A significant increase in the renal cortical mature form of Cath B and Cath L were measured in E-64 treated rats. |
References: [1]Wilder C L, Walton C, Watson V, et al. Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells[J]. The international journal of biochemistry & cell biology, 2016, 79: 199-208. [2]Blass G, Levchenko V, Ilatovskaya D V, et al. Chronic cathepsin inhibition by E‐64 in Dahl salt‐sensitive rats[J]. Physiological reports, 2016, 4(17): e12950. | |
E-64 is a potent and irreversible inhibitor of cysteine proteases with an IC50 of 9nM for papain[1]. E-64 can inhibit the cysteine proteases cathepsin B, H, L and papain, but has no effect on serine proteases or metalloproteinases[2]. E-64 has antiparasitic activity in vitro and can induce oxidative stress and apoptosis in filarial parasites[3]. E-64 can improve the preimplantation development of bovine somatic cell nuclear transfer embryos[4].
In vitro, E-64 (0-50μM) treatment of breast cancer MDA-MB-231 cells for 24h increased the amount of intracellular active cathepsin S and reduced the amount of cathepsin L in a dose-dependent manner[5]. E-64 (4mM) treatment of porcine alveolar macrophages (PAM) for 48h upregulated the mRNA levels of IFNγ, IL-12 and IFN-α in cells without cytotoxicity[6].
In vivo, daily intravenous infusion of E-64 (1mg) in Dahl salt-sensitive rats significantly increased the mature forms of the lysosomal proteases Cath B and Cath L in the renal cortex of rats, but had no effect on high-salt diet-induced hypertension and renal damage[7].
References:
[1] Matsumoto K, Mizoue K, Kitamura K, et al. Structural basis of inhibition of cysteine proteases by E‐64 and its derivatives[J]. Peptide Science, 1999, 51(1): 99-107.
[2] Barrett A J, Kembhavi A A, Brown M A, et al. L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L[J]. Biochemical Journal, 1982, 201(1): 189-198.
[3] Wadhawan M, Singh N, Rathaur S. Inhibition of cathepsin B by E-64 induces oxidative stress and apoptosis in filarial parasite[J]. PLoS One, 2014, 9(3): e93161.
[4] Min S H, Song B S, Yeon J Y, et al. A cathepsin B inhibitor, E-64, improves the preimplantation development of bovine somatic cell nuclear transfer embryos[J]. Journal of Reproduction and Development, 2014, 60(1): 21-27.
[5] Wilder C L, Walton C, Watson V, et al. Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells[J]. The international journal of biochemistry & cell biology, 2016, 79: 199-208.
[6] Liu B, Cui Y, Lu G, et al. Small molecule inhibitor E-64 exhibiting the activity against African swine fever virus pS273R[J]. Bioorganic & Medicinal Chemistry, 2021, 35: 116055.
[7] Blass G, Levchenko V, Ilatovskaya D V, et al. Chronic cathepsin inhibition by E‐64 in Dahl salt‐sensitive rats[J]. Physiological reports, 2016, 4(17): e12950.
E-64是一种有效的不可逆的半胱氨酸蛋白酶抑制剂，对木瓜蛋白酶的IC50为9nM[1]。E-64能够抑制半胱氨酸蛋白酶组织蛋白酶B、H、L和木瓜蛋白酶，但是对丝氨酸蛋白酶或金属蛋白酶没有作用[2]。E-64具有体外抗寄生虫活性，可以诱导丝虫寄生虫氧化应激和细胞凋亡[3]。E-64能够改善牛体细胞核移植胚胎的植入前发育[4]。
在体外，E-64（0-50μM）处理乳腺癌MDA-MB-231细胞24h，以剂量依赖性方式升高了细胞内活性组织蛋白酶S的量，减少了组织蛋白酶L的量[5]。E-64（4mM）处理猪肺泡巨噬细胞（PAM）48h，上调了细胞中IFNγ、IL-12和IFN-α的mRNA水平，且没有细胞毒性[6]。
在体内，E-64（1mg）通过每天静脉导管输注治疗Dahl盐敏感大鼠，显著增加了大鼠体内肾皮质成熟形式的溶酶体蛋白酶Cath B和Cath L，但是对高盐饮食诱导的高血压和肾损害没有效果[7]。
| Cas No. | 66701-25-5 | SDF | |
| 别名 | N-(反式-环氧丁二酰基)-L-亮氨酸-4-胍基丁基酰胺,Proteinase inhibitor E 64 | ||
| 化学名 | (2S,3S)-3-[[(2S)-1-[4-(diaminomethylideneamino)butylamino]-4-methyl-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic acid | ||
| Canonical SMILES | CC(C)CC(C(=O)NCCCCN=C(N)N)NC(=O)C1C(O1)C(=O)O | ||
| 分子式 | C15H27N5O5 | 分子量 | 357.41 |
| 溶解度 | ≥ 53.6mg/mL in DMSO | 储存条件 | Store at 2-8°C |
| General tips | 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；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.7979 mL | 13.9895 mL | 27.9791 mL |
| 5 mM | 0.5596 mL | 2.7979 mL | 5.5958 mL |
| 10 mM | 0.2798 mL | 1.399 mL | 2.7979 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes
Genes (Basel) 2022 Feb 10;13(2):324.35205369 PMC8872247
Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.
Small molecule inhibitor E-64 exhibiting the activity against African swine fever virus pS273R
Bioorg Med Chem 2021 Apr 1;35:116055.33607487 10.1016/j.bmc.2021.116055
African swine fever (ASF) is a viral disease in swine that results in high mortality in domestic pigs and causes considerable economic losses. Currently, there is no effective vaccine or drugs available for treatment. Identification of new anti-ASFV drugs is urgently needed. Here, the pS273R protein of the African swine fever virus (ASFV) is a specific SUMO-1-like cysteine protease that plays an important role in its replication process. To inhibit virus replication and improve treatment options, a set of small-molecule compounds, targeted inhibitors against the ASFV pS273R protease, were obtained through molecular screening by homology modeling and molecular docking based on structural information of pS273R. Our results clearly demonstrated that the 14th carbon atom of the cysteinase inhibitor E-64 could form one CS covalent bond with the Cys 232 amino acid of the pS273R protease and seven additional hydrogen bonds to maintain a stable binding state. Simultaneously, cell viability, immunophenotyping, and in vitro enzyme activity inhibition assays were performed to comprehensively evaluate E-64 characteristics. Our findings demonstrated that 4 mmol/L E-64 could effectively inhibit the enzyme activity center of the pS273R protease by preventing pS273R protease from lysing pp62, while promoting the upregulation of immune-related cytokines at the transcription level. Moreover, cell viability results revealed that 4 mmol/L E-64 was not cytotoxic. Taken together, we identified a novel strategy to potentially prevent ASFV infection in pigs by blocking the activity of pS273R protease with a small-molecule inhibitor.
Chronic cathepsin inhibition by E-64 in Dahl salt-sensitive rats
Physiol Rep 2016 Sep;4(17):e12950.27597769 PMC5027357
Cysteine cathepsins are lysosomal enzymes expressed in the kidneys and other tissues, and are involved in the maturation and breakdown of cellular proteins. They have been shown to be integrally involved in the progression of many cardiovascular and renal diseases. The goal of this study was to determine the involvement of cysteine cathepsins in the development of salt-sensitive hypertension and associated kidney damage. In our experiments, Dahl salt-sensitive (SS) rats were fed an 8% high salt NaCl diet and intravenously infused with the irreversible cysteine cathepsin inhibitor E-64 (1 mg/day) or the vehicle (control). Both the control and E-64 infused groups developed significant hypertension and kidney damage, and no difference of the mean arterial pressure and the hypertension-associated albuminuria was observed between the groups. We next tested basal calcium levels in the podocytes of both control and infused groups using confocal calcium imaging. Basal calcium did not differ between the groups, indicative of the lack of a protective or aggravating influence by the cathepsin inhibition. The efficacy of E-64 was tested in Western blotting. Our findings corresponded to the previously reported, E-64 induced increase in cathepsin B and L abundance. We conclude that the inhibition of cysteine cathepsins by E-64 does not have any effects on the blood pressure development and kidney damage, at least under the studied conditions of this model of SS hypertension.
Crystal structure of an actinidin-E-64 complex
Biochemistry 1992 Jun 9;31(22):5172-6.1606141 10.1021/bi00137a012
E-64, 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane, is a potent and highly selective irreversible inhibitor of cysteine proteases. The crystal structure of a complex of actinidin and E-64 has been determined at 1.86-A resolution by using the difference Fourier method and refined to an R-factor of 14.5%. The electron density map clearly shows that the C2 atom of the E-64 epoxide ring is covalently bonded to the S atom of the active-site cysteine 25. The charged carboxyl group of E-64 forms four H-bonds with the protein and thus may play an important role in favorably positioning the inhibitor molecule for nucleophilic attack by the active-site thiolate anion. The interaction features between E-64 and actinidin are very similar to those seen in the papain-E-64 complex; however, the amino-4-guanidinobutane group orients differently. The crystals of the actinidin-E-64 complex diffracted much better than the papain-E-64 complex, and consequently the present study provides more precise geometrical information on the binding of the inhibitor. Moreover, this study provides yet another confirmation that the binding of E-64 is at the S subsites and not at the S' subsites as has been previously proposed. The original actinidin structure has been revised using the new cDNA sequence information.
Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo
J Biochem 1980 Dec;88(6):1805-11.7462205 10.1093/oxfordjournals.jbchem.a133155
The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).