Cathepsin B (Ki = 2 ~ 5 nM )

通过巧妙的药物设计，CA-074 是 E-64 的合成类似物，E-64 是一种天然存在的肽基环氧化物，可不可逆地阻断大多数已知的溶酶体半胱氨酸蛋白酶。利用组织蛋白酶 B 的二肽基羧肽酶活性创建。在活细胞中，CA-074 可用于特异性抑制组织蛋白酶 B，前提是实验设置允许大量的液相药物内吞作用 [2]。组织蛋白酶 B 被 CA-074 抑制，Ki 为 2 至 5 nM，而组织蛋白酶 H 和 L 的初始 Kis 范围为 40 至 200 μM。对于纯化的大鼠组织蛋白酶 B，CA-074 的抑制作用比对组织蛋白酶 H 和 L 的抑制作用强 10,000-30,000 倍 [1]。

腹腔注射化合物CA-074可有效、选择性地抑制大鼠组织蛋白酶B活性[1]。八只脑缺血持续 20 分钟的猴子在受伤后立即接受静脉注射 CA-074，这防止了 67% 的 CA1 神经元在第五天晚些时候死亡。在八只猴子中，三只具有非常好的抑制水平，五只具有良好的抑制[3]。

在体外和体内测试了E-64的新衍生物（化合物CA-030和CA-074）对组织蛋白酶B的选择性抑制作用。它们对纯化的大鼠组织蛋白酶B的抑制作用是对组织酶H和L的抑制作用的10000-30000倍：它们对组织素B的初始Ki值约为2-5nM，与E-64-c的Ki值相似，而它们对组组织蛋白酶H和L的Ki初始值约为40200 microM[1]。

据报道，使用区分组织蛋白酶B和相关溶酶体半胱氨酸蛋白酶的抑制剂的研究表明，该酶参与了广泛的生理和病理过程。在体内选择性抑制组织蛋白酶B最常用的物质是CA-074Me，它是E-64衍生物CA-074的甲酯。然而，我们现在发现CA-074Me使小鼠成纤维细胞内的组织蛋白酶B和组织蛋白酶L失活。相反，这些细胞暴露于亲代化合物CA 074导致内源性组织蛋白酶B的选择性抑制，而细胞内组织蛋白酶L不受影响。这些结果表明，应使用CA-074而不是CA-074Me来特异性灭活活细胞内的组织蛋白酶B[2]。

Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS [3].

[1]. Novel epoxysuccinyl peptides. A selective inhibitor of cathepsin B, in vivo. FEBS Lett. 1991 Mar 25;280(2):311-5.

[2]. CA-074, but not its methyl ester CA-074Me, is a selective inhibitor of cathepsin B within living cells. Biol Chem. 2002 Jul-Aug;383(7-8):1305-8.

[3]. Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'. Eur J Neurosci. 1998 May;10(5):1723-33.

New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10,000-30,000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L: their initial Ki values for cathepsin B were about 2-5 nM, like that of E-64-c, whereas their initial Ki values for cathepsins H and L were about 40 200 microM. In in vivo conditions, such as intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.[1]

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Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA-074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.[2]

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Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.[3]

C18H29N3O6

383.45

383.206

C, 56.38; H, 7.62; N, 10.96; O, 25.03

134448-10-5

147859-80-1

9821383

White to yellow solid

1.267g/cm3

728.9ºC at 760mmHg

394.7ºC

0mmHg at 25°C

1.539

0.606

128.34

3

6

9

27

596

5

O=C(N1[C@@H](CCC1)C(O)=O)[C@@]([C@@H](C)CC)([H])NC([C@@H]2[C@@H](C(NCCC)=O)O2)=O

ZEZGJKSEBRELAS-PEDHHIEDSA-N

InChI=1S/C18H29N3O6/c1-4-8-19-15(22)13-14(27-13)16(23)20-12(10(3)5-2)17(24)21-9-6-7-11(21)18(25)26/h10-14H,4-9H2,1-3H3,(H,19,22)(H,20,23)(H,25,26)/t10-,11-,12-,13-,14-/m0/s1

(2S-trans)-1-(N-((3-((Propylamino)carbonyl)oxiranyl)carbonyl)-L-isoleucyl)-L-proline; (2S)-1-[(2S,3S)-3-Methyl-2-[[(3S)-3-(propylcarbamoyl)oxirane-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carboxylic acid

CA 074; CA074; 134448-10-5; MFCD00797531; CHEMBL490920; Cathepsin B Inhibitor III; (2S)-1-[(2S,3S)-3-methyl-2-{[(2S,3S)-3-(propylcarbamoyl)oxiran-2-yl]formamido}pentanoyl]pyrrolidine-2-carboxylic acid; PrNH-tES-Ile-Pro-OH; CA 074 TFA; CA-074

2934.99.03.00

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)

DMSO : ~125 mg/mL (~326.00 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.42 mM) (饱和度未知) 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), 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400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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: ≥ 2.08 mg/mL (5.42 mM) (饱和度未知) in 10% DMSO + 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 20.8 mg/mL澄清DMSO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: ≥ 2.08 mg/mL (5.42 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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。