E-64 (Proteinase inhibitor E 64) is a potent irreversible inhibitor against general cysteine proteases with IC₅₀ of 9 nM for papain.

IC50: 9 nM (Papain)^[1]

E-64 (Proteinase inhibitor E 64) is a cathepsin B-specific inhibitor, and its binding modes with papain, actinidin, cathepsin L, and cathepsin K have been reviewed at the atomic level. E-64 has been widely used as a potent and irreversible (covalent-type) inhibitor for many cysteine proteases such as papain, ficin, actinidin, cathepsin B and L^[1]. The S.cervi adult parasites are incubated in the Kreb’s Ringer bicarbonate (KRB) maintenance medium for 8 h at 37°C, 5% CO₂ with 5, 10, 20 and 40 μM concentration of E-64. E-64 shows a concentration and time dependent decrease in motility and viability of the parasites (EC₅₀=16 μM)^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

A broad spectrum of expression of CD4 and CD19 is found exists in both the islets and pancreatic lymph nodes (PLNs) and that anti-serpin B13 mAb exposure causes a significant shift that favored cells expressing low-to-intermediate amounts of these markers. However, this shift is abolished in animals that receive anti-serpin B13 mAb in the presence of the protease inhibitor E-64 (Proteinase inhibitor E 64), which maintains its blocking activity under the experimental conditions used^[3]. Dahl salt-sensitive (SS) rats are 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 develope significant hypertension and kidney damage, and no difference of the mean arterial pressure and the hypertension-associated albuminuria is observed between the groups^[4].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

357.41

C₁₅H₂₇N₅O₅

66701-25-5

Room temperature in continental US; may vary elsewhere.

DMSO : 125 mg/mL (349.74 mM; Need ultrasonic)

H₂O : 12.5 mg/mL (34.97 mM; Need ultrasonic)

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, 6 months; -20°C, 1 month。-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。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方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；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    90% saline

  Solubility: ≥ 2.5 mg/mL (6.99 mM); Clear solution
• 2.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.08 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (5.82 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 3.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.08 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (5.82 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 4.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.08 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (5.82 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

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

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

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

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

The Cathepsin B activity is determined using Z-Arg-Arg-4mβNA as substrate with slight modifications. The crude extract is pre-incubated at 37°C for 5 min in 50 mM sodium acetate buffer, pH 5.0 containing 1 mM EDTA and 5 mM DTT. The substrate (final concentration, 100 μM) is added to make the final assay volume of 1 mL. The reaction mixture is incubated at 37°C for 30 min. The reaction is terminated by adding equal volume of stopping reagent containing Fast Garnet GBC salt (1 mg/mL), 10 mM pHMB and 50 mM EDTA, pH 6.0. The extraction of product, β-napthylamine (β-NA), is carried out with n-butanol. After complete layer separation, the absorbance is measured in n-butanol layer and activity is calculated using molar extinction coefficient of β-napthylamine solution as 31.5 M/cm per sec at 520 nm. One unit of enzyme activity is defined as the amount of enzyme liberating 1 μmol of βNA per minute at 37°C. The half maximal inhibitory concentration (IC₅₀) is calculated by plotting the graph between the different concentration of E-64 and the % inhibition in cathepsin B activity. Here, IC₅₀ indicates the concentration of the E-64 required to inhibit the parasitic cathepsin B activity by half^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
The NOD/LtJ and BDC2.5 T cell receptor (TCR) transgenic NOD mice are used to study the effects of treatment with anti-serpin B13 monoclonal antibody (mAb). Four-week-old female NOD/LtJ mice are injected intravenously four times over a period of 10 days with anti-serpin B13 mAb (100 μg/injection). In addition, during the same period, some animals are also injected intraperitoneally with the protease inhibitor E64 at 10 mg/kg/day for several days. Control mice are treated with diluent (a sterilized PBS solution containing 10% DMSO) and control IgG. The solutions containing E64 or DMSO are prepared immediately before use. Twenty-four hours after the last injection, the mice are killed, and cells from their lymphoid organs and pancreatic islets are subjected to FACS analysis.
Rats^[4]
Seven-week old male Dahl Salt Sensitive rats (SS/JrHsdMcwi) are used. Briefly, 8-week old anesthetized SS rats have their left femoral artery and vein catheterized. Both catheters are fixed and exteriorized from the back of the neck and 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. Animals are allowed 360° movement using a tether-swivel system. This preparation allowed chronic venous infusion and arterial blood pressure measurement in conscious, freely moving rats. A stable baseline blood pressure is obtained for 4 days prior to switching both groups to an 8.0% NaCl diet and the simultaneous addition of E-64 (1 mg/day; 280 mM stock in DMSO) or the vehicle (DMSO in saline) control to the venous catheter. Daily MAP is calculated by averaging MAP taken every min over the beginning 3 h period of the rat sleep cycle.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

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

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

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

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