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E-64 (also known as Proteinase inhibitor E 64), a naturally occuring compound isolated from cultures of Aspergillus, is a novel, potent, irreversible and selective cysteine protease inhibitor with potential antineoplastic and antiparasitic activity. Its IC50 of 9 nM indicates that it inhibits papain, a cysteine protease. The thiol group of papain vanished when E-64 was demonstrated to inhibit ficin, papain, and the fruit and stem bromelains. Two additional mammalian cysteine proteinases have been shown to be inhibited by E-64: calpain, a calcium-dependent proteinase from chicken muscle, and cathepsin L, a proteinase from human breast tumor tissue. These attributes collectively implied that E-64 could prove to be a useful inhibitor in the field of cysteine proteinase research.
| Targets |
Papain (IC50 = 9 nM)
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| ln Vitro |
E-64 (Proteinase inhibitor E 64) is a cathepsin B-specific inhibitor whose atomic binding modes with actinidin, papain, cathepsin K, and cathepsin L have all been examined. Many cysteine proteases, including papain, ficin, actinidin, cathepsin B, and L, have been effectively and irreversibly inhibited (covalently) by E-64[1]. For eight hours at 37°C and 5% CO2, the adult S. Cervi parasites are cultured in Kreb's Ringer bicarbonate (KRB) maintenance medium containing 5, 10, 20, and 40 μM concentrations of E-64. E-64 exhibits a concentration- and time-dependent decline in the parasites' motility and viability (EC50=16 μM)[2].
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| ln Vivo |
Both the islets and pancreatic lymph nodes (PLNs) exhibit a wide range of CD4 and CD19 expression, and exposure to anti-serpin B13 mAb significantly shifted the balance in favor of cells expressing low-to-intermediate levels of these markers. The protease inhibitor E-64 (Proteinase inhibitor E 64), which retains its blocking activity under the utilized experimental conditions, eliminates this shift in animals that receive anti-serpin B13 mAb[3]. Rats with Dahl salt sensitivity (SS) are given an 8% high-salt NaCl diet and given either the vehicle (control) or the irreversible cysteine cathepsin inhibitor E-64 (1 mg/day) intravenously. Significant hypertension and renal damage occur in both the E-64-infused and control groups, and there is no difference in the groups' mean arterial pressure or albuminuria linked to hypertension[4].
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| Enzyme Assay |
Z-Arg-Arg-4mβNA, with a few modifications, is used as the substrate to measure the activity of Cathepsin B. The 50 mM sodium acetate buffer, pH 5.0, containing 1 mM EDTA and 5 mM DTT, is pre-incubated with the crude extract for 5 minutes at 37°C. The assay volume is increased to 1 mL by adding the substrate (final concentration, 100 μM). The reaction mixture is incubated for thirty minutes at 37°C. An equal volume of stopping reagent containing 50 mM EDTA, pH 6.0, and 10 mM pHMB, Fast Garnet GBC salt (1 mg/mL), is added to end the reaction. N-butanol is used in the extraction process to yield the product, β-napthylamine (β-NA). The absorbance in the n-butanol layer is measured after the layers have completely separated, and the activity is computed using the molar extinction coefficient of the β-napthylamine solution, which is 31.5 M/cm per sec at 520 nm. An enzyme's activity is measured in units of 1 μmol of βNA released per minute at 37°C. Plotting the graph between the various E-64 concentrations and the percentage inhibition in cathepsin B activity yields the half maximal inhibitory concentration, or IC50. Here, IC50 denotes the E-64 concentration needed to reduce parasitic cathepsin B activity by half[2].
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| Cell Assay |
E-64 exhibited a dose-dependent inhibition of H-59 invasion, reaching a maximal inhibition of 97% at a non-toxic concentration of 10 μg/ml. When measuring cell migration using filters coated with 7.5 μg/filter type IV collagen, the reduction was only 25%, indicating that cysteine proteinases had a relatively insignificant effect on cell migration in the absence of a basement membrane barrier. However, treatment with E-64 did not significantly alter M-27 invasion, even at concentrations up to 100 μg/ml.
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| Animal Protocol |
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. |
| References |
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| Additional Infomation |
E64 is an epoxy monocarboxylic acid, a dicarboxylic acid monoamide, a member of guanidines and a L-leucine derivative. It has a role as a protease inhibitor, an antimalarial and an antiparasitic agent. It is a tautomer of an E64 zwitterion.
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| Molecular Formula |
C15H27N5O5
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| Molecular Weight |
357.41
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| Exact Mass |
357.201
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| Elemental Analysis |
C, 50.41; H, 7.61; N, 19.60; O, 22.38
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| CAS # |
66701-25-5
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| Related CAS # |
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| PubChem CID |
123985
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Melting Point |
182ºC
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| Index of Refraction |
1.618
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| LogP |
-1.46
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
25
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| Complexity |
518
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| Defined Atom Stereocenter Count |
3
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| SMILES |
O=C([C@H]1O[C@@H]1C(N[C@H](C(NCCCCNC(N)=N)=O)CC(C)C)=O)O
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| InChi Key |
LTLYEAJONXGNFG-DCAQKATOSA-N
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| InChi Code |
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
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| Chemical Name |
(2S,3S)-3-[[(2S)-1-[4-(diaminomethylideneamino)butylamino]-4-methyl-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic acid
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| Synonyms |
E 64; E64; Proteinase inhibitor E 64; (2S,3S)-3-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid; CHEMBL374508; E 64; CHEBI:30270; E64; E-64
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. View More
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. 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. 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. |
| Preparing Stock Solutions | 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 | 1.3990 mL | 2.7979 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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