Papain (IC50 = 9 nM)

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].

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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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 [4].

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However, this shift was abolished in animals that received anti-serpin B13 mAb in the presence of the protease inhibitor E-64 (Fig. 3, A and B), which maintained its blocking activity under the experimental conditions used (supplemental Fig. S3). These changes were observed in the pancreas and PLNs but not in the distant lymphoid organs (e.g. inguinal lymph nodes). Together, these data suggest that the protease activity is important for anti-serpin antibodies to cause the shift toward cells expressing low levels of CD4 and CD19. It is possible that the effects of anti-serpin B13 mAb do not exactly reflect the effects of elevated levels of natural anti-serpin B13 autoantibodies. To address this problem, we compared NOD mice with distinct levels of anti-serpin B13 autoantibodies. We found that young animals with high levels of endogenous anti-serpin B13 autoantibodies (SBAhigh) had a markedly reduced population of islet-associated CD4high cells compared with animals with low levels of these autoantibodies (SBAlow). The number of islet-associated CD4high cells did not decrease, however, in SBAhigh mice that received the protease inhibitor E-64 (Fig. 4). This observation suggests that natural anti-serpin autoantibodies regulate proteases in vivo in a fashion similar to that described for anti-serpin B13 mAb [3].

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].

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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Exposure of worms to E-64 [2]
Equal number (n = 10) of adult female S. cervi were incubated in the 20 ml KRB maintenance medium containing different concentrations of E-64 (10 μM, 20 μM and 40 μM final concentration) for 8 h at 37°C and 5% CO2. The worms incubated in the maintenance medium only served as control. After 8 h worms were recovered and washed thoroughly with PBS, homogenized and assayed for various enzyme activities.

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Effect on parasite motility [2]
The motility of parasites was performed by visual inspection. The parasites incubated in E-64 containing medium were assessed visually till 8 h and scored either positive or negative (+/-) depending on their motility. After 8 h the recovery of motility was recorded by keeping the worms in fresh medium (devoid of E-64) for 1 h. Parasite motility was scored as -, no movement; +, least active; ++, less active; +++, moderately active; and ++++, highly active. To check the effect of E-64 on microfilariae (mf), adult female parasites were dissected longitudinally and released mf were collected in KRB maintenance medium and visualized under microscope at 40× (Motic B1 series).

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Effect on parasite viability [2]
The MTT assay was carried out to determine parasite viability according to the method of Mosmann et al 1988 with slight modifications. The treated worms were incubated in phosphate buffered saline (PBS) containing 1.0 ml of 0.5 mg/ml MTT [3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide] for 2 h at 37°C. The worms were then transferred to a fresh eppendorf containing 200 μl of dimethyl sulphoxide (DMSO) to solubilize the formazan crystals. After 1 h the medium was then carefully removed without disturbing the dark blue formazan crystals. The optical density of the resulting formazan solution was determined on a microplate reader (BioRad) at a wavelength of 540 nm. The half maximal effective concentration (EC50) of E-64 was calculated by plotting the graph between the concentration of E-64 and % viability of the adult parasites after 8 h of treatment. Here, EC50 refers to the concentration of the inhibitor where 50% of its maximal effect is observed or 50% of the parasite viability was reduced after specified incubation duration.

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Assay for cathepsin B cysteine proteases [2]
The Cathepsin Β activity was determined by the colorimetric method of Barrett (1977) using Z-Arg-Arg-4mβNA as substrate with slight modifications. The crude extract was 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) was added to make the final assay volume of 1.0 ml. The reaction mixture was incubated at 37°C for 30 min. The reaction was 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), was carried out with n-butanol. After complete layer separation, the absorbance was measured in n-butanol layer and activity was calculated using molar extinction coefficient of β-napthylamine solution as 31.5 M−1cm−1sec−1 at 520 nm. One unit of enzyme activity was defined as the amount of enzyme liberating 1 μmol of βNA per minute at 37°C. The half maximal inhibitory concentration (IC50) was 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.

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In vitro effect of E-64 on GSH [2]
To see the effect of E-64 on GSH, 10 μM GSH was incubated with different concentration of E-64 (5, 10, 20, 40 μM) for 20 minutes at 37°C prior to the addition of DTNB. A set without E-64 was taken as the control. Further, 1.88 ml 0.1 M potassium phosphate buffer, pH 8.0 and 0.02 ml of 4% DTNB was added to make the reaction volume upto 2 ml and incubated at RT for 15 min for color development. The absorbance of color developed was recorded at 412 nm.

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.

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Treatment of NOD/LtJ Mice with Anti-serpin B13 mAb and Protease Inhibitor [3]
Four-week-old female NOD/LtJ mice were 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 were also injected intraperitoneally with the protease inhibitor E-64 at 10 mg/kg/day for several days. Control mice were treated with diluent (a sterilized PBS solution containing 10% dimethyl sulfoxide) and control IgG. The solutions containing E-64 or dimethyl sulfoxide were prepared immediately before use. Twenty-four hours after the last injection, the mice were killed, and cells from their lymphoid organs and pancreatic islets were subjected to FACS analysis.

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Chronic instrumentation for venous infusion and blood pressure measurement [4]
Chronic instrumentation of rats for venous infusion and blood pressure measurements was done as described previously. Briefly, 8‐week old anesthetized SS rats 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. This preparation allowed chronic venous infusion and arterial blood pressure measurement in conscious, freely moving rats. 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, 1 mg/day; 280 mmol/L stock in DMSO) or the vehicle (DMSO in saline) control to the venous catheter. Daily MAP was calculated by averaging MAP taken every min over the beginning 3 h period 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.

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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Background: Current available antifilarial drug strategies only eliminate the larval stages of filarial parasites. Therefore, there is an urgent need of drugs which are macrofilaricidals. Identification of molecular targets crucial for survival of parasite is a prerequisite for drug designing. Cathepsin B, a cysteine protease family member is known to play crucial role in the normal growth, digestion of nutrients, exsheathment of the helminth parasites. Therefore, we targeted this enzyme in the filarial parasite using its specific inhibitor, E-64. Methods and findings: We have exposed the parasites to E-64 and observed their motility and viability at various time intervals. It caused marked decrease in the motility and viability of the parasites ultimately leading to their death after 8 hours. It is well known that E-64 protects the cell from apoptosis, however, it causes apoptotic effect in carcinoma cell lines. To understand the mechanism of action of E-64 on parasite survival, we have measured levels of different apoptotic markers in the treated parasites. E-64 significantly reduced the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also activated ced-3, homolog of mammalian caspase 3 suggesting initiation of an apoptotic like event in the filarial parasites. Different antioxidant enzymes were also evaluated to further explore the mechanism behind the death of the parasites. There was marked decrease in the level of GSH and activity of Glutathione reductase and glutathione-s-transferase leading to increased generation of reactive oxygen species. This led to the induced oxidation of fatty acids and protein which might alter the mitochondrial membrane permeability. Conclusion: This study suggests that inhibition of cathepsin B by E-64 generates oxidative stress followed by mitochondrial mediated apoptotic like event in filarial parasites leading to their death. Hence, suggesting filarial cathepsin B as a potential chemotherapeutic target for lymphatic filariasis. [2]

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This paper focuses on the inhibitory mechanism of E-64 and its derivatives (epoxysuccinyl-based inhibitors) with some cysteine proteases, based on the binding modes observed in the x-ray crystal structures of their enzyme-inhibitor complexes. E-64 is a potent irreversible inhibitor against general cysteine proteases, and its binding modes with papain, actinidin, cathepsin L, and cathepsin K have been reviewed at the atomic level. E-64 interacts with the Sn subsites of cysteine proteases. Although the Sn-Pn (n = 1-3) interactions of the inhibitor with the main chains of the active site residues are similar in respective complexes, the significant difference is observed in the side-chain interactions of S2-P2 and S3-P3 pairs because of different residues constituting the respective subsites. E-64-c and CA074 are representative derivatives developed from E-64 as a clinical usable and a cathepsin B-specific inhibitors, respectively. In contrast with similar binding/inhibitory modes of E-64-c and E-64 for cysteine proteases, the inhibitory mechanism of cathepsin B-specific CA074 results from the binding to the Sn' subsite.[1]

C15H27N5O5

357.41

357.201

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

66701-25-5

123985

White to off-white solid powder

1.4±0.1 g/cm3

182ºC

1.618

-1.46

5

6

11

25

518

3

O=C([C@H]1O[C@@H]1C(N[C@H](C(NCCCCNC(N)=N)=O)CC(C)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; 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

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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 (Please use freshly prepared in vivo formulations for optimal results.)