Protease

Leupeptin Ac is a dipeptide. L-Leucinamide, N-acetyl-L-leucyl-N-[4-[(aminoiminomethyl)amino]-1-formylbutyl]- is a natural product found in Streptomyces roseus and Xenorhabdus bovienii with data available.

The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial.[3]

[1]. Leupeptins, new protease inhibitors from Actinomycetes. J Antibiot (Tokyo). 1969 Jun;22(6):283-6.
[2]. The structure and activity of leupeptins and related analogs. J Antibiot (Tokyo). 1971 Jun;24(6):402-4.
[2]. Strong Binding of Leupeptin with TMPRSS2 Protease May Be an Alternative to Camostat and Nafamostat for SARS-CoV-2 Repurposed Drug: Evaluation from Molecular Docking and Molecular Dynamics Simulations. Appl Biochem Biotechnol. 2021 Jan 29;193(6):1909–1923.

Leupeptin is a tripeptide composed of N-acetylleucyl, leucyl, and arginine residues linked by peptide bonds. It is an inhibitor of calpapsin, a class of calcium-activated proteases that promote cell death. Leupeptin has multiple functions, including being an inhibitor of serine proteases, a bacterial metabolite, a cathepsin B inhibitor, a calpapsin inhibitor, and an EC 3.4.21.4 (trypsin) inhibitor. It is a tripeptide and also an aldehyde. It is the conjugate base of Leupeptin (1+). Leupeptin has been reported to exist in Streptomyces lavenderensis, Streptomyces exfoliatus, and several other organisms with relevant data. To address the urgent need for drugs to treat the COVID-19 pandemic, drug reuse is one of the best solutions compared to other technologies such as vaccine development and new plasma transfusion. In our drug screening, we investigated three drug molecules—carmostat, naftomostat, and Leupeptin—all of which inhibit the TMPRSS2 serine protease. TMPRSS2 is an activator for the fusion of the SARS-CoV-2 virus into host cells. Molecular docking studies have shown that all three drug molecules can interact with TMPRSS2 and have binding affinity. However, leucopeptide forms a strong interaction with the key amino acids Ser186, His41 and Asp180 in the catalytic triplet of the active site of TMPRSS2, while the other two drug molecules lack interaction with Ser186 and His41. In addition, leucopeptide showed high stability (low RMSD and RMSF) when the three complexes were molecularly dynamically simulated, and the molecule also formed a stable interaction with the key amino acids Ser186, His41 and Asp186 of TMPRSS2 during a 100-nanosecond molecular dynamics simulation. By comparing the binding free energies of the three molecules, it can be found that leucopeptide has a high binding affinity for TMPRSS2. The above static and dynamic studies confirm that leucopeptide is very stable and can strongly inhibit the TMPRSS2 serine 2 protease, thereby preventing the SARS-CoV-2 virus from fusing with the host human cells. Therefore, it is considered a drug to be reused after clinical studies. [3]

C20H38N6O4

426.55

426.295

C, 56.32; H, 8.98; N, 19.70; O, 15.00

24365-47-7

Leupeptin hemisulfate;103476-89-7;Leupeptin;55123-66-5; Leupeptin;55123-66-5;Leupeptin Ac-LL;24365-47-7; Leupeptin hemisulfate;103476-89-7; 39740-82-4 (HCl); 1082207-96-2 (hemisulfate hydrate)

72429

Typically exists as solid at room temperature

1.21g/cm3

1.557

2.378

5

5

14

30

602

3

N/C(=N/CCCC(NC([C@@H](NC([C@@H](NC(=O)C)CC(C)C)=O)CC(C)C)=O)C=O)/N

GDBQQVLCIARPGH-BSOSBYQFSA-N

InChI=1S/C20H38N6O4/c1-12(2)9-16(24-14(5)28)19(30)26-17(10-13(3)4)18(29)25-15(11-27)7-6-8-23-20(21)22/h11-13,15-17H,6-10H2,1-5H3,(H,24,28)(H,25,29)(H,26,30)(H4,21,22,23)/t15?,16-,17-/m0/s1

(2S)-2-acetamido-N-[(2S)-1-[[5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]-4-methylpentanamide

Leupeptin Ac; 24365-47-7; Leupeptin Ac-LL; C01591; (2S)-2-acetamido-N-[(2S)-1-[[5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]-4-methylpentanamide; L-Leucinamide, N-acetyl-L-leucyl-N-[4-[(aminoiminomethyl)amino]-1-formylbutyl]-; AC1NR4EG; SCHEMBL4669504;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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