protease: Cathepsin B; Cathepsin L; Cathepsin H; Ser/Thr Protease; Mpro
Target: Inhibits bovine trypsin (Ki = 72 nM), bovine α-chymotrypsin (weaker inhibition, no specific Ki reported), human plasmin (Ki = 35 nM), bovine thrombin (weak inhibition, no specific Ki reported), and bovine spleen cathepsin B (Ki = 3.4 μM) [1]
Inhibits proteases involved in tubulin degradation in plant cells (no specific Ki/IC50 reported) [2]
Inhibits SARS-CoV-2 main protease (Mpro, IC50 = 127.2 μM) [5]

Leupeptin, produced by a variety of actinomycetes, which effectively prevent proteolysis.[1] Tubulin purity is raised when leupeptin hemisulfate shields it from endogenous proteolytic activities during the isolation process.[2] Leupeptin hemisulfate has the potential to restore up to 50% of the expression of the hepatitis B surface antigen (HBsAg) in cell suspension cultures. [3]

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- Inhibited bovine trypsin activity with a Ki of 72 nM; at 10 μg/ml, it completely inhibited human plasmin-induced fibrinolysis. It showed weaker inhibition against bovine α-chymotrypsin and thrombin. At 100 μg/ml, it did not affect the growth of rabbit kidney cells or mouse fibroblasts [1]
- Inhibited proteolytic activities in tobacco BY-2 suspension cells that degrade tubulin, as demonstrated by reduced tubulin breakdown in cell extracts treated with 100 μM leupeptin over 24 hours [2]
- Inhibited growth of human prostate cancer PC-3 cells in vitro, with reduced cell proliferation and decreased expression of angiogenic factors (VEGF) and osteolytic factors (MMP-9) when treated with leupeptin (concentrations not specified) [3]
- Reduced SARS-CoV-2 viral RNA replication in Vero cells with an EC50 of 42.34 μM after 72 hours of treatment; inhibited Mpro activity with an IC50 of 127.2 μM [5]

Leupeptin was well accepted by the animals and resulted in a significant dose-dependent rise in LC3b-II in the lysosome enriched fraction (LE fraction) as well as total tissue extracts. Leupeptin caused electron-dense vesicular structures to accumulate at the electron microscopy (EM) level. In hepatocytes, these structures became visible 60 minutes after treatment (40 mg/kg). The findings indicated that leupeptin prevented LC3b-II from being broken down inside lysosomes, increasing its levels in vivo. As a result, the leupeptin-based assay has the potential to be used to investigate the dynamics of macroautophagy in mice.

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Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice. [4]

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- In a mouse model of bone metastasis of human prostate cancer (PC-3 cells injected into tibia), leupeptin (10 mg/kg, intraperitoneal injection, daily for 4 weeks) reduced tumor growth, decreased osteolytic lesions, and inhibited angiogenesis (reduced CD31-positive vessels) [3]
- In C57BL/6 mice, intraperitoneal injection of leupeptin (9, 18, or 36 mg/kg, single dose) increased the LC3B-II/LC3B-I ratio in liver, kidney, and heart tissues, indicating inhibited autophagic flux; the effect was dose-dependent [4]

Mpro enzyme activity inhibition test. [5]
A total of 20 mM leupeptin hemisulfate in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0). A 30-μl inhibitor solution with a series of concentrations in 25 mM Tris buffer (pH 8.0) was first mixed with 10 μl 100 μM peptide substrate (Dabcyl-TSAVLQ↓SGFRKMK-Edans; GenScript). Next, 10 μl of a final concentration of 200 nM Mpro was added to the plate. The relative fluorescence unit (RFU) value was measured with an excitation wavelength of 360 nm and an emission wavelength of 490 nm at 37°C for 1 h by using a SpectraMax Paradigm multimode detection platform (Molecular Devices, USA). Experiments were performed in triplicate. The enzyme activity reaction rate and inhibition rate were calculated by using MS Excel. The inhibition curve was plotted by using GraphPad Prism 8.0.
In vitro antiviral assays. [5]
A total of 20 mM leupeptin hemisulfate in deionized water was diluted to 200 μM to 0.06 μM with DMEM containing 1% FBS. Vero cells cultured overnight in 96-well plates were infected by virus at a multiplicity of infection (MOI) of 0.01 for 2 h. The medium was removed, and fresh drug-containing medium was then added to the cells. After 48 h, the cells were lysed in lysis buffer. The viral RNA in 100 μl of the cell supernatant was quantified by reverse transcription-PCR (RT-PCR). Seventy-two hours later, the changes of cytopathic effect were also observed by microscopy. Experiments were performed in triplicate. The experimental results were processed using MS Excel and GraphPad Prism 8.0.

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- Trypsin inhibition assay: Bovine trypsin was incubated with substrate (benzoyl-L-arginine-p-nitroanilide) in buffer at 37°C, and the rate of p-nitroaniline release was measured spectrophotometrically. Leupeptin was added to the reaction mixture, and the inhibition constant (Ki) was calculated from the dose-response curve [1]
- Plasmin inhibition assay: Human plasmin was mixed with fibrinogen, and fibrinolysis was monitored. Leupeptin was added at varying concentrations, and the concentration required to inhibit 50% of fibrinolysis was determined [1]
- SARS-CoV-2 Mpro assay: Recombinant Mpro was incubated with fluorogenic substrate (Dabcyl-KTSAVLQSGFR-Edans) and leupeptin at varying concentrations. Fluorescence intensity was measured, and IC50 was calculated based on reduced substrate cleavage [5]

Leupeptin inhibited human coronavirus strain 229E multiplication in MRC-C cell cultures. Leupeptin's IC50 value in plaque tests was 0.4 μg/mL, and at 50 μg/mL, it had no effect on the host cells' ability to grow. Leupeptin (100 μg/mL) only inhibited virus yield in single-cycle growth experiments when added within two hours of infection, suggesting that it acts on the early stages of virus replication.[5]

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- Plant cell protease assay: Tobacco BY-2 suspension cells were lysed, and cell extracts were incubated with purified tubulin in the presence or absence of leupeptin (100 μM). Tubulin degradation was analyzed by SDS-PAGE and densitometry [2]
- Prostate cancer cell assay: PC-3 cells were cultured in vitro and treated with leupeptin (concentrations not specified). Cell proliferation was measured by MTT assay, and VEGF/MMP-9 expression was analyzed by Western blot and RT-PCR [3]
- SARS-CoV-2 replication assay: Vero cells were infected with SARS-CoV-2 and treated with leupeptin (0.06–200 μM) for 72 hours. Viral RNA was extracted, and viral load was quantified by RT-PCR [5]

C57BL/6NCrl male mice
20 mg/kg
i.p.

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Mice received i.p. injections of 0.5 ml sterile Phosphate Buffered Saline (PBS, GIBCO 10010) or 0.5 ml PBS containing 9–40 mg/kg leupeptin hemisulfate. In other experiments (Fig. 1), mice alternatively received 28–112 mg/kg chloroquine in PBS or 0.1–0.3 mg/kg Bafilomycin B1 in PBS. After injection, the mice were returned to their cages and provided free access to food and water unless they were being subjected to calorie-starvation for experimental purposes. At specified time points after injection, the mice were euthanized and their solid organs were manually dissected and flash frozen in liquid nitrogen. In experiments in which macroautophagic flux was compared between treatments (for example starvation versus fed; Fig. 7) or genotypes (beclin 1+/+ versus beclin 1−/−, Fig. 8), care was taken to dissect the different experimental groups in parallel to ensure they were exposed to leupeptin for equal amounts of time. [4]

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- Bone metastasis model: Nude mice were anesthetized, and PC-3 cells (1×10⁵) were injected into the left tibia. After 1 week, leupeptin (10 mg/kg) or vehicle was administered via intraperitoneal injection once daily for 4 weeks. Mice were euthanized, and tibias were harvested for histopathological analysis and quantification of tumor area [3]
- Autophagy flux assay: C57BL/6 mice were injected intraperitoneally with a single dose of leupeptin (9, 18, or 36 mg/kg) dissolved in saline. After 4 hours, mice were euthanized, and liver, kidney, and heart tissues were collected for Western blot analysis of LC3B-II/LC3B-I ratio [4]

[1]. Biological activities of leupeptins. J Antibiot (Tokyo). 1969 Nov;22(11):558-68.

[2]. Inhibition of plant cell proteolytic activities that degrade tubulin. Cell Biol Int Rep. 1985 Sep;9(9):849-57.

[3]. Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metaPlant Cell Rep. 2007 Sep;26(9):1575-84.

[4]. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011 Jun;7(6):629-42.
[5]. Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs. mBio. 2021 Oct 26;12(5):e0222021.

Leupeptin is a tripeptide composed of N-acetylleucyl, leucyl and argininal residues joined in sequenceby peptide linkages. It is an inhibitor of the calpains, a family of calcium-activated proteases which promote cell death. It has a role as a serine protease inhibitor, a bacterial metabolite, a cathepsin B inhibitor, a calpain inhibitor and an EC 3.4.21.4 (trypsin) inhibitor. It is a tripeptide and an aldehyde. It is a conjugate base of a leupeptin(1+).
Leupeptin has been reported in Streptomyces lavendulae, Streptomyces exfoliatus, and other organisms with data available.

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The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.[2]

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Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.[3]

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Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to be an ideal drug target for treating COVID-19. Leupeptin, a broad-spectrum covalent inhibitor of serine, cysteine, and threonine proteases, showed inhibitory activity against Mpro, with a 50% inhibitory concentration (IC50) value of 127.2 μM in vitro in our study here. In addition, leupeptin can also inhibit SARS-CoV-2 in Vero cells, with 50% effective concentration (EC50) values of 42.34 μM. More importantly, various strains of streptomyces that have a broad symbiotic relationship with medicinal plants can produce leupeptin and leupeptin analogs to regulate autogenous proteases. Fingerprinting and structure elucidation using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), respectively, further proved that the Qing-Fei-Pai-Du (QFPD) decoction, a traditional Chinese medicine (TCM) formula for the effective treatment of COVID-19 during the period of the Wuhan outbreak, contains leupeptin. All these results indicate that leupeptin at least contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This also reminds us to pay attention to the microbiomes in TCM herbs as streptomyces in the soil might produce leupeptin that will later infiltrate the medicinal plant. We propose that plants, microbiome, and microbial metabolites form an ecosystem for the effective components of TCM herbs.[5]

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- Leupeptin is a tripeptide protease inhibitor isolated from Streptomyces roseus. It is soluble in water and stable in acidic conditions [1]
- It acts as a reversible competitive inhibitor of serine and cysteine proteases by binding to the active site of target enzymes [1]

C20H38N6O4.1/2H2SO4

475.59

950.56

C, 50.51; H, 8.27; N, 17.67; O, 20.18; S, 3.37

103476-89-7

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

72429

N-acetyl-L-leucyl-L-leucyl-L-argininal compound with N-acetyl-L-leucyl-L-leucyl-L-argininal sulfuric acid

Ac-LLR-CHO; Ac-Leu-Leu-Arg-al.Ac-Leu-Leu-Arg-al.H2SO4

White to off-white solid powder

1.2±0.1 g/cm3

1.557

Microbial Metabolite

1.16

5

5

14

30

602

3

S(=O)(=O)(O[H])O[H].O=C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])([H])[H])=O)N([H])[C@]([H])(C(N([H])[C@]([H])(C([H])=O)C([H])([H])C([H])([H])C([H])([H])/N=C(\N([H])[H])/N([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H].O=C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])([H])[H])=O)N([H])[C@]([H])(C(N([H])[C@]([H])(C([H])=O)C([H])([H])C([H])([H])C([H])([H])/N=C(\N([H])[H])/N([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H]

CIPMKIHUGVGQTG-VFFZMTJFSA-N

InChI=1S/2C20H38N6O4.H2O4S/c2*1-12(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;1-5(2,3)4/h2*11-13,15-17H,6-10H2,1-5H3,(H,24,28)(H,25,29)(H,26,30)(H4,21,22,23);(H2,1,2,3,4)/t2*15-,16-,17-;/m00./s1

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

NK-381; Leupeptin hemisulfate; 103476-89-7; Leupeptin; Leupeptin hemisulfate anhydrous; Leupeptin hemisulfate salt; UNII-05V9Y5208M; 05V9Y5208M; L-Leucinamide, N-acetyl-L-leucyl-N-((1S)-4-((aminoiminomethyl)amino)-1-formylbutyl)-, sulfate (2:1); NK 381; Leupeptin hemisulfate; NK381;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: 100 mg/mL (210.27 mM) in PBS, clear solution; with sonication (<60°C).

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Solubility in Formulation 2: ~83 mg/mL (175 mM) in H2O

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