Leupeptin, Microbial
(Synonyms: 亮肽素) 目录号 : GC10027
Leupeptin, Microbial 是一种广谱丝氨酸和半胱氨酸蛋白酶抑制剂。
Cas No.:103476-89-7
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Leupeptin analysis [1]: | |
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Preparation Method |
The concentration of Leupeptin, Microbial was calculated by enzyme inhibition assay using 50 pg of papain and 0.2 mM of N-benzoylL-arginine ethyl ester as the target protease and papain substrate, respectively. Enzyme reactions were carried out at 25 ℃ and pH 6.2. |
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Applications |
Leupeptin, Microbial is a leucine-specific protease and a metalloprotease. |
| Cell experiment [2]: | |
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Cell lines |
MRC-C cell |
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Preparation Method |
To investigate the effect of Leupeptin, Microbial on virus yield, MRC-C cultures in 35 mm dishes were infected by adsorption of virus from 0T ml concentrated suspension of HCV 229E for 1 h at 36 ℃. The cultures were washed twice and overlaid with 2 ml Eagle’s MEM plus 0.2% bovine serum albumin and 20 mm-HEPES buffer pH 7.4. Leupeptin, Microbial at a range of concentrations was added to the virus inoculum, to the maintenance medium and also to maintenance medium used to treat the cultures for 1 h before infection. After 24 h at 36 ℃ in air, the cells were scraped into the medium and disrupted by sonication. |
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Reaction Conditions |
0.4 to l00 µg/ml for 25 h（1 h before infection to 24 h after infection） |
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Applications |
The protease inhibitor Leupeptin, Microbial prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells. |
| Animal experiment [3]: | |
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Animal models |
C57BL/6NCrl male mouse |
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Preparation Method |
Mice received i.p. injections of 0.5 ml sterile Phosphate Buffered Saline or 0.5 ml PBS containing 9–40 mg/kg Leupeptin, Microbial hemisulfate. |
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Dosage form |
0, 9, 18 36 and 40 mg/kg Leupeptin, Microbial; Intraperitoneal injection |
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Applications |
Rate of LC3b accumulation after Leupeptin, Microbial treatment was greatest in the liver and lowest in spleen. |
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References: [1]. Kim IS, Kim YB, et,al. Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin. Biochem J. 1998 Apr 15;331 (Pt 2)(Pt 2):539-45. doi: 10.1042/bj3310539. PMID: 9531495; PMCID: PMC1219386. [2]. Appleyard G, Tisdale M. Inhibition of the growth of human coronavirus 229E by leupeptin. J Gen Virol. 1985 Feb;66 (Pt 2):363-6. doi: 10.1099/0022-1317-66-2-363. PMID: 3968542. [3]. Haspel J, Shaik RS, et,al. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011 Jun;7(6):629-42. doi: 10.4161/auto.7.6.15100. Epub 2011 Jun 1. PMID: 21460622; PMCID: PMC3127049. |
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Leupeptin, Microbial is a broad-spectrum serine and cysteine protease inhibitor [1]. Leupeptin, Microbial is a reversible protease inhibitor, It acts on bovine trypsin, human plasminase, bovine splenic cathepsin B, and recombinant human caltrypsin with Ki values of 35 nM, 3.4 μM, 6 nM, and 72 nM[7].
The protease inhibitor Leupeptin, Microbial prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells [3]. Leupeptin, Microbial-mediated inhibition of trypsin-like proteases maintains substrate mycelium development, whereas proteolytic degradation of Leupeptin, Microbial in stationary phase cultures derepresses the trypsin-like proteases, leading to the digestion of substrate mycelium and promotion of aerial mycelium formation [2]. As the calpain inhibitor Leupeptin, Microbial primarily protected hair cells from neomycin[5]. The expression of hepatitis B surface antigen (HBsAg) from Leupeptin recovered up to 50% of the cell suspension culture [6].
Leupeptin, Microbial was well tolerated in mice. Leupeptin, Microbial significantly increased LC3b-II in a dose-dependent way in the total tissue extract and lysosomal enrichment portion (LE portion). At the level of electron microscopy (EM), Leupeptin, Microbial induced the accumulation of electron-dense vesicle structures [4].
References:
[1]. Aoyagi T, Miyata S, et,al. Biological activities of leupeptins. J Antibiot (Tokyo). 1969 Nov;22(11):558-68. doi: 10.7164/antibiotics.22.558. PMID: 4243683.
[2]. Kim IS, Kim YB, et,al. Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin. Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):539-45. doi: 10.1042/bj3310539. PMID: 9531495; PMCID: PMC1219386.
[3]. Appleyard G, Tisdale M. Inhibition of the growth of human coronavirus 229E by leupeptin. J Gen Virol. 1985 Feb;66 ( Pt 2):363-6. doi: 10.1099/0022-1317-66-2-363. PMID: 3968542.
[4]. Haspel J, Shaik RS, et,al. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011 Jun;7(6):629-42. doi: 10.4161/auto.7.6.15100. Epub 2011 Jun 1. PMID: 21460622; PMCID: PMC3127049.
[5]. Aoyagi T, Miyata S, et,al. Biological activities of leupeptins. J Antibiot (Tokyo). 1969 Nov;22(11):558-68. doi: 10.7164/antibiotics.22.558. PMID: 4243683.
[6]. Coffin AB, Williamson KL, et,al. Profiling drug-induced cell death pathways in the zebrafish lateral line. Apoptosis. 2013 Apr;18(4):393-408. doi: 10.1007/s10495-013-0816-8. PMID: 23413197; PMCID: PMC3627356.
[7]. Ganapathi TR, Sunil Kumar GB, et,al. Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures. Plant Cell Rep. 2007 Sep;26(9):1575-84. doi: 10.1007/s00299-007-0379-7. Epub 2007 May 30. PMID: 17534624.
Leupeptin, Microbial 是一种广谱丝氨酸和半胱氨酸蛋白酶抑制剂[1]。 Leupeptin, Microbial 是一种可逆的蛋白酶抑制剂，它作用于牛胰蛋白酶、人纤溶酶、牛脾脏组织蛋白酶 B 和重组人钙蛋白酶，Ki 值为 35 nM、3.4 μM、6 nM 和 72 nM[7].
蛋白酶抑制剂 Leupeptin Microbial 阻止了 MRC-C 细胞培养物中人类冠状病毒株 229E 的增殖[3]。亮肽素，微生物介导的胰蛋白酶样蛋白酶抑制维持底物菌丝体发育，而亮肽素的蛋白水解降解，微生物在稳定期培养物中去抑制胰蛋白酶样蛋白酶，导致底物菌丝体的消化和气生菌丝体形成的促进 [2].作为钙蛋白酶抑制剂 Leupeptin，Microbial 主要保护毛细胞免受新霉素的侵害[5]。 Leupeptin的乙型肝炎表面抗原（HBsAg）的表达在细胞悬浮培养物中恢复了50%[6]。
Leupeptin, Microbial 在小鼠中具有良好的耐受性。 Leupeptin, Microbial 在总组织提取物和溶酶体富集部分（LE 部分）中以剂量依赖性方式显着增加 LC3b-II。在电子显微镜（EM）水平，亮肽素、微生物诱导电子致密囊泡结构的积累[4]。
| Cas No. | 103476-89-7 | SDF | |
| 别名 | 亮肽素 | ||
| 化学名 | 2-acetamido-N-(1-((5-((diaminomethylene)amino)-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-4-methylpentanamide | ||
| Canonical SMILES | O=C(C(C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H])([H])N([H])C(C([H])([H])[H])=O)N([H])C(C(N([H])C(C([H])=O)([H])C([H])([H])C([H])([H])C([H])([H])/N=C(N([H])[H])/N([H])[H])=O)([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] | ||
| 分子式 | C20H38N6O4.1/2H2SO4 | 分子量 | 493.6 |
| 溶解度 | ≥ 24.7mg/mL in DMSO， ≥ 20mg/mL in Water | 储存条件 | Store at -20°C |
| General tips | 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 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.0259 mL | 10.1297 mL | 20.2593 mL |
| 5 mM | 0.4052 mL | 2.0259 mL | 4.0519 mL |
| 10 mM | 0.2026 mL | 1.013 mL | 2.0259 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs
mBio2021 Oct 26;12(5):e0222021.PMID: 34579576DOI: 10.1128/mBio.02220-21
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 ¦̍ 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 ¦̍. 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. IMPORTANCE A TCM formula has played an important role in the treatment of COVID-19 in China. However, the mechanism of TCM action is still unclear. In this study, we identified Leupeptin, a metabolite produced by plant-symbiotic actinomyces (PSA), which showed antiviral activity in both cell culture and enzyme assays. Moreover, Leupeptin found in the QFPD decoction was confirmed by both HPLC fingerprinting and HRMS. These results suggest that Leupeptin likely contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This result gives us important insight into further studies of the PSA metabolite and medicinal plant ecosystem for future TCM modernization research.
Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria
Angew Chem Int Ed Engl2020 Oct 5;59(41):17872-17880.PMID: 32609431DOI: 10.1002/anie.202005506
Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of Leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of "pro-pyrazinones" featuring a hetero-tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of Leupeptins can be associated with animal colonization phenotypes.
Natural products from thioester reductase containing biosynthetic pathways
Nat Prod Rep2018 Sep 19;35(9):847-878.PMID: 29916519DOI: 10.1039/c8np00013a
Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in Microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.
Effects of Microbial proteinase inhibitors on the degradation of endogenous and internalized proteins by rat yolk sacs
Biochem J1981 Apr 15;196(1):41-8.PMID: 7306078DOI: 10.1042/bj1960041
1. The effects of Leupeptin and other Microbial proteinase inhibitors were measured in rat yolk sacs on the uptake and degradation of formaldehyde-denatured 125I-labelled bovine serum albumin as well as on the degradation of 3H-labelled endogenous protein. 2. Leupeptin, at concentrations between 1 and 100 micrograms/ml, inhibits the degradation of added albumin without affecting pinocytic uptake. Accordingly large amounts of undegraded albumin accumulate within the tissue. 3. Removal of Leupeptin produces a rapid recovery of the capacity to degrade albumin. 4. Endogenous protein degradation is rapidly inhibited by Leupeptin, but to a far lesser extent than the breakdown of albumin. However, the inhibition is only slightly reversed on removal of Leupeptin. 5. Degradation of both albumin and endogenous protein in intact yolk sacs is inhibited by the Microbial proteinase inhibitors in the order: Leupeptin greater than antipain greater than chymostatin; elastatinal, pepstatin and bestatin are ineffective. 6. Similar results are found when albumin is incubated in yolk-sac homogenates at pH 4 with the inhibitors. 7. The marked inhibitory effects of Leupeptin, antipain and chymostatin suggest that cathepsin B and possibly cathepsin L participate in the degradation of 125I-labelled albumin in yolk sacs. By comparison, the smaller inhibitory effects of the proteinase inhibitors on endogenous protein breakdown imply a minor role of lysosomal cathepsins in this process.