Protease

Leupeptin Ac是一种二肽。L-亮氨酸酰胺，N-乙酰基-L-亮氨酸-N-[4-[（氨基亚氨基甲基）氨基]-1-甲酰基丁基]-是玫瑰链霉菌和牛爪蟾中发现的一种天然产物。

中国爆发了前所未有的冠状病毒SARS-CoV-2，导致人类急性呼吸道感染。目前还没有精确的疫苗/治疗剂可用于对抗COVID-19疾病。一些改头换面的药物挽救了病人的生命，但完全治愈的机会相对较少。几种药物靶点已被报道可抑制SARS-CoV-2病毒感染，其中TMPRSS2（跨膜蛋白酶丝氨酸2）是潜在靶点之一；抑制这种蛋白酶可以阻止病毒进入宿主人类细胞。甲磺酸卡莫司他、那莫司他和白细胞介素是药物，其中前两种药物用于治疗COVID-19，白细胞介素也进行了测试。为了考虑这些药物作为COVID-19的重新用途药物，有必要了解它们与TMPRSS2的结合亲和力和稳定性。在本研究中，我们与TMPRSS2进行了这些分子的分子对接和分子动力学（MD）模拟。对接研究表明，与其他两种药物分子相比，lepeptin分子与TMPRSS2蛋白的结合较强。MD模拟的RMSD和RMSF值证实了胰肽和TMPRSS2的氨基酸比其他两个分子更稳定。此外，胰肽与TMPRSS2的关键氨基酸形成相互作用，并且在MD模拟中保持了这种相互作用。这些结构和动力学信息有助于评估这些药物是否可以作为再用途药物使用，然而，胰肽与TMPRSS2的强结合谱表明，在临床试验后可能会考虑将其作为COVID-19疾病的再用途药物。[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 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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To address the urgent need of drugs to treat the COVID-19 pandemic disease, repurposing drugs is one of the best solutions comparing with the other techniques like vaccine development and new plasma transfusion. In the line of drug searching process, we have undertaken camostat, nafamostat, and leupeptin drug molecules to inhibit the TMPRSS2 serine 2 protease, which is the activator of SARS-CoV-2 for the fusion of the virus enter into the host human cell. The molecular docking study reveals that these three drug molecules form interactions and has binding affinity towards TMPRSS2. However, the leupeptin forms strong interactions with the key amino acids Ser186, His41, and Asp180 of catalytic triad present in the active site of TMPRSS2 as the other two molecules lacking Ser186 and His41 interactions, whereas in the molecular dynamics simulations of these three complexes, leupeptin is highly stable (RMSD & RMSF) and this molecule also forms stable interactions with the key amino acids Ser186, His41, and Asp186 of TMPRSS2 during the 100-ns MD simulations. On comparing the binding-free energy of all the three molecules, notably, leupeptin has high binding affinity towards TMPRSS2. From the above static and dynamic studies, it is confirmed that leupeptin is very stable and it could strongly inhibit the TRMPRSS2 serine 2 protease and this leads to stop the fusion of SARS-CoV-2 virus onto the host human cell, hence it is considered as a repurposed drug 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

166.27

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。