Zonulin; paracellular permeability and apical-basolateral polarity

larazotideacetate（1-100μM；5天）对Vero细胞增殖有影响[1]。醋酸拉扎肽（1-100 μM；3 d）可抑制水痘带状疱疹病毒（VZV）； OKA 菌株和 07-1 菌株的 EC50 值分别为 44.14 和 59.06 μM [1]。在 Caco-2 细胞中，larazotideacetate（1 和 3 mM；72 小时）可降低细胞因子引起的紧密连接的通透性 [2]。在 IEC6 细胞中，PTG 诱导的 ZO-1 易位和肌动蛋白细胞骨架重排可被 larazotideacetate (12.5 mM；1 h) 抑制 [2]。

醋酸拉拉佐肽（250 μg；腹腔注射每周两次，持续 7 周）可抑制麸质敏感转基因小鼠的肠道通透性[1]。

将重组严重急性呼吸系统综合征冠状病毒2 Mpro（Proteros）（终浓度为20nM）与AT1001和Dabcyl-KTSAVLQSGFRKM-E（Edans）-NH2底物（5μM）的系列稀释液在20μL（反应体积）测定缓冲溶液（20mM HEPES，pH 7.5，1mM DTT，1mM EDTA，100mM NaCl，0.01%Tween20）中混合。将适当体积的底物与42.5nL化合物在100%DMSO中一起加入反应缓冲液中。最后，加入适当体积的靶酶，并以10分钟的孵育时间开始反应。通过Pherastar FSX微孔板读取器在360nm激发，在500nm的发射波长下监测Edans的荧光信号。使用Calpeptin作为参考建立实验[1]。

细胞毒性测定 [1]
细胞类型： Vero 细胞系
测试浓度： 1-100 μM
孵育时间： 5天
实验结果：抑制Vero细胞生长，CC50值为82.5 μM。

Animal/Disease Models: HLA-HCD4/DQ8 double transgenic mice [2]
Doses: 250 μg
Route of Administration: intraperitoneal (ip) injection; 250 μg twice a week for 7 weeks
Experimental Results: Barrier function parameters improved, macrophages in lamina propria Cell count diminished to control levels.

[1]. Peptide Derivatives of the Zonulin Inhibitor Larazotide (AT1001) as Potential Anti SARS-CoV-2: Molecular Modelling, Synthesis and Bioactivity Evaluation. Int J Mol Sci. 2021 Aug 30;22(17):9427.

[2]. Larazotide acetate regulates epithelial tight junctions in vitro and in vivo. Peptides. 2012 May;35(1):86-94.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.[1]

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Tight junctions (TJs) control paracellular permeability and apical-basolateral polarity of epithelial cells, and can be regulated by exogenous and endogenous stimuli. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. Herein we studied the mechanism by which larazotide acetate, an 8-mer peptide and TJ regulator, inhibits the cellular changes elicited by gliadin fragments, AT-1002, and cytokines. Previously, we demonstrated that AT-1002, a 6-mer peptide derived from the Vibrio cholerae zonula occludens toxin ZOT, caused several biochemical changes in IEC6 and Caco-2 cells resulting in decreased transepithelial electrical resistance (TEER) and increased TJ permeability. In this study, larazotide acetate inhibited the redistribution and rearrangement of zonula occludens-1 (ZO-1) and actin caused by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, larazotide acetate inhibited the AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. Additionally, larazotide acetate inhibited the translocation of a gliadin 13-mer peptide, which has been implicated in celiac disease, across Caco-2 cell monolayers. Further, apically applied larazotide acetate inhibited the increase in TJ permeability elicited by basolaterally applied cytokines. Finally, when tested in vivo in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, larazotide acetate inhibited gliadin-induced macrophage accumulation in the intestine and preserved normal TJ structure. Taken together, our data suggest that larazotide acetate inhibits changes elicited by AT-1002, gliadin, and cytokines in epithelial cells and preserves TJ structure and function in vitro and in vivo.[2]

C34H59N9O12

785.9

785.428

C, 51.96; H, 7.57; N, 16.04; O, 24.43

881851-50-9

Larazotide;258818-34-7

44146842

White to off-white solid powder

3.736

360.55

10

13

21

55

1320

5

CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@H]1C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)CN.CC(=O)O

NYGCNONRVCGHAT-UFIKZEAMSA-N

InChI=1S/C32H55N9O10.C2H4O2/c1-16(2)12-20(38-30(49)26(17(3)4)39-24(44)14-35-23(43)13-33)28(47)40-27(18(5)6)31(50)37-19(9-10-22(34)42)32(51)41-11-7-8-21(41)29(48)36-15-25(45)461-2(3)4/h16-21,26-27H,7-15,33H2,1-6H3,(H2,34,42)(H,35,43)(H,36,48)(H,37,50)(H,38,49)(H,39,44)(H,40,47)(H,45,46)1H3,(H,3,4)/t19-,20-,21-,26-,27-/m0./s1

2-[[(2S)-1-[(2S)-5-Amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-3-methylbutanoyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]acetic acid Acetate

Larazotide Acetate; AT-1001; AT 1001; AT1001;Larazotide acetate; 881851-50-9; Larazotide (acetate); AT-1001; Larazotide acetate [USAN]; Larazotide acetate (USAN); UNII-FO8S2IW40N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~16.67 mg/mL (~21.21 mM)
DMSO : ~3.2 mg/mL (~4.07 mM)

配方 1 中的溶解度: 100 mg/mL (127.24 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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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。