Zonulin; paracellular permeability and apical-basolateral polarity

On Vero cell proliferation, larazotideacetate (1-100μM; 5 days) had an effect [1]. The varicella zoster virus (VZV) can be inhibited by larazotide acetate (1-100 μM; 3 d); the EC50 values for the OKA strain and the 07-1 strain, respectively, are 44.14 and 59.06 μM [1]. In Caco-2 cells, larazotideacetate (1 and 3 mM; 72 hours) reduces the permeability of tight junctions caused by cytokines [2]. In IEC6 cells, PTG-induced ZO-1 translocation and actin cytoskeleton rearrangement are inhibited by larazotideacetate (12.5 mM; 1 h) [2].

Larazotide acetate (250 μg; intraperitoneally injected twice weekly for 7 weeks) inhibits intestinal permeability in gluten-sensitive transgenic mice [1].

The recombinant SARS-CoV-2 Mpro (Proteros) (20 nM at a final concentration) was mixed with serial dilutions of AT1001 and Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH2 substrate (5 μM) in 20 μL (reaction volume) assay buffer solution (20 mM HEPES, pH 7.5, 1 mM DTT, 1 mM EDTA, 100 mM NaCl, 0.01% Tween20). The appropriate volume of substrate was added in reaction buffer along with 42.5 nL compound in 100% DMSO. Finally, the appropriate volume of target enzyme was added, and the reaction started with an incubation time of 10 min. The fluorescence signal of the Edans was monitored at an emission wavelength of 500 nm by exciting at 360 nm, by means of Pherastar FSX microplate Reader. Calpeptin was used as reference to set up the experiments[1].

Cytotoxicity assay [1]
Cell Types: Vero cell line
Tested Concentrations: 1-100 μM
Incubation Duration: 5 days
Experimental Results: Inhibited Vero cell growth, CC50 value was 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.

novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen causing a severe and rapidly developing pneumonia (COVID-19) outbreak. The virus’s enzyme, called 3CLpro or main protease (Mpro), is crucial for viral replication, making it a promising target for antiviral drug development. Recently, we have used a drug repositioning strategy to demonstrate that the zonulin octapeptide inhibitor AT1001 (lalazolyl acetate) can bind to the Mpro catalytic domain, thus rapidly responding to the global COVID-19 pandemic. Therefore, in this study, we attempted to investigate the antiviral activity of AT1001 and its five derivatives through cell-based experiments. Our results identified the molecular framework of the AT1001 peptide, which can be used for lead compound optimization to develop a new generation of SARS-CoV-2 antiviral drugs with higher bioactivity and improve the success rate of clinical trials. [1]

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Tight junctions (TJs) control the paracellular permeability and apical-basal lateral polarity of epithelial cells and can be regulated by exogenous and endogenous stimuli. Permeability dysregulation is associated with a variety of pathological conditions, such as celiac disease and inflammatory bowel disease. This study investigated the mechanism by which laprazole peptide acetate (an octapeptide and a tight junction regulator) inhibits cellular changes induced by gliadin fragments, AT-1002, and cytokines. We previously demonstrated that the hexapeptide AT-1002, derived from the Vibrio cholerae tight junction toxin ZOT, induces various biochemical changes in IEC6 and Caco-2 cells, leading to decreased transepithelial electrical resistance (TEER) and increased tight junction (TJ) permeability. This study found that laprazole peptide acetate inhibits the redistribution and rearrangement of tight junction protein-1 (ZO-1) and actin induced by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, laprazole peptide acetate inhibits AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. In addition, laprazole acetate inhibited the translocation of gliadin 13 peptide associated with celiac disease on the Caco-2 cell monolayer. Furthermore, apical application of laprazole acetate inhibited the increase in tight junction permeability induced by cytokine application on the basolateral side. Finally, in in vivo experiments in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, laprazole acetate inhibited gliadin-induced intestinal macrophage aggregation and maintained normal tight junction structure. In summary, our data suggest that laprazole acetate can inhibit the changes induced by AT-1002, gliadin, and cytokines in epithelial cells and maintain the structure and function of tight junctions 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

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

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)

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

Solubility in Formulation 1: 100 mg/mL (127.24 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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