AT-1002

Mechanism of action of ZOT-derived peptide AT-1002, a tight junction regulator and absorption enhancer

Tight junctions (TJs) are intercellular structures that control paracellular permeability and epithelial polarity. It is now accepted that TJs are highly dynamic structures that are regulated in response to exogenous and endogenous stimuli. Here, we provide details on the mechanism of action of AT-1002, the active domain of Vibrio cholerae's second toxin, zonula occludens toxin (ZOT). AT-1002, a hexamer peptide, caused the redistribution of ZO-1 away from cell junctions as seen by fluorescence microscopy. AT-1002 also activated src and mitogen activated protein (MAP) kinase pathways, increased ZO-1 tyrosine phosphorylation, and rearrangement of actin filaments. Functionally, AT-1002 caused a reversible reduction in transepithelial electrical resistance (TEER) and an increase in lucifer yellow permeability in Caco-2 cell monolayers. In vivo, co-administration of salmon calcitonin with 1 mg of AT-1002 resulted in a 5.2-fold increase in AUC over the control group. Our findings provide a mechanistic explanation for AT-1002-induced tight junction disassembly, and demonstrate that AT-1002 can be used for delivery of other agents in vivo.

Structure-activity relationship studies of permeability modulating peptide AT-1002

AT-1002 a 6-mer synthetic peptide belongs to an emerging novel class of compounds that reversibly increase paracellular transport of molecules across the epithelial barrier. The aim of this project was to elaborate on the structure-activity relationship of this peptide with the specific goal to replace the P2 cysteine amino acid. Herein, we report the discovery of peptides that exhibit reversible permeability enhancement properties with an increased stability profile.

Development of an efficient transdermal delivery system of small interfering RNA using functional peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.

Label-free discrimination of tumorigenesis stages using in vitro prostate cancer bone metastasis model by Raman imaging

Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm^-1, which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.

Type 2 diabetes detection based on serum sample Raman spectroscopy

In this work, we propose to the Raman spectroscopy as a new technique for the detection of the type 2 diabetes using blood serum samples. The serum samples were obtained from 15 patients who were clinically diagnosed with type 2 diabetes mellitus and 20 healthy volunteers. The average spectra showed equally intense peaks as, 695 cm^-1, the doublet of tyrosine at 828 and 853 cm^-1, phenylalanine at 1002 and 1028 cm^-1, the phospholipid shoulder at 1300-1345 cm^-1, and proteins (amide I) at 1654 cm^-1. The major differences were found at 661 and 1404 cm^-1 (glutathione), 714 (polysaccharides), 605 (Phe), 545 cm^-1 (tryptophan), and the shoulder of amide III at 1230-1282 cm^-1, where seem to disappear in the diabetes spectrum. On the contrary, the region that is more highlighted due to that diabetes peaks are clearly more intense was 897-955 cm^-1. Principal component analysis and linear discriminate analysis were employed for developing discrimination method. The first three principal components provided a classification of the samples from healthy and diabetes patients with high sensitivity and specificity. In addition, when the first principal component was plotted as a function of the Raman shift, it revealed these shifts accounted for the greatest differences between control and diabetes samples, which coincided with the shifts of spectral differences shown by mean spectra. Our results demonstrated that serum sample Raman spectroscopy promises to become a non-invasive support tool of the currently applied techniques for type 2 diabetes detection, decreasing the false-positive cases.