Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure.

The gastrointestinal tract (GIT) is exposed to xenobiotics, including drugs, through both: local (oral) and systemic routes. Despite the advances in drug discovery and in vitro pre-clinical models, there is a lack of appropriate translational models to distinguish the impact of these routes of exposure. Changes in intestinal permeability has been observed in different gastrointestinal and systemic diseases. This study utilized one such xenobiotic, arsenic, to which more than 200 million people around the globe are exposed via their food, drinking water, work environment, soil, and air. The purpose of this study was to establish an in vitro model to mimic gastrointestinal tract exposure to xenobiotics via oral or intravenous routes. To achieve this, we compared the route (mimicking oral and intravenous exposure to GIT and the dose response (using threshold approach) of trivalent and pentavalent inorganic arsenic species on the permeability of in vitro cultured polarized T84 cells, an example of intestinal epithelial cells. Arsenic treatment to polarized T84 cells via the apical and basolateral compartment of the trans-well system reflected oral or intravenous routes of exposure in vivo, respectively. Sodium arsenite, sodium arsenate, dimethyl arsenic acid sodium salt (DMAV), and disodium methyl arsonate hydrate (MMAV) were assessed for their effects on intestinal permeability by measuring the change in trans-epithelial electrical resistance (TEER) of T-84 cells. Polarized T-84 cells exposed to 12.8 µM of sodium arsenite from the basolateral side showed a marked reduction in TEER. Cytotoxicity of sodium arsenite, as measured by release of lactate dehydrogenase (LDH), was increased when cells were exposed via the basolateral side. The mRNA expression of genes related to cell junctions in T-84 cells was analyzed after exposure with sodium arsenite for 72 h. Changes in TEER correlated with mRNA expression of focal-adhesion-, tight-junction- and gap-junction-related genes (upregulation of Jam2, Itgb3 and Notch4 genes and downregulation of Cldn2, Cldn3, Gjb1, and Gjb2). Overall, exposure to sodium arsenite from the basolateral side was found to have a differential effect on monolayer permeability and on cell-junction-related genes as compared to apical exposure. Most importantly, this study established a preclinical human-relevant in vitro translational model to assess the changes in permeability and cytotoxicity during exposure, mimicking oral or intravenous routes.

Targeting protein-protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model.

Protein-protein interactions (PPI) of co-stimulatory molecules CD2-CD58 are important in the early stage of an immune response, and increased expression of these co-stimulatory molecules is observed in the synovial region of joints in rheumatoid arthritis (RA) patients. A CD2 epitope region that binds to CD58 was grafted on to sunflower trypsin inhibitor (SFTI) template structure to inhibit CD2-CD58 PPI. The peptide was incorporated with an organic moiety dibenzofuran (DBF) in its structure. The designed peptidomimetic was studied for its ability to inhibit CD2-CD58 interactions in vitro, and its thermal and enzymatic stability was evaluated. Stability studies indicated that the grafted peptidomimetic was stable against trypsin cleavage. In vivo studies using the collagen-induced arthritis (CIA) model in mice indicated that the peptidomimetic was able to slow down the progress of arthritis, an autoimmune disease in the mice model. These studies suggest that with the grafting of organic functional groups in the stable peptide template SFTI stabilizes the peptide structure, and these peptides can be used as a template to design stable peptides for therapeutic purposes.

Conformational Changes of the Receptor Binding Domain of SARS-CoV-2 Spike Protein and Prediction of a B-Cell Antigenic Epitope Using Structural Data.

COVID-19, the illness caused by the SARS-CoV-2 virus, is now a worldwide pandemic with mortality in hundreds of thousands as infections continue to increase. Containing the spread of this viral infection and decreasing the mortality rate is a major challenge. Identifying appropriate antigenic epitopes from the viral proteins is a very important task for vaccine production and the development of diagnostic kits and antibody therapy. A novel antigenic epitope would be specific to the SARS-CoV-2 virus and can distinguish infections caused by common cold viruses. In this study two approaches are employed to identify both continuous and conformational B-cell antigenic epitopes. To achieve this goal, we modeled a complete structure of the receptor binding domain (RBD) of the spike protein using recently deposited coordinates (6vxx, 6vsb, and 6w41) in the protein data bank. In addition, we also modeled the RBD-ACE2 receptor complex for SARS-CoV-2 using the SARS-CoV RBD-ACE2 complex (3D0J) as a reference model. Finally, structure based predicted antigenic epitopes were compared to the ACE2 binding region of RBD of SARS-CoV-2. The identified conformational epitopes show overlaps with the ACE2-receptor binding region of the RBD of SARS-CoV-2. Strategies defined in the current study identified novel antigenic epitope that is specific to the SARS-CoV-2 virus. Integrating such approach in the diagnosis can distinguish infections caused by common cold viruses from SARS-CoV-2 virus.

Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide.

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.

In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC.

Studies related to lung cancer have shown a link between human epidermal growth factor receptor-2 (HER2) expression and poor prognosis in patients with non-small cell lung cancer (NSCLC). HER2 overexpression has been observed in 3-38% of NSCLC, while strong HER2 protein overexpression is found in 2.5% of NSCLC. However, HER2 dimerization is important in lung cancer, including EGFR mutated NSCLC. Since HER2 dimerization leads to cell proliferation, targeting the dimerization of HER2 will have a significant impact on cancer therapies. A peptidomimetic has been designed that can be used as a therapeutic agent for a subset of NSCLC patients overexpressing HER2 or possessing HER2 as well as EGFR mutation. A cyclic peptidomimetic (18) has been designed to inhibit protein-protein interactions of HER2 with its dimerization partners EGFR and HER3. Compound 18 exhibited antiproliferative activity in HER2-positive NSCLC cell lines at nanomolar concentrations. Western blot analysis showed that 18 inhibited phosphorylation of HER2 and Akt in vitro and in vivo. Stability studies of 18 at various temperature and pH (pH 1 and pH 7.6), and in the presence of liver microsomes indicated that 18 was stable against thermal and chemical degradation. Pharmacokinetic parameters were evaluated in nude mice by administrating single doses of 4 mg/kg and 6 mg/kg of 18 via IV. The anticancer activity of 18 was evaluated using an experimental metastasis lung cancer model in mice. Compound 18 suppressed the tumor growth in mice when compared to control. A proximity ligation assay further proved that 18 inhibits HER2:HER3 and EGFR: HER2 dimerization. Overall, these results suggest that 18 can be a potential treatment for HER2-dimerization related NSCLC.

Peptides, Peptidomimetics, and Polypeptides from Marine Sources: A Wealth of Natural Sources for Pharmaceutical Applications.

Nature provides a variety of peptides that are expressed in most living species. Evolutionary pressure and natural selection have created and optimized these peptides to bind to receptors with high affinity. Hence, natural resources provide an abundant chemical space to be explored in peptide-based drug discovery. Marine peptides can be extracted by simple solvent extraction techniques. The advancement of analytical techniques has made it possible to obtain pure peptides from natural resources. Extracted peptides have been evaluated as possible therapeutic agents for a wide range of diseases, including antibacterial, antifungal, antidiabetic and anticancer activity as well as cardiovascular and neurotoxin activity. Although marine resources provide thousands of possible peptides, only a few peptides derived from marine sources have reached the pharmaceutical market. This review focuses on some of the peptides derived from marine sources in the past ten years and gives a brief review of those that are currently in clinical trials or on the market.