RESUMO
Introduction: Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa. Methods: We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays. Results: Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.
RESUMO
This work describes fabrication of gold electrodes modified with peptide conjugate DAL-PEG-DK5-PEG-OH that enables ultra-sensitive detection of lipopolysaccharide (LPS) isolated from the reference strain of Escherichia coli O26:B6. The initial step of the established procedure implies immobilization of the fully protected DAL-PEG-DK5-PEG-OH peptide on the surface of the gold electrode previously modified by cysteamine. Then side chain- and Fmoc-deprotection was performed in situ on the electrode surface, followed by its incubation in 1 % of BSA solution to block non-specific bindings sites before LPS detection. The efficiency of the modification was confirmed by X-ray Photoelectron Spectroscopy (XPS) measurements. Additionally, the cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS) were employed to monitor the effectiveness of each step of the modification. The obtained results confirmed that the presence of the surface-attached covalently bound peptide DAL-PEG-DK5-PEG-OH enables LPS detection by means of CV technique within the range from 5 × 10-13 to 5 × 10-4 g/mL in PBS solution. The established limit of detection (LOD) for EIS measurements was 4.93 × 10-21 g/mL with wide linear detection range from 5 × 10-21 to 5 × 10-14 g/mL in PBS solution. Furthermore, we confirmed the ability of the electrode to detect LPS in a complex biological samples, like mouse urine and human serum. The effectiveness of the electrodes in identifying LPS in both urine and serum matrices was confirmed for samples containing LPS at both 2.5 × 10-15 g/mL and 2.5 × 10-9 g/mL.