Phosphonate inhibitors of West Nile virus NS2B/NS3 protease

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M−1s−1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease

Berberine hampers influenza A replication through inhibition of MAPK/ERK pathway

Background: Berberine (BBR) is an isoquinoline alkaloid which exhibits a variety of biological and therapeutic properties, and has been reported by some to block replication of the influenza virus. However, contradictory results have also been presented, and the mechanistic explanation is lacking. Methods: A panel of cell lines (Madin–Darby canine kidney (MDCK), adenocarcinoma human alveolar basal epithelial cells (A549), lung epithelial type I (LET1)) and primary human airway epithelial cells (HAE) susceptible to influenza virus infection were infected with a seasonal influenza A virus in the presence or absence of BBR. Cytotoxicity towards cell lines was measured using XTT assay. The yield of the virus was analyzed using RT-qPCR. To study the molecular mechanism of BBR, confocal microscopy and Western blot analyses of cellular fractions were applied. Results and conclusions: Our results show cell-type-dependent anti-influenza properties of BBR in vitro which suggests that the compound acts on the cell and not the virus. Importantly, BBR hampers influenza replication in primary human airway epithelium 3D cultures that mimic the natural replication site of the virus. Studies show that the influenza A virus upregulates the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway and hijacks this pathway for nucleolar export of the viral ribonucleoprotein. Our results suggest that BBR interferes with this process and hampers influenza A replication

Novel coronavirus-like particles targeting cells lining the respiratory tract

<div><p>Virus like particles (VLPs) produced by the expression of viral structural proteins can serve as versatile nanovectors or potential vaccine candidates. In this study we describe for the first time the generation of HCoV-NL63 VLPs using baculovirus system. Major structural proteins of HCoV-NL63 have been expressed in tagged or native form, and their assembly to form VLPs was evaluated. Additionally, a novel procedure for chromatography purification of HCoV-NL63 VLPs was developed. Interestingly, we show that these nanoparticles may deliver cargo and selectively transduce cells expressing the ACE2 protein such as ciliated cells of the respiratory tract. Production of a specific delivery vector is a major challenge for research concerning targeting molecules. The obtained results show that HCoV-NL63 VLPs may be efficiently produced, purified, modified and serve as a delivery platform. This study constitutes an important basis for further development of a promising viral vector displaying narrow tissue tropism.</p></div

In vitro testing the potential of a novel chimeric IgG variant for inhibiting collagen fibrils formation in recurrent hereditary gingival fibromatosis: chimeric antibody in a gingival model

Gingival fibromatosis is a progressive enlargement of the gingiva. It may hinder oral cavity hygiene and result in underlying bone loss. The long-term benefits of surgery cannot be predicted. On the other hand, alternative, efficient and non-invasive methods are not available at present. The aim of this study was to test the inhibitory effects of a chimeric IgG variant on collagen fibril formation in the cell culture of gingival fibroblasts taken from a patient with hereditary gingival fibromatosis with a high propensity for recurrence. Gingival biopsies were collected from the mandibular gingiva and used for histological evaluation as well as to establish a fibroblast culture. A histological evaluation was made in haematoxylin-eosin and Heidenhain's trichrome stained tissue sections. The inhibitory effect of a chimeric antibody on collagen fibril formation was determined in fibroblast cultures by using a collagen-specific Western blot and immunofluorescent staining. A histological evaluation revealed epithelial acanthosis with singular elongated rete pegs extending into the underlying connective tissue stroma that consisted of locally abundant, irregular collagen bundles. Based on observations with an in vitro model we conclude that a chimeric anti-collagen antibody efficiently inhibits collagen fibril accumulation in cell culture derived from diffuse, hereditary gingival fibromatosis that is characterized by a high propensity for recurrence (high proliferation index). Employing cell cultures from standardized group of patients with recurrent hereditary gingival fibromatosis as well as standarizing relevant 3D (tissue-like) models will be crucial for further tests of the antibody

Characterization of SARS-CoV-2 replication complex elongation and proofreading activity

The replication complex (RC) of SARS-CoV-2 was recently shown to be one of the fastest RNA-dependent RNA polymerases of any known coronavirus. With this rapid elongation, the RC is more prone to incorporate mismatches during elongation, resulting in a highly variable genomic sequence. Such mutations render the design of viral protein targets difficult, as drugs optimized for a given viral protein sequence can quickly become inefficient as the genomic sequence evolves. Here, we use biochemical experiments to characterize features of RNA template recognition and elongation fidelity of the SARS-CoV-2 RdRp, and the role of the exonuclease, nsp14. Our study highlights the 2′OH group of the RNA ribose as a critical component for RdRp template recognition and elongation. We show that RdRp fidelity is reduced in the presence of the 3′ deoxy-terminator nucleotide 3′dATP, which promotes the incorporation of mismatched nucleotides (leading to U:C, U:G, U:U, C:U, and A:C base pairs). We find that the nsp10–nsp14 heterodimer is unable to degrade RNA products lacking free 2′OH or 3′OH ribose groups. Our results suggest the potential use of 3′ deoxy-terminator nucleotides in RNA-derived oligonucleotide inhibitors as antivirals against SARS-CoV-2