Development of a novel drug candidate with improved potency against influenza virus neuraminidase compared with currently available therapeutics, and high activity against oseltamivir-resistant viruses. A number of synthetic compounds were evaluated for antiviral properties in vitro and in vivo. Three-dimensional molecular docking, assisted by a pharmacophore model, was applied to classify compounds within the series by their inhibitory potency. Compound stability in blood and in animal models was determined. Pharmacokinetic studies in dogs and rats after oral or intravenous administration were performed. A novel highly potent drug candidate [(3R,4R,5S)-5-[(diaminomethylene)amino]-3-(1-ethylpropoxy)-4-[(fluoroacetyl)amino]cyclohex-1-ene-1-carboxylic acid; AV5080] was synthesized and tested. AV5080 exhibited high activity against influenza virus neuraminidase in vitro, with IC₅₀ values of 0.03 nM and 0.07 nM against the neuraminidase of A/Duck/Minnesota/1525/1981/H5N1 and A/Perth/265/2009/H1N1 (wild-type), respectively. Notably, AV5080 was highly active against oseltamivir-resistant influenza viruses. Based on the results presented in this study, AV5080 is a promising novel oral drug candidate for the treatment of influenza, including oseltamivir-resistant types. Further pre-clinical development of AV5080 is warranted.

Introduction. The ability of SARS-CoV-2 antibodies to neutralize the virus is the primary indicator of their specific activity. The test for virus neutralizing antibodies (NAbs) is much needed in different biomedical studies.The aim of the study is to find optimum conditions for microscopic and spectrophotometric detection of SARSCoV-2 NAbs by inhibition of cytopathic effect (CPE) in cell cultures.Materials and methods. Blood sera collected from COVID-19 convalescent patients and healthy individuals (n = 96) were tested using the ELISA method. The SARS-CoV-2 coronavirus, Dubrovka strain (GenBank accession no. MW514307.1) was grown in culture medium of Vero cell line CCL-81 (ATCC). Real-time RT-PCR, ELISA, and Sanger sequencing were used for detection of the virus. The results of the neutralization test (NT) were assessed through the microscopic examination for CPE and by the methyl thiazolyl tetrazolium (MTT) assay.Results. SARS-CoV-2 was isolated from a COVID-19 patient and adapted to grow in cell culture. At a low dose of infection (MOI = 0.00001), the virus caused a pronounced CPE with the cell viability less than 3%, thus making it possible to assess NT results by CPE inhibition. The NT and ELISA-based comparative study of sera showed positive correlation between virus NAb titers and Nab titers to S-protein RBD (Spearman’s r = 0.714; p 0.001). The results of NAbs microscopic and spectrophotometric detection (the MTT assay) also demonstrated positive correlation (Spearman’s r = 0.963; p 0.05).Conclusion. The SARS-CoV-2 virus adapted to Vero cell culture served to develop a NAb titer assessment system, which can be used both in microscopic studies and for an MTT assay in spectrophotometric studies. The MTT assay provides automated reading of NT results, optimizes the statistical analysis of the obtained data, and minimizes subjectivity in assessment of results. Being a vital dye, MTT penetrates only viable cells, thus contributing to the reliability of the obtained results compared to other dyes.

This article reexamines some opinions concerning pH requirements for optimal immobilization of monoclonal antibodies (mAbs) by passive adsorption in antigen capture ELISA. It was discovered that substitution of “classical” sodium phosphate (pH 7.5) and carbonate (pH 9.5) coating solutions by acid (pH 2.8) buffers maximized antigen capture 4 out of 10 different tested anti-HBsAg mAbs, resulting in a 1.5–2.5 increase of binding curve coefficients. By measuring both mAbs amounts and functionality, the enhancement effect was attributed to the better preservation of solid phase antibodies activity.