Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Immunogenic and Protective Properties of Recombinant Hemagglutinin of Influenza A (H5N8) Virus.

In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus-the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy.

The Potential of Usnic-Acid-Based Thiazolo-Thiophenes as Inhibitors of the Main Protease of SARS-CoV-2 Viruses.

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (-)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle-just as pure (+)- and (-)-usnic acids-showed no anti-3CLpro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand-protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.

Design, synthesis and antiviral evaluation of novel conjugates of the 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold and saturated N-heterocycles via 1,2,3-triazole linker.

A new series of heterocyclic derivatives with a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment was designed, synthesised and biologically evaluated. Synthesis of the target compounds was performed using the Cu(I) catalysed cycloaddition reaction. The key starting substances in the click reaction were an alkyne containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment and a series of azides with saturated nitrogen-containing heterocycles. Some of the derivatives were found to exhibit strong antiviral activity against Marburg and Ebola pseudotype viruses. Lysosomal trapping assays revealed the derivatives to possess lysosomotropic properties. The molecular modelling study demonstrated the binding affinity between the compounds investigated and the possible active site to be mainly due to hydrophobic interactions. Thus, combining a natural hydrophobic structural fragment and a lysosome-targetable heterocycle may be an effective strategy for designing antiviral agents.

The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper.

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.

Molecular Modeling of Viral Type I Fusion Proteins: Inhibitors of Influenza Virus Hemagglutinin and the Spike Protein of Coronavirus.

The fusion of viral and cell membranes is one of the basic processes in the life cycles of viruses. A number of enveloped viruses confer fusion of the viral envelope and the cell membrane using surface viral fusion proteins. Their conformational rearrangements lead to the unification of lipid bilayers of cell membranes and viral envelopes and the formation of fusion pores through which the viral genome enters the cytoplasm of the cell. A deep understanding of all the stages of conformational transitions preceding the fusion of viral and cell membranes is necessary for the development of specific inhibitors of viral reproduction. This review systematizes knowledge about the results of molecular modeling aimed at finding and explaining the mechanisms of antiviral activity of entry inhibitors. The first section of this review describes types of viral fusion proteins and is followed by a comparison of the structural features of class I fusion proteins, namely influenza virus hemagglutinin and the S-protein of the human coronavirus.

(+)-Usnic Acid and Its Derivatives as Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses.

In order to test the antiviral activity, a series of usnic acid derivatives were synthesized, including new, previously undescribed compounds. The activity of the derivatives against three strains of SARS-CoV-2 virus was studied. To understand the mechanism of antiviral action, the inhibitory activity of the main protease of SARS-CoV-2 virus was studied using the developed model as well as the antiviral activity against the pseudoviral system with glycoprotein S of SARS-CoV-2 virus on its surface. It was shown that usnic acid exhibits activity against three strains of SARS-CoV-2 virus: Wuhan, Delta, and Omicron. Compounds 10 and 13 also showed high activity against the three strains. The performed biological studies and molecular modeling allowed us to assume that the derivatives of usnic acid bind in the N-terminal domain of the surface glycoprotein S at the binding site of the hemoglobin decay metabolite.

Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses?

Flaviviruses are single-stranded RNA viruses that have emerged in recent decades and infect up to 400 million people annually, causing a variety of potentially severe pathophysiological processes including hepatitis, encephalitis, hemorrhagic fever, tissues and capillaries damage. The Flaviviridae family is represented by four genera comprising 89 known virus species. There are no effective therapies available against many pathogenic flaviviruses. One of the promising strategies for flavivirus infections prevention and therapy is the use of neutralizing antibodies (NAb) that can disable the virus particles from infecting the host cells. The envelope protein (E protein) of flaviviruses is a three-domain structure that mediates the fusion of viral and host membranes delivering the infectious material. We previously developed and characterized 10H10 mAb which interacts with the E protein of the tick-borne encephalitis virus (TBEV) and many other flaviviruses' E proteins. The aim of this work was to analyze the structure of E protein binding sites recognized by the 10H10 antibody, which is reactive with different flavivirus species. Here, we present experimental data and 3D modeling indicating that the 10H10 antibody recognizes the amino acid sequence between the two cysteines C92-C116 of the fusion loop (FL) region of flaviviruses' E proteins. Overall, our results indicate that the antibody-antigen complex can form a rigid or dynamic structure that provides antibody cross reactivity and efficient interaction with the fusion loop of E protein.

Borneol Ester Derivatives as Entry Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses.

In the present work we studied the antiviral activity of the home library of monoterpenoid derivatives using the pseudoviral systems of our development, which have glycoproteins of the SARS-CoV-2 virus strains Wuhan and Delta on their surface. We found that borneol derivatives with a tertiary nitrogen atom can exhibit activity at the early stages of viral replication. In order to search for potential binding sites of ligands with glycoprotein, we carried out additional biological tests to study the inhibition of the re-receptor-binding domain of protein S. For the compounds that showed activity on the pseudoviral system, a study using three strains of the infectious SARS-CoV-2 virus was carried out. As a result, two leader compounds were found that showed activity on the Wuhan, Delta, and Omicron strains. Based on the biological results, we searched for the potential binding site of the leader compounds using molecular dynamics and molecular docking methods. We suggested that the compounds can bind in conserved regions of the central helices and/or heptad repeats of glycoprotein S of SARS-CoV-2 viruses.

Simulation of Molecular Dynamics of SARS-CoV-2 S-Protein in the Presence of Multiple Arbidol Molecules: Interactions and Binding Mode Insights.

In this work, we evaluated the antiviral activity of Arbidol (Umifenovir) against SARS-CoV-2 using a pseudoviral system with the glycoprotein S of the SARS-CoV-2 virus on its surface. In order to search for binding sites to protein S of the virus, we described alternative binding sites of Arbidol in RBD and in the ACE-2-RBD complex. As a result of our molecular dynamics simulations combined with molecular docking data, we note the following fact: wherever the molecules of Arbidol bind, the interaction of the latter affects the structural flexibility of the protein. This interaction may result both in a change in the shape of the domain-enzyme binding interface and simply in a change in the structural flexibility of the domain, which can subsequently affect its affinity to the enzyme. In addition, we examined the possibility of Arbidol binding in the stem part of the surface protein. The possibility of Arbidol binding in different parts of the protein is not excluded. This may explain the antiviral activity of Arbidol. Our results could be useful for researchers searching for effective SARS-CoV-2 virus inhibitors targeting the viral entry stage.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

Synthesis and Antiviral Activity of Camphene Derivatives against Different Types of Viruses.

To date, the 'one bug-one drug' approach to antiviral drug development cannot effectively respond to the constant threat posed by an increasing diversity of viruses causing outbreaks of viral infections that turn out to be pathogenic for humans. Evidently, there is an urgent need for new strategies to develop efficient antiviral agents with broad-spectrum activities. In this paper, we identified camphene derivatives that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses, including influenza virus A/PR/8/34 (H1N1), Ebola virus (EBOV), and the Hantaan virus. The lead-compound 2a, with pyrrolidine cycle in its structure, displayed antiviral activity against influenza virus (IC50 = 45.3 µM), Ebola pseudotype viruses (IC50 = 0.12 µM), and authentic EBOV (IC50 = 18.3 µM), as well as against pseudoviruses with Hantaan virus Gn-Gc glycoprotein (IC50 = 9.1 µM). The results of antiviral activity studies using pseudotype viruses and molecular modeling suggest that surface proteins of the viruses required for the fusion process between viral and cellular membranes are the likely target of compound 2a. The key structural fragments responsible for efficient binding are the bicyclic natural framework and the nitrogen atom. These data encourage us to conduct further investigations using bicyclic monoterpenoids as a scaffold for the rational design of membrane-fusion targeting inhibitors.

Monoterpenoid-based inhibitors of filoviruses targeting the glycoprotein-mediated entry process.

In this study, we screened a large library of (+)-camphor and (-)-borneol derivatives to assess their filovirus entry inhibition activities using pseudotype systems. Structure-activity relationship studies revealed several compounds exhibiting submicromolar IC50 values. These compounds were evaluated for their effect against natural Ebola virus (EBOV) and Marburg virus. Compound 3b (As-358) exhibited the good antiviral potency (IC50 = 3.7 µM, SI = 118) against Marburg virus, while the hydrochloride salt of this compound 3b·HCl had a strong inhibitory effect against Ebola virus (IC50 = 9.1 µM, SI = 31) and good in vivo safety (LD50 > 1000 mg/kg). The results of molecular docking and in vitro mutagenesis analyses suggest that the synthesized compounds bind to the active binding site of EBOV glycoprotein similar to the known inhibitor toremifene.