In Vitro Antiviral Activity of a New Indol-3-carboxylic Acid Derivative Against SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012-2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 µM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 106 TCID50/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility - high activity (IC50 = 1.06 µg/mL) and high selectivity index (SI = 78.6) - this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

[Interferon-regulating activity of the CelAgrip drug and its influence on the formation of reactive oxygen species and expression of innate immunity genes in Burkitt's lymphome cells cultures.]

INTRODUCTION: Interferons (IFN) and IFN inducers are effective in suppressing viral reproduction and correcting of the innate immunity mechanisms. The aim of the study was to test the hypothesis of the possible involvement of the IFN inducer CelAgrip (CA) as an activator or suppressor of antiviral effects in Burkitt's lymphoma (LB) cell cultures with different ability to produce Epstein-Barr virus antigens (EBV). MATERIAL AND METHODS: The kinetic analysis of the dynamics of reactive oxygen species (ROS) production and determination of gene group expression by real-time PCR in response to CA treatment were done in human cell lines LB P3HR-1 and Namalva, spontaneously producing and not producing EBV antigens. RESULTS AND DISCUSSION: When treating CA in Namalva cells, a decrease in the ROS activation index was found; in P3HR-1 cells, an increase was observed. After treatment with CA, there was no reliable activation of the IFN-α, IFN-ß and IFN-λ genes in Namalva cells, but the expression of the ISG15 and P53(TP53) genes was increased more than 1200 times and 4.5 times, respectively. When processing the CA of P3HR-1 cells, the expression of IFN-α genes increased by more than 200 times, IFN-λ - 100 times, and the ISG15 gene - 2.2 times. The relationship between IFN-inducing action of CA and the activity of ISG15 and ROS in LB cell cultures producing and not producing EBV antigens is supposed. CONCLUSION: In Namalva cells that do not produce EBV antigens the treatment of CA results in suppression of ROS generation and activation of the expression of genes ISG15 and P53 (TP53); in P3HR-1 cells producing EBV antigens, the opposite picture is observed - the formation of ROS and the expression of the IFN-α and IFN-λ genes are activated and the activity of the ISG15 and P53 (TP53) genes is suppressed.

[Interferon-regulating activity of the celagrip antiviral drug and its influence on formation of reactive oxygen species and expression of innate immunity genes in the follicular lymphoma patients].

INTRODUCTION: Medicines from the group of interferon inducers (IFNs) "swith on" the synthesis of type 1 interferons (IFN-I) and induce the expression of IFN-stimulated genes (ISGs) that regulate innate immunity reactions and protect the host from infectious agents and the tumour pathology.The purpose of the study was to determine the role of the drug celagrip (CA) in the activation of innate immunity genes and the effect on the production of reactive oxygen species (ROS) in patients with follicular lymphoma (FL). OBJECTIVES:  to study the intensity of ROS production and the level of expression of the IFN-α2, IFN-λ1, ISG15, BCL2, P53(TP53) and USP18 genes in response to the treatment of blood cells of patients with FL with the preparation of CA. MATERIAL AND METHODS: The study involved primary cancer patients diagnosed with follicular lymphoma (FL) and healthy volunteers. A kinetic analysis of the dynamics of production of reactive oxygen species (ROS) was performed in whose blood cells, and the expression of the group of genes was determined by real-time PCR in response to CA processing. RESULTS AND DISCUSSION: ROS production by blood cells of patients with FL and volunteers in the presence of CA significantly decreased (P < 0.05). The level of gene expression of ISG15, P53(TR53) and USP 18 in the group of patients with FL was significantly higher than that in the group of volunteers. When treating blood cells with CA, it becomes possible to divide patients with FL into groups with a positive and negative response in accordance with the level of expression of the USP18 gene. We divided FL patients into groups with a positive and negative response in accordance with the level of USP18 gene expression after treatment of blood cells with CA. CONCLUSIONS: The CA drug reduces the production of ROS and simultaneously stimulates the activity of the innate immunity genes ISG15, P53(TP53) and USP18 in the blood cells of patients with FL.

[Cytokine-regulating activity of anti-virus preparation CelAgripus in Burkitt's lymphoma stable B-cell lines.]

INTRODUCTION: Cytokines activated in response to immunosuppressive viral infections can directly or indirectly affect the neoplastic transformation of B cells. In this study, we studied a new substance designed to produce the antiviral drug CelAgrip (CA, CelAgripus), which exhibits interferon (IFN) and cytokine-inducing activity and, apparently, can be used as an activator of antiviral immunity. Purpose - is to evaluate the cytokine-regulating effect of CA in Burkitt's lymphoma (LB) cell lines latently infected with the Epstein-Barr virus (EBV). OBJECTIVES: to study the CA-induced expression of the cytokine genes IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IFN-α, IFN -γ, IFN-ß, IFN-λ1, IFN-λ2, IFN-λ3, TNF-α in normal and EBV transformed LB cells. MATERIAL AND METHODS: Cell line: the human embryo fibroblasts (HEF), Namalva, Daudi, Raji, P3HR-1. Preparations: CA, gossypol-acetic acid (GAA), sodium carboxymethyl cellulose (Na-CMC). METHODS: RT-PCR and methods for assessing cytotoxicity (MTT and Scepter 2.0 Merck cell counter). RESULTS: The effect of the CA preparation on the expression of IFN-λ, IL-1ß, IL-6, IL-8 and IL-10 genes was revealed. DISCUSSION: We observed the activation of gene expression of IFN-λ, IL-1ß, IL-6, IL-8 and suppression of IL-10 gene activity when treatment CA of LB cells. CONCLUSION: The substance CA has new effects on the activation of the expression of a number of key cytokine genes in stable Burkitt lymphoma cell lines.

Signal Immune Reactions of Macrophages Differentiated from THP-1 Monocytes to Infection with Pandemic H1N1PDM09 Virus and H5N2 and H9N2 Avian Influenza A Virus.

In culture of THP-1 cells differentiated into macrophages with PMA (THP-PMA macrophages) infected with influenza viruses of subtypes H1, H5 and H9, we measured the expression of TLR7 and RIG1 receptor genes, sensors of viral RNA and ribonucleoprotein, and the levels of production of inflammatory cytokines IL-1ß, TNFα, IL-10, and IFNα. The sensitivity and inflammatory response of THP-PMA macrophages to pandemic influenza A virus H1N1pdm09 and avian influenza H5N2 and H9N2 viruses correlate with the intracellular level of their viral RNA and activation of the RIG1 gene. Abortive infection is accompanied by intensive macrophage secretion of TNFα, IL-1ß, and toxic factors inducing cell death. Activity of endosomal TLR7 receptor gene changed insignificantly in 24 h after infection and significantly decreased in 48 and 72 h under the action of H5N2 and H9N2, which correlated with manifestation of the cytopathogenic effect of these viruses. H5N2 and H9N2 avian viruses in THP-PMA macrophages are strong activators of the expression of the gene of the cytoplasmic RIG1 receptor 24 and 48 h after infection, and the pandemic virus H1N1pdm09 is a weak stimulator of RIG1 gene. Avian influenza H5N2 and H9N2 viruses are released by rapid induction of the inflammatory response in macrophages. At the late stages of infection, we observed a minor increase in IL-10 secretion in macrophages and, probably, the polarization of a part of the population in type M2. The studied influenza A viruses are weak inductors of IFN in THP-PMA macrophages. In the culture medium of THP-PMA macrophages infected with H9N2 and H5N2 viruses, MTT test revealed high levels of toxic factors causing the death of Caco-2 cells. In contrast to avian viruses, pandemic virus H1N1pdm09 did not induce production of toxic factors.

[AVIAN RECOMBINANT VIRUS H5N1 INFLUENZA (A/VIETNAM/1203/04) AND ITS ESCAPE-MUTANT m13(13) INDUCE EARLY SIGNALING REACTIONS OF THE IMMUNITY IN HUMAN LYMPHOCYTES].

The innate immune receptors TLR4, TLR7, TLR8, and RIG1 recognized the structures of the influenza viruses in human lymphocytes and were activated by the recombinant avian influenza virus A/Vietnam/1203/04 and its escape-mutant m13(13) during early period of interaction. The stimulated levels are not connected with viral reproduction. Donor cells with the low constitutive immune receptors gene expression levels showed higher stimulation. Inflammation virus effects resulted in. increasing production of TNF-alpha and IFN-gamma by lymphocytes. Signaling gene reactions of the parent and mutant viruses endosomal as well as cytoplasmic receptors are very similar. The mutant virus A/Vietnam/1203/04 (HA S145F), stimulated an increase in the transcription level of the membrane receptor gene TLR4 and a decrease in the level of activation of TNF-alpha gene. Further studies of natural influenza virus isolates are necessary to estimate the role of HA antigenic changes on immune reactions in humans.

[[Antiviral activity of the interferon beta 1a].

The antiviral activity of the interferon beta Ia was studied using the example of the antiviral activity of the drugs interferon beta Ia Genfaxon and Rebif for the influenza and herpes. A pronounced antiviral effect of the drugs against influenza and herpes viruses was-shown far the first time.

[Grippol, Vaxigrip and influvac vaccines--inductors of innate and adaptive immunity factor genes in human blood cells].

AIM: Study the effect of inactivated influenza vaccines on the activity of innate and adaptive immunity genes (TLR3, TLR4 and B2M), RNA-interference Dicer1-gene, production of cytokines (antiviral IFN type I and II, regulatory IL10, IL17) and pro-inflammatory factors IL1-ß, TNFα. MATERIALS AND METHODS: Gene expression was determined by rRT-PCR with authors' primers in human blood cells treated with various doses of the vaccines. Concentration of cytokines by enzyme immunoassay was measured in cultural fluid using "Vector-best" kits. RESULTS: The studied vaccines have characteristic effects on genetic level. Grippol vaccine predominately stimulates TLR4 gene, activates TLR3, B2M and Dicer1 genes. Influvac vaccine mostly induces TLR3 gene and to a lesser extent TLR4 gene, does not influence the expression of B2M gene and inhibits Dicer1 gene. Vaxigrip split vaccine--the most potent stimulator of gene activity at low doses. Its main targets are TLR3 and B2M genes. All the inactivated vaccines--inductors of high level of IFNγ, low level of TNFα and do not induce IL17. Grippol additionally stimulates secretion of IL1-ß, and Vaxigrip - IFNα. Subunit vaccines Grippol and Influvac that contain purified influenza virus hemagglutinins induce IL10 synthesis in blood cells. CONCLUSION: Immunogenetic characteristics of the inactivated influenza vaccines administered nowadays are obtained.

[Modern Russian Etiotropic Antiinfluenza Drugs].

Two main groups of the Russian etiotropic drugs recommended by the Ministry of Health of the Russian Federation for prophylaxis or treatment of influenza are described in the review, i.e. chemotherapeutics whose targets are various stages of the influenza virus reproduction and interferons and their inductors engaging innate immunity patterns.