Antiviral screening of natural, anti-inflammatory compound library against African swine fever virus.

BACKGROUND: African swine fever virus (ASFV) is a major threat to pig production and the lack of effective vaccines underscores the need to develop robust antiviral countermeasures. Pathologically, a significant elevation in pro-inflammatory cytokine production is associated with ASFV infection in pigs and there is high interest in identifying dual-acting natural compounds that exhibit antiviral and anti-inflammatory activities. METHODS: Using the laboratory-adapted ASFV BA71V strain, we screened a library of 297 natural, anti-inflammatory compounds to identify promising candidates that protected Vero cells against virus-induced cytopathic effect (CPE). Virus yield reduction, virucidal, and cell cytotoxicity experiments were performed on positive hits and two lead compounds were further characterized in dose-dependent assays along with time-of-addition, time-of-removal, virus entry, and viral protein synthesis assays. The antiviral effects of the two lead compounds on mitigating virulent ASFV infection in porcine macrophages (PAMs) were also tested using similar methods, and the ability to inhibit pro-inflammatory cytokine production during virulent ASFV infection was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The screen identified five compounds that inhibited ASFV-induced CPE by greater than 50% and virus yield reduction experiments showed that two of these compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. Mechanistic analysis confirmed that both compounds potently inhibited early stages of ASFV infection and that the compounds also inhibited infection of PAMs by the virulent ASFV Arm/07 isolate. Importantly, during ASFV infection in PAM cells, both compounds markedly reduced the production of pro-inflammatory cytokines involved in disease pathogenesis while tetrandrine had a greater and more sustained anti-inflammatory effect than berbamine. CONCLUSIONS: Together, these findings support that dual-acting natural compounds with antiviral and anti-inflammatory properties hold promise as preventative and therapeutic agents to combat ASFV infection by simultaneously inhibiting viral replication and reducing virus-induced cytokine production.

Natural antimicrobial peptides as a source of new antiviral agents.

Current antiviral drugs are limited because of their adverse side effects and increased rate of resistance. In recent decades, much scientific effort has been invested in the discovery of new synthetic and natural compounds with promising antiviral properties. Among this new generation of compounds, antimicrobial peptides with antiviral activity have been described and are attracting attention due to their mechanism of action and biological properties. To understand the potential of antiviral peptides (AVPs), we analyse the antiviral activity of well-known AVP families isolated from different natural sources, discuss their physical-chemical properties, and demonstrate how AVP databases can guide us to design synthetic AVPs with better therapeutic properties. All considerations in this sphere of antiviral therapy clearly demonstrate the remarkable contribution that AVPs may make in conquering old as well as newly emerging viruses that plague humanity.

Synthesis, Antitumor Activity, and Docking Analysis of New Pyrido[3',2':4,5]furo(thieno)[3,2-d]pyrimidin-8-amines.

Continuing our research in the field of new heterocyclic compounds, herein we report on the synthesis and antitumor activity of new amino derivatives of pyrido[3',2':4,5](furo)thieno[3,2-d]pyrimidines as well as of two new heterocyclic systems: furo[2-e]imidazo[1,2-c]pyrimidine and furo[2,3-e]pyrimido[1,2-c]pyrimidine. Thus, by refluxing the 8-chloro derivatives of pyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidines with various amines, the relevant pyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidin-8-amines were obtained. Further, the cyclization of some amines under the action of phosphorus oxychloride led to the formation of new heterorings: imidazo[1,2-c]pyrimidine and pyrimido[1,2-c]pyrimidine. The possible antitumor activity of the newly synthesized compounds was evaluated in vitro. The biological tests evidenced that some of them showed pronounced antitumor activity. A study of the structure-activity relationships revealed that the compound activity depended mostly on the nature of the amine fragments. A docking analysis was also performed for the most active compounds.

Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus.

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

Flavonoids: promising natural compounds against viral infections.

Flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. Different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. This review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. We also present future perspectives on therapeutic applications of flavonoids against viral infections.

Apigenin inhibits African swine fever virus infection in vitro.

African swine fever virus (ASFV) is one of the most devastating diseases of domestic pigs for which no effective vaccines are available. Flavonoids, natural products isolated from plants, have been reported to have significant in vitro and in vivo antiviral activity against different viruses. Here, we tested the antiviral effect of five flavonoids on the replication of ASFV in Vero cells. Our results showed a potent, dose-dependent anti-ASFV effect of apigenin in vitro. Time-of-addition experiments revealed that apigenin was highly effective at the early stages of infection. Apigenin reduced the ASFV yield by more than 99.99 % when it was added at 1 hpi. The antiviral activity of apigenin was further investigated by evaluation of ASFV protein synthesis and viral factories. This flavonoid inhibited ASFV-specific protein synthesis and viral factory formation. ASFV-infected cells continuously treated with apigenin did not display a cytopathic effect. Further studies addressing the use of apigenin in vivo are needed.

A study of lymphoid organs and serum proinflammatory cytokines in pigs infected with African swine fever virus genotype II.

African swine fever virus (ASFV), the causative agent of one of the most important viral diseases of domestic pigs for which no vaccine is available, causes immune system disorders in infected animals. In this study, the serum levels of proinflammatory cytokines, as well as the histological and cellular constitution of lymphoid organs of pigs infected with ASFV genotype II were investigated. The results showed a high degree of lymphocyte depletion in the lymphoid organs, particularly in the spleen and lymph nodes, where ASFV infection led to a twofold decrease in the number of lymphocytes on the final day of infection. Additionally, ASFV-infected pigs had atypical forms of lymphocytes found in all lymphoid organs. In contrast to lymphocytes, the number of immature immune cells, particularly myelocytes, increased dramatically and reached a maximum on day 7 postinfection. The serum levels of TNF-α, IL-1ß, IL-6, and IL-8 were evaluated. Proinflammatory cytokines showed increased levels after ASFV infection, with peak values at 7 days postinfection, and this highlights their role in the pathogenesis of ASFV. In conclusion, this study showed that ASFV genotype II, like other highly virulent strains, causes severe pathological changes in the immune system of pigs.

Discovery of new antiviral agents through artificial intelligence: In vitro and in vivo results.

In this research, we employed a deep reinforcement learning (RL)-based molecule design platform to generate a diverse set of compounds targeting the neuraminidase (NA) of influenza A and B viruses. A total of 60,291 compounds were generated, of which 86.5 % displayed superior physicochemical properties compared to oseltamivir. After narrowing down the selection through computational filters, nine compounds with non-sialic acid-like structures were selected for in vitro experiments. We identified two compounds, DS-22-inf-009 and DS-22-inf-021 that effectively inhibited the NAs of both influenza A and B viruses (IAV and IBV), including H275Y mutant strains at low micromolar concentrations. Molecular dynamics simulations revealed a similar pattern of interaction with amino acid residues as oseltamivir. In cell-based assays, DS-22-inf-009 and DS-22-inf-021 inhibited IAV and IBV in a dose-dependent manner with EC50 values ranging from 0.29 µM to 2.31 µM. Furthermore, animal experiments showed that both DS-22-inf-009 and DS-22-inf-021 exerted antiviral activity in mice, conferring 65 % and 85 % protection from IAV (H1N1 pdm09), and 65 % and 100 % protection from IBV (Yamagata lineage), respectively. Thus, these findings demonstrate the potential of RL to generate compounds with promising antiviral properties.

Computational evaluation and benchmark study of 342 crystallographic holo-structures of SARS-CoV-2 Mpro enzyme.

The coronavirus disease 19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global health crisis with millions of confirmed cases and related deaths. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication and presents an attractive target for drug development. Despite the approval of some drugs, the search for effective treatments continues. In this study, we systematically evaluated 342 holo-crystal structures of Mpro to identify optimal conformations for structure-based virtual screening (SBVS). Our analysis revealed limited structural flexibility among the structures. Three docking programs, AutoDock Vina, rDock, and Glide were employed to assess the efficiency of virtual screening, revealing diverse performances across selected Mpro structures. We found that the structures 5RHE, 7DDC, and 7DPU (PDB Ids) consistently displayed the lowest EF, AUC, and BEDROCK scores. Furthermore, these structures demonstrated the worst pose prediction results in all docking programs. Two structural differences contribute to variations in docking performance: the absence of the S1 subsite in 7DDC and 7DPU, and the presence of a subpocket in the S2 subsite of 7DDC, 7DPU, and 5RHE. These findings underscore the importance of selecting appropriate Mpro conformations for SBVS, providing valuable insights for advancing drug discovery efforts.

Glycerol Monolaurate Inhibits Wild-Type African Swine Fever Virus Infection in Porcine Macrophages.

Naturally abundant antimicrobial lipids, such as fatty acids and monoglycerides, that disrupt membrane-enveloped viruses are promising mitigants to inhibit African swine fever virus (ASFV). Among mitigant candidates in this class, glycerol monolaurate (GML) has demonstrated particularly high antiviral activity against laboratory-adapted ASFV strains. However, there is an outstanding need to further determine the effects of GML on wild-type ASFV strains, which can have different virulence levels and sensitivities to membrane-disrupting compounds as compared to laboratory-adapted strains. Herein, we investigated the antiviral effects of GML on a highly virulent strain of a wild-type ASFV isolate (Armenia/07) in an in vitro porcine macrophage model. GML treatment caused a concentration-dependent reduction in viral infectivity, and there was a sharp transition between inactive and active GML concentrations. Low GML concentrations had negligible effect on viral infectivity, whereas sufficiently high GML concentrations caused a >99% decrease in viral infectivity. The concentration onset of antiviral activity matched the critical micelle concentration (CMC) value of GML, reinforcing that GML micelles play a critical role in enabling anti-ASFV activity. These findings validate that GML can potently inhibit wild-type ASFV infection of porcine macrophages and support a biophysical explanation to guide antimicrobial lipid performance optimization for pathogen mitigation applications.

Design of new chemical entities targeting both native and H275Y mutant influenza a virus by deep reinforcement learning.

Influenza virus remains a major public health challenge due to its high morbidity and mortality and seasonal surge. Although antiviral drugs against the influenza virus are widely used as a first-line defense, the virus undergoes rapid genetic changes, resulting in the emergence of drug-resistant strains. Thus, new antiviral drugs that can outwit resistant strains are of significant importance. Herein, we used deep reinforcement learning (RL) algorithm to design new chemical entities (NCEs) that are able to bind to the native and H275Y mutant (oseltamivir-resistant) neuraminidases (NAs) of influenza A virus with better binding energy than oseltamivir. We generated more than 66211 NCEs, which were prioritized based on the filtering rules, structural alerts, and synthetic accessibility. Then, 18 NCEs with better MM/PBSA scores than oseltamivir were further analyzed in molecular dynamics (MD) simulations conducted for 100 ns. The MD experiments showed that 8 NCEs formed very stable complexes with the binding pocket of both native and H275Y mutant NAs of H1N1. Furthermore, most NCEs demonstrated much better binding affinity to group 2 (N2, N3, and N9) and influenza B virus NAs than oseltamivir. Although all 8 NCEs have non-sialic acid-like structures, they showed a similar binding mode as oseltamivir, indicating that it is possible to find new scaffolds with better binding and antiviral properties than sialic acid-like inhibitors. In conclusion, we have designed potential compounds as antiviral candidates for further synthesis and testing against wild and mutant influenza virus.Communicated by Ramaswamy H. Sarma.

Advanced virtual screening enables the discovery of a host-targeting and broad-spectrum antiviral agent.

We employed an advanced virtual screening (AVS) approach to identify potential inhibitors of human dihydroorotate dehydrogenase (DHODH), a validated target for development of broad-spectrum antivirals. We screened a library of 495118 compounds and identified 495 compounds that exhibited better binding scores than the reference ligands involved in the screening. From the top 100 compounds, we selected 28 based on their consensus docking scores and structural novelty. Then, we conducted in vitro experiments to investigate the antiviral activity of selected compounds on HSV-1 infection, which is susceptible to DHODH inhibitors. Among the tested compounds, seven displayed statistically significant antiviral effects, with Comp 19 being the most potent inhibitor. We found that Comp 19 exerted its antiviral effect in a dose-dependent manner (IC50 = 1.1 µM) and exhibited the most significant antiviral effect when added before viral infection. In the biochemical assay, Comp 19 inhibited human DHODH in a dose-dependent manner with the IC50 value of 7.3 µM. Long-timescale molecular dynamics simulations (1000 ns) revealed that Comp 19 formed a very stable complex with human DHODH. Comp 19 also displayed broad-spectrum antiviral activity and suppressed cytokine production in THP-1 cells. Overall, our study provides evidence that AVS could be successfully implemented to discover novel DHODH inhibitors with broad-spectrum antiviral activity.

Pathology of porcine peripheral white blood cells during infection with African swine fever virus.

BACKGROUND: African swine fever virus (ASFV) is the causative agent of African swine fever (ASF) that is the significant disease of domestic pigs. Several studies showed that ASFV can influence on porcine blood cells in vitro. Thus, we asked ourselves whether ASFV infection results in changes in porcine blood cells in vivo. A series of experiments were performed in order to investigate the effects of ASFV infection on porcine peripheral white blood cells. Nine pigs were inoculated by intramuscular injection with 104 50% hemadsorbing doses of virus (genotype II) distributed in Armenia and Georgia. The total number of fifteen cell types was calculated during experimental infection. RESULTS: Although band-to-segmented neutrophils ratio became much higher (3.5) in infected pigs than in control group (0.3), marked neutropenia and lymphopenia were detected from 2 to 3 days post-infection. In addition to band neutrophils, the high number of other immature white blood cells, such as metamyelocytes, was observed during the course of infection. From the beginning of infection, atypical lymphocytes, with altered nuclear shape, arose and became 15% of total cells in the final phase of infection. Image scanning cytometry revealed hyperdiploid DNA content in atypical lymphocytes only from 5 days post-infection, indicating that DNA synthesis in pathological lymphocytes occurred in the later stages of infection. CONCLUSION: From this study, it can be concluded that ASFV infection leads to serious changes in composition of white blood cells. Particularly, acute ASFV infection in vivo is accompanied with the emergence of immature cells and atypical lymphocytes in the host blood. The mechanisms underlying atypical cell formation remain to be elucidated.

Antiviral activity of brequinar against African swine fever virus infection in vitro.

African swine fever virus (ASFV) is a double-stranded DNA virus that causes an acute and hemorrhagic disease in domestic swine, resulting in significant economic losses to the global porcine industry. The lack of vaccines and antiviral drugs highlights the urgent need for antiviral studies against ASFV. Here, we report that brequinar (BQR), which is a specific inhibitor of dihydroorotate dehydrogenase, robustly inhibits ASFV replication in Vero cells, as well as in porcine macrophages. We demonstrate that BQR exerts its antiviral activity in a dose-dependent manner through the depletion of pyrimidine pool. Although BQR does not affect the synthesis of an early viral protein, pI215L, the synthesis of late viral proteins, p17 and p72, is suppressed in the presence of BQR. We also show that BQR is able to induce cellular antiviral response in ASFV-infected macrophages by enhancing the expression of interferon-stimulated genes. Taken together, our study reveals that targeting nucleotide biosynthesis represents a promising strategy for developing antiviral agents against ASFV.

Combination of consensus and ensemble docking strategies for the discovery of human dihydroorotate dehydrogenase inhibitors.

The inconsistencies in the performance of the virtual screening (VS) process, depending on the used software and structural conformation of the protein, is a challenging issue in the drug design and discovery field. Varying performance, especially in terms of early recognition of the potential hit compounds, negatively affects the whole process and leads to unnecessary waste of the time and resources. Appropriate application of the ensemble docking and consensus-scoring approaches can significantly increase reliability of the VS results. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway. It is considered as a valuable therapeutic target in cancer, autoimmune and viral diseases. Based on the conducted benchmark study and analysis of the effect of different combinations of the applied methods and approaches, here we suggested a structure-based virtual screening (SBVS) workflow that can be used to increase the reliability of VS.

Flavonoid Library Screening Reveals Kaempferol as a Potential Antiviral Agent Against African Swine Fever Virus.

Naturally occurring plant flavonoids are a promising class of antiviral agents to inhibit African swine fever virus (ASFV), which causes highly fatal disease in pigs and is a major threat to the swine industry. Currently known flavonoids with anti-ASFV activity demonstrate a wide range of antiviral mechanisms, which motivates exploration of new antiviral candidates within this class. The objective of this study was to determine whether other flavonoids may significantly inhibit ASFV infection in vitro. We performed a cell-based library screen of 90 flavonoids. Our screening method allowed us to track the development of virus-induced cytopathic effect by MTT in the presence of tested flavonoids. This screening method was shown to be robust for hit identification, with an average Z-factor of 0.683. We identified nine compounds that inhibit ASFV Ba71V strain in Vero cells. Among them, kaempferol was the most potent and exhibited dose-dependent inhibition, which occurred through a virostatic effect. Time-of-addition studies revealed that kaempferol acts on the entry and post-entry stages of the ASFV replication cycle and impairs viral protein and DNA synthesis. It was further identified that kaempferol induces autophagy in ASFV-infected Vero cells, which is related to its antiviral activity and could be partially abrogated by the addition of an autophagy inhibitor. Kaempferol also exhibited dose-dependent inhibition of a highly virulent ASFV Arm/07 isolate in porcine macrophages. Together, these findings support that kaempferol is a promising anti-ASFV agent and has a distinct antiviral mechanism compared to other anti-ASFV flavonoids.

A new microtubule-stabilizing agent shows potent antiviral effects against African swine fever virus with no cytotoxicity.

African swine fever virus (ASFV) is the causal agent of a fatal disease of domestic swine for which no effective antiviral drugs are available. Recently, it has been shown that microtubule-targeting agents hamper the infection cycle of different viruses. In this study, we conducted in silico screening against the colchicine binding site (CBS) of tubulin and found three new compounds with anti-ASFV activity. The most promising antiviral compound (6b) reduced ASFV replication in a dose-dependent manner (IC50 = 19.5 µM) with no cellular (CC50 > 500 µM) and animal toxicity (up to 100 mg/kg). Results also revealed that compound 6b interfered with ASFV attachment, internalization and egress, with time-of-addition assays, showing that compound 6b has higher antiviral effects when added within 2-8 h post-infection. This compound significantly inhibited viral DNA replication and disrupted viral protein synthesis. Experiments with ASFV-infected porcine macrophages disclosed that antiviral effects of the compound 6b were similar to its effects in Vero cells. Tubulin polymerization assay and confocal microscopy demonstrated that compound 6b promoted tubulin polymerization, acting as a microtubule-stabilizing, rather than a destabilizing agent in cells. In conclusion, this work emphasizes the idea that microtubules can be targets for drug development against ASFV.

Inhibition of African swine fever virus in liquid and feed by medium-chain fatty acids and glycerol monolaurate.

BACKGROUND: The ongoing African swine fever virus (ASFv) epidemic has had a major impact on pig production globally and biosecurity efforts to curb ASFv infectivity and transmission are a high priority. It has been recently identified that feed and feed ingredients, along with drinking water, can serve as transmission vehicles and might facilitate transboundary spread of ASFv. Thus, it is important to test the antiviral activity of regulatory compatible, antiviral feed additives that might inhibit ASFv infectivity in feed. One promising group of feed additive candidates includes medium-chain fatty acids (MCFA) and monoglyceride derivatives, which are known to disrupt the lipid membrane surrounding certain enveloped viruses and bacteria. RESULTS: The antiviral activities of selected MCFA, namely caprylic, capric, and lauric acids, and a related monoglyceride, glycerol monolaurate (GML), to inhibit ASFv in liquid and feed conditions were investigated and suitable compounds and inclusion rates were identified that might be useful for mitigating ASFv in feed environments. Antiviral assays showed that all tested MCFA and GML inhibit ASFv. GML was more potent than MCFA because it worked at a lower concentration and inhibited ASFv due to direct virucidal activity along with one or more other antiviral mechanisms. Dose-dependent feed experiments further showed that sufficiently high GML doses can significantly reduce ASFv infectivity in feed in a linear manner in periods as short as 30 min, as determined by infectious viral titer measurements. Enzyme-linked immunosorbent assay (ELISA) experiments revealed that GML treatment also hinders antibody recognition of the membrane-associated ASFv p72 structural protein, which likely relates to protein conformational changes arising from viral membrane disruption. CONCLUSION: Together, the findings in this study indicate that MCFA and GML inhibit ASFv in liquid conditions and that GML is also able to reduce ASFv infectivity in feed, which may help to curb disease transmission.

Antiviral agents against African swine fever virus.

African swine fever virus (ASFV) is a significant transboundary virus that continues to spread outside Africa in Europe and most recently to China, Vietnam and Cambodia. Pigs infected with highly virulent ASFV develop a hemorrhagic fever like illness with high lethality reaching up to 100%. There are no vaccines or antiviral drugs available for the prevention or treatment of ASFV infections. We here review molecules that have been reported to inhibit ASFV replication, either as direct-acting antivirals or host-targeting drugs as well as those that act via a yet unknown mechanism. Prospects for future antiviral research against ASFV are also discussed.

Inhibition of African swine fever virus infection by genkwanin.

African swine fever virus (ASFV) is the causative agent of an economically important disease of pigs for which no effective vaccines or antiviral drugs are available. Recent outbreaks in EU countries and China have highlighted the critical role of antiviral research in combating this disease. We have previously shown that apigenin, a naturally occurring plant flavone, possesses significant anti-ASFV activity. However, apigenin is practically insoluble in highly polar solvents and it occurs typically in derivative forms in plants. Here we screened several commercially available apigenin derivatives for their ability to inhibit ASFV Ba71V strain in Vero cells. Among them, genkwanin showed significant inhibition of ASFV, reducing viral titer from 6.5 ±â€¯0.1 to 4.75 ±â€¯0.25 log TCID/ml in a dose-dependent manner (IC50 = 2.9 µM and SI = 205.2). Genkwanin reduced the levels of ASFV early and late proteins, as well as viral DNA synthesis. Our further experiments indicated that genkwanin is able to inhibit ASFV infection at entry and egress stages. Finally, genkwanin displayed potent antiviral activity against highly virulent ASFV isolate currently circulating in Europe and China, emphasizing its value as candidate for antiviral drug development.