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Background: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines., Methods: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model., Findings: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3D pol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice., Interpretation: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application., Funding: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC)., Competing Interests: Declaration of interests HF received grants from Synthetic Biology Translational Research Program, School of Medicine, National University of Singapore., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness generally manifests as a mild disease in young children and immunocompromised adults. It has however emerged as a significant public health threat in recent years as outbreaks have been occurring regularly, especially in the Asia-Pacific. The disease can result from infections by a wide variety of human enteroviruses, particularly, Enterovirus A71 (EV-A71) has garnered more attention due to its association with severe disease in infected patients. Despite the potential to result severe neurological complications or even fatality, there is currently no effective antiviral for treatment of EV-A71 infections and the only vaccines available are restricted to distribution in China. In this study, we report the in vitro and in vivo evaluation of two candidate antiviral compounds active against EV-A71, a viral capsid inhibitor (G197) and a novel host-targeting phosphatidylinositol 4-kinase III beta inhibitor (N373) which, especially when used in combination, can significantly improve the survival and pathology of infected mice.

Dengue infection is one of the most deleterious public health concerns for two-billion world population being at risk. Plasma leakage, hemorrhage, and shock in severe cases were caused by immunological derangement from secondary heterotypic infection. Flavanone, commonly found in medicinal plants, previously showed potential as anti-dengue inhibitors for its direct antiviral effects and suppressing the pro-inflammatory cytokine from dengue immunopathogenesis. Here, we chemically modified flavanones, pinocembrin and pinostrobin, by halogenation and characterized them as potential dengue 2 inhibitors and performed toxicity tests in human-derived cells and in vivo animal model. Dibromopinocembrin and dibromopinostrobin inhibited dengue serotype 2 at the EC 50 s of 2.0640 ± 0.7537 and 5.8567 ± 0.5074 µM with at the CC 50 s of 67.2082 ± 0.9731 and >100 µM, respectively. Both of the compounds also showed minimal toxicity against adult C57BL/6 mice assessed by ALT and Cr levels in day one, three, and eight post-intravenous administration. Computational studies suggested the potential target be likely the NS5 methyltransferase at SAM-binding pocket. Taken together, these two brominated flavanones are potential leads for further drug discovery investigation.

COVID-19 was declared a pandemic by the World Health Organization on March 11, 2020. This novel coronavirus disease, caused by the SARS-CoV-2 virus, has resulted in severe and unprecedented social and economic disruptions globally. Since the discovery of COVID-19 in December 2019, numerous antivirals have been tested for efficacy against SARS-CoV-2 in vitro and also clinically to treat this disease. This review article discusses the main antiviral strategies currently employed and summarizes reported in vitro and in vivo efficacies of key antiviral compounds in use.

Over the course of the last 50 years, the emergence of several arboviruses have resulted in countless outbreaks globally. With a high proportion of infections occurring in tropical and subtropical regions where arthropods tend to be abundant, Asia in particular is a region that is heavily affected by arboviral diseases caused by dengue, Japanese encephalitis, West Nile, Zika, and chikungunya viruses. Major gaps in protection against the most significant emerging arboviruses remains as there are currently no antivirals available, and vaccines are only available for some. A potential source of antiviral compounds could be discovered in natural products-such as vegetables, fruits, flowers, herbal plants, marine organisms and microorganisms-from which various compounds have been documented to exhibit antiviral activities and are expected to have good tolerability and minimal side effects. Polyphenols and plant extracts have been extensively studied for their antiviral properties against arboviruses and have demonstrated promising results. With an abundance of natural products to screen for new antiviral compounds, it is highly optimistic that natural products will continue to play an important role in contributing to antiviral drug development and in reducing the global infection burden of arboviruses.

Chikungunya virus (CHIKV) is an alphavirus that causes a febrile illness accompanied by myalgia and arthralgia. Despite having re-emerged as a significant public health threat, there are no approved therapeutics or prophylactics for CHIKV infection. In this study, we explored the anti-CHIKV effects of proteasome inhibitors and their potential mechanism of antiviral action. A panel of proteasome inhibitors with different functional groups reduced CHIKV infectious titers in a dose-dependent manner. Bortezomib, which has been FDA-approved for multiple myeloma and mantle cell lymphoma, was further investigated in downstream studies. The inhibitory activities of bortezomib were confirmed using different cellular models and CHIKV strains. Time-of-addition and time-of-removal studies suggested that bortezomib inhibited CHIKV at an early, post-entry stage of replication. In western blot analysis, bortezomib treatment resulted in a prominent decrease in structural protein levels as early as 6 hpi. Contrastingly, nsP4 levels showed strong elevations across all time-points. NsP2 and nsP3 levels showed a fluctuating trend, with some elevations between 12 to 20 hpi. Finally, qRT-PCR data revealed increased levels of both positive- and negative-sense CHIKV RNA at late stages of infection. It is likely that the reductions in structural protein levels is a major factor in the observed reductions in virus titer, with the alterations in non-structural protein ratios potentially being a contributing factor. Proteasome inhibitors like bortezomib likely disrupt CHIKV replication through a variety of complex mechanisms and may display a potential for use as therapeutics against CHIKV infection. They also represent valuable tools for studies of CHIKV molecular biology and virus-host interactions., Competing Interests: The authors have declared that no competing interests exist.

Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine's synergistic effects with interferon-β at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.

Dengue virus, the causative agent for the dengue fever, infects approximately 50-100 million people worldwide per year. The high incidence of dengue fever, along with its potential to develop into a severe, life-threatening form, resulted in great interest in the discovery of an antiviral against it. In this study, we constructed a DENV2-EGFP infectious clone, established a fluorescence-based, high-throughput screening platform, and conducted a screen for anti-DENV compounds on a flavonoid-derivative library, Amongst the hits identified, ST081006 was found to be a strong inhibitor of the DENV replication. Time-course studies suggest that the presence of ST081006 is necessary to inhibit successive rounds of virus replication. Further investigations demonstrated that ST081006 affects the synthesis of both viral protein and viral RNA, and one anti-DENV mechanism is the direct inhibition of viral protein synthesis. The replication of all dengue serotypes, along with that of the enterovirus EV-A71, was shown to be affected by ST081006. Attempts to generate ST081006-resistant DENV were unsuccessful, and thus hints at host factors as potential drug target. Together, these results suggest that ST081006 affect DENV replication, likely by acting on a target involved in the viral protein and/or RNA synthesis pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) disease outbreaks continue to develop across the globe with significant associated mortality and economic burden, yet no treatment has been approved to combat this virus. In an attempt to identify novel drug candidates as therapeutics for DENV infection, we evaluated four US Food and Drug Administration (FDA) approved drugs including aminolevullic acid, azelaic acid, mitoxantrone hydrochloride, and quinine sulfate, and tested their ability to inhibit DENV replication using focus-forming unit assay to quantify virus production. Of the four investigated compounds, quinine was found to have the most pronounced anti-DENV activity. Quinine inhibited DENV production of DENV by about 80% compared to untreated controls, while the other three drugs decreased virus production by only about 50%. Moreover, quinine inhibited DENV production of all four serotypes of DENV. Reduction in virus production was documented in three different cell lines of human origin. Quinine significantly inhibited DENV replication by reducing DENV RNA and viral protein synthesis in a dose-dependent manner. In addition, quinine ameliorated expression of genes related to innate immune response. These findings suggest the efficacy of quinine for stimulating antiviral genes to reduce DENV replication. The antiviral activity of quinine observed in this study may have applicability in the development of new drug therapies against DENV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) has re-emerged as one of the many medically important arboviruses that have spread rampantly across the world in the past decade. Infected patients come down with acute fever and rashes, and a portion of them suffer from both acute and chronic arthralgia. Currently, there are no targeted therapeutics against this debilitating virus. One approach to develop potential therapeutics is by understanding the viral-host interactions. However, to date, there has been limited research undertaken in this area. In this review, we attempt to briefly describe and update the functions of the different CHIKV proteins and their respective interacting host partners. In addition, we also survey the literature for other reported host factors and pathways involved during CHIKV infection. There is a pressing need for an in-depth understanding of the interaction between the host environment and CHIKV in order to generate potential therapeutics., Competing Interests: The authors declare no conflict of interest.

Hand Foot Mouth Disease (HFMD), resulting from human enterovirus A71 (HEVA71) infection can cause severe neurological complications leading to fatality in young children. Currently, there is no approved antiviral for therapeutic treatment against HEVA71 infection. In this study, a 500-compound flavonoid library was screened to identify potential inhibitors of HEVA71 using high-throughput immunofluorescence-based phenotypic screening method. Two lead flavonoid compounds, ST077124 and ST024734 at the non-cytotoxic concentration of 50 μM were found to be effective antivirals that inhibited replication of HEVA71, reducing infectious viral titers by 3.5 log 10  PFU/ml and 2.5 log 10  PFU/ml respectively. Our study revealed that ST077124 is a specific antiviral compound that inhibits human enteroviruses while ST024734 exhibited antiviral activity against human enteroviruses as well as dengue virus type-2. We also identified that both compounds affected the viral RNA transcription and translation machinery of HEVA71 but did not interfere with the viral internal ribosomal entry site (IRES) activity. Hence, our findings strongly suggest that ST077124 and ST024734 are effective antiviral compounds of minimal cytotoxicity and could serve as promising therapeutic agents against HEVA71 infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1 log PFU/ml reduction of the virus infectious titre., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A common childhood affliction of viral origin in young children and immunocompromised adults, the Hand, Foot and Mouth Disease (HFMD) has become a significant public health concern in the Asia-Pacific Region. Characterized by the appearance of vesiculopapular rashes on the hands, feet and mouth, the disease is generally mild and self-limiting. In a minority of cases, patients can develop neurological complications that could result in permanent morbidity or even fatality. In the absence of a specific antiviral for treatment, medical care is limited to supportive and symptomatic relief, presenting a need for more research into an effective antiviral to be used in the management of the disease. In this study, we evaluated the efficacy of chloroquine, a FDA-approved lysosomotropic agent, against several serotypes of HFMD-associated enteroviruses, including EV-A71, in reducing infectious virus production. We have also evaluated chloroquine in a murine model of EV-A71 infection to ascertain its antiviral efficacy in vivo. The results suggest that chloroquine could be a broad-acting antiviral effective against HFMD-associated enteroviruses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.

The Hand, Foot and Mouth Disease (HFMD) can result from infections by a plethora of human enteroviruses of the species Enterovirus A and B. These infections are highly contagious, resulting in regular outbreaks especially in the Asia-Pacific Region in the recent decade. Although this disease is generally a childhood affliction which manifests as a mild, febrile illness accompanied by the vesicles on the hands, feet and mouth, permanent morbidity or even fatality can result from severe forms of the disease in a subset of the infected patients. The N-terminal myristoylation signal (MGXXXS) of viral capsid protein VP4, one of the four viral structural proteins, is an extremely well conserved feature of enteroviruses, a potential antiviral target that may yield broad-spectrum inhibitors of HFMD. In this study, we have confirmed through the use of small interfering RNAs, human N-myristoyltransferase 1 plays an integral role in human Enterovirus 71 replication. Subsequent studies by inhibition of myristoylation using different myristic acid analogues elicited differential effects on the virus replication in human rhabdomyosarcoma cells. In particular, 2-hydroxymyristic acid specifically inhibited the cleavage between VP4 and VP2, part of the virion maturation process required to ensure infectivity of progeny virions while 4-oxatetradecanoic acid reduced the synthesis of viral RNA. These findings suggest that the requirement of a myristate moiety in viral structural protein precursor cleavage can serve as a viable antiviral target for further research., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study focuses on the antiviral activity of selected flavonoids against the Chikungunya virus (CHIKV), a mosquito-transmitted virus that can cause incapacitating arthritis in infected individuals. Based on the results of screening on Vero cells, the tested compounds were evaluated further with various assays, including cytotoxicity assay, virus yield assay by quantitative reverse transcription polymerase chain reaction (qRT-PCR), virus RNA replication assay with a CHIKV replicon cell line, Western blotting, and quantitative immunofluorescence assay. Baicalein, fisetin, and quercetagetin displayed potent inhibition of CHIKV infection, with 50% inhibitory concentrations [IC50] of 1.891 μg/ml (6.997 μM), 8.444 μg/ml (29.5 μM), and 13.85 μg/ml (43.52 μM), respectively, and with minimal cytotoxicity. The time-of-addition studies and various antiviral assays demonstrated that baicalein and quercetagetin mainly inhibited CHIKV binding to the Vero cells and displayed potent activity against extracellular CHIKV particles. The qRT-PCR, immunofluorescence assay, and Western blot analyses indicated that each of these flavonoids affects CHIKV RNA production and viral protein expression. These data provide the first evidence of the intracellular anti-CHIKV activity of baicalein, fisetin, and quercetagetin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has recently engendered large epidemics around the world. There is no specific antiviral for treatment of patients infected with CHIKV, and development of compounds with significant anti-CHIKV activity that can be further developed to a practical therapy is urgently required. Andrographolide is derived from Andrographis paniculata, a herb traditionally used to treat a number of conditions including infections. This study sought to determine the potential of andrographolide as an inhibitor of CHIKV infection. Andrographolide showed good inhibition of CHIKV infection and reduced virus production by approximately 3log10 with a 50% effective concentration (EC50) of 77 μM without cytotoxicity. Time-of-addition and RNA transfection studies showed that andrographolide affected CHIKV replication and the activity of andrographolide was shown to be cell type independent. This study suggests that andrographolide has the potential to be developed further as an anti-CHIKV therapeutic agent.

Chikungunya virus (CHIKV) infection in human is associated with debilitating and persistent arthralgia and arthritis. Currently, there is no specific vaccine or effective antiviral available. Anti-CHIKV Phosphorodiamidate Morpholino Oligomer (CPMO) was evaluated for its antiviral efficacy and cytotoxcity in human cells and neonate murine model. Two CPMOs were designed to block translation initiation of a highly conserved sequence in CHIKV non-structural and structural polyprotein, respectively. Pre-treatment of HeLa cells with CPMO1 significantly suppressed CHIKV titre, CHIKV E2 protein expression and prevented CHIKV-induced CPE. CPMO1 activity was also CHIKV-specific as shown by the lack of cross-reactivity against SINV or DENV replication. When administered prophylactically in neonate mice, 15 μg/g CPMO1v conferred 100% survival against CHIKV disease. In parallel, these mice demonstrated significant reduction in viremia and viral load in various tissues. Immunohistological examination of skeletal muscles and liver of CPMO1v-treated mice also showed healthy tissue morphology, in contrast to evident manifestation of CHIKV pathogenesis in PBS- or scrambled sCPMO1v-treated groups. Taken together, our findings highlight for the first time that CPMO1v has strong protective effect against CHIKV infection. This warrants future development of morpholino as an alternative antiviral agent to address CHIKV infection in clinical applications.

The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection.

The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 μM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 μM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dengue infection poses a serious threat globally due to its recent rapid spread and rise in incidence. Currently, there is no approved vaccine or effective antiviral drug for dengue virus infection. In response to the urgent need for the development of an effective antiviral for dengue virus, the US Drug Collection library was screened in this study to identify compounds with anti-dengue activities. Lanatoside C, an FDA approved cardiac glycoside was identified as a candidate anti-dengue compound. Our data revealed that lanatoside C has an IC50 of 0.19μM for dengue virus infection in HuH-7 cells. Dose-dependent reduction in dengue viral RNA and viral proteins synthesis were also observed upon treatment with increasing concentrations of lanatoside C. Time of addition study indicated that lanatoside C inhibits the early processes of the dengue virus replication cycle. Furthermore, lanatoside C can effectively inhibit all four serotypes of dengue virus, flavivirus Kunjin, alphavirus Chikungunya and Sindbis virus as well as the human enterovirus 71. These findings suggest that lanatoside C possesses broad spectrum antiviral activity against several groups of positive-sense RNA viruses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC50 values of 1.3 μM and 2.7 μM, respectively, and selectivity indices of 10.9 and 9.2, respectively.

Chikungunya virus (CHIKV) has re-emerged as a significant public health threat since the 2005 chikungunya fever epidemic in La Réunion. Driven by the medical importance of this virus, as well as the lack of approved antivirals, research into the field of CHIKV antivirals has recently intensified. Potential therapeutics that have been reported to show anti-CHIKV activity in vitro range from known broad-spectrum antivirals like chloroquine to novel strategies involving RNA silencing technology. Although most of the earlier efforts focused on compounds that target host components, some recent studies have reported viral targets such as nonstructural proteins. This article examines the reported in vitro and in vivo efficacies, as well as the therapeutic potential of these antiviral compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting ≥70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC(50)] = 0.24 μM) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogenic virus in the swine production. Current vaccines against PRRSV do not induce sterile immunity and the virus evolves at a rapid rate with frequent appearances of new strains. In this study, we screened a library of 502 highly purified natural product compounds to identify specific inhibitors of PRRSV replication cycle. Our observations showed that many of the inhibitory compounds identified have activity on the cellular ion transport mechanisms. We identified for the first time, four compounds which inhibit the PRRSV replication cycle at micro molar concentration or less, namely, 12-deoxyphorbol 13-phenylacetate 20-acetate, ouabain, bufalin and valinomycin. Further, we have identified 15 other compounds which can inhibit the PRRSV replication at the concentration of 8μM. This study provides a basis for further development of pharmacological agents to inhibit PRRSV replication., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A small molecule library containing 480 known bioactive compounds was screened for antiviral activity against poliovirus (PV) using a cellular fluorescence resonance energy transfer (FRET) assay for viral protease activity. The infected reporter cells treated with the viral replication-suppressing compounds were examined via fluorescence microscope 7.5 h postinfection. Twelve molecules showed moderate to potent antiviral activity at concentrations less than 32 microM during the primary screening. Three compounds, anisomycin, linoleic acid, and lycorine, were chosen for validation. A dose-dependent cytotoxicity assay and a secondary screening using conventional plaque assay were conducted to confirm the results. The developed method can be used for rapid screening for molecules with antiviral activity.

The COVID-19 pandemic has brought about unprecedented medical and healthcare challenges worldwide. With the continual emergence and spread of new COVID-19 variants, four drug compound libraries were interrogated for their antiviral activities against SARS-CoV-2. Here, we show that the drug screen has resulted in 121 promising anti-SARS-CoV-2 compounds, of which seven were further shortlisted for hit validation: citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate. In particular, the active form of vitamin D, calcitriol, exhibits strong potency against SARS-CoV-2 on cell-based assays and is shown to work by modulating the vitamin D receptor pathway to increase antimicrobial peptide cathelicidin expression. However, the weight, survival rate, physiological conditions, histological scoring, and virus titre between SARS-CoV-2 infected K18-hACE2 mice pre-treated or post-treated with calcitriol were negligible, indicating that the differential effects of calcitriol may be due to differences in vitamin D metabolism in mice and warrants future investigation using other animal models. [ABSTRACT FROM AUTHOR]

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using an in vitro model of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10 and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) – above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10 of live virus and 2.5-log10 of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10 and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as future in vivo experiments and clinical trials for the treatment of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy. [ABSTRACT FROM AUTHOR]

Background: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. Methods: Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. Results: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. Conclusion: Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection. [ABSTRACT FROM AUTHOR]

Plants have had historical significance in medicine since the beginning of civilization. The oldest medical pharmacopeias of the African, Arabian, and Asian countries solely utilize plants and herbs to treat pain, oral diseases, skin diseases, microbial infections, multiple types of cancers, reproductive disorders among a myriad of other ailments. The World Health Organization (WHO) estimates that over 65% of the world population solely utilize botanical preparations as medicine. Due to the abundance of plants, plant-derived medicines are more readily accessible, affordable, convenient, and have safer side-effect profiles than synthetic drugs. Plant-based decoctions have been a significant part of Jamaican traditional folklore medicine. Jamaica is of particular interest because it has approximately 52% of the established medicinal plants that exist on earth. This makes the island particularly welcoming for rigorous scientific research on the medicinal value of plants and the development of phytomedicine thereof. Viral infections caused by the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), hepatitis virus B and C, influenza A virus, and the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) present a significant global burden. This is a review of some important Jamaican medicinal plants, with particular reference to their antiviral activity. [ABSTRACT FROM AUTHOR]

Curcumin is a natural compound that has many medical applications. However, its low solubility and poor stability could impede its clinical applications. The present study aimed to formulate dry proniosomes to overcome these pitfalls and improve the therapeutic efficacy of Curcumin. Curcumin-loaded proniosomes were fabricated by the slurry method according to 32 factorial design using Design-Expert software to demonstrate the impact of different independent variables on entrapment efficiency (EE%) and % drug released after 12 h (Q12h). The optimized formula (F5) was selected according to the desirability criteria. F5 exhibited good flowability and appeared, after reconstitution, as spherical nanovesicles with EE% of 89.94 ± 2.31% and Q12h of 70.89 ± 1.62%. F5 demonstrated higher stability and a significant enhancement of Q12h than the corresponding niosomes. The docking study investigated the ability of Curcumin to bind effectively with the active site of DNA polymerase of Herpes simplex virus (HSV). The antiviral activity and the safety of F5 were significantly higher than Curcumin. F5 improved the safety of Acyclovir (ACV) and reduced its effective dose that produced a 100% reduction of viral plaques. Proniosomes could be promising stable carriers of Curcumin to be used as a safe and efficient antiviral agent. [ABSTRACT FROM AUTHOR]

Mosquito-borne Zika virus (ZIKV) is a Flavivirus that came under intense study from 2014 to 2016 for its well-known ability to cause congenital microcephaly in fetuses and neurological Guillain–Barré disease in adults. Substantial research on screening antiviral agents against ZIKV and preventing ZIKV infection are globally underway, but Food and Drug Administration (FDA)-approved treatments are not available yet. Compounds from Chinese medicinal herbs may offer an opportunity for potential therapies for anti-ZIKV infection. In this study, we evaluated the antiviral efficacy of harringtonine against ZIKV. Harringtonine possessed anti-ZIKV properties against the binding, entry, replication, and release stage through the virus life cycle. In addition, harringtonine have strong virucidal effects in ZIKV and exhibited prophylaxis antiviral ability prior ZIKV infection. The antiviral activity also observed in the treatment against Japanese encephalitis reporter virus (RP9-GFP strain). Overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-ZIKV infection therapies. [ABSTRACT FROM AUTHOR]

In invertebrate eukaryotes and prokaryotes, respectively, the RNAi and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) pathways are highly specific and efficient RNA and DNA interference systems, and are well characterised as potent antiviral systems. It has become possible to recruit or reconstitute these pathways in mammalian cells, where they can be directed against desired host or viral targets. The RNAi and CRISPR-Cas systems can therefore yield ideal antiviral therapeutics, capable of specific and efficient viral inhibition with minimal off-target effects, but development of such therapeutics can be slow. This review covers recent advances made towards developing RNAi or CRISPR-Cas strategies for clinical use. These studies address the delivery, toxicity or target design issues that typically plague the in vivo or clinical use of these technologies. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne virus that has risen to prominence as a significant threat to public health in the recent decade. Since its re-emergence in 2007, ZIKV has spread at an alarming rate and has since become endemic to multiple regions around the world. Infections are primarily asymptomatic, however the virus has become associated with the development of severe neurological complications such as Guillain–Barré syndrome (GBS) and congenital microcephaly. At present, there are currently no approved antivirals for ZIKV infections. In this study, we utilised a phenotype-based screening platform to perform a high-throughput screen on a 1172-compound US FDA-approved drug library to identify potential novel inhibitors against ZIKV. Candesartan cilexetil, an angiotensin II receptor inhibitor, displayed potent inhibition effects against ZIKV and subsequent downstream time-course studies revealed that it targets a post-entry stage(s) of the ZIKV replication cycle. Moreover, candesartan cilexetil also inhibited viral RNA production and viral protein synthesis. Candesartan cilexetil also exhibited antiviral effects against Dengue virus serotype-2 (DENV2), Kunjin virus (KUNV) and Chikungunya virus (CHIKV), indicating that its antiviral properties may not be restricted to ZIKV. Our study has demonstrated for the first time the potential application of candesartan cilexetil as an antiviral. • A high-throughput screen was performed on a 1172-compound FDA-approved library to identify potential inhibitors of ZIKV. • Candesartan cilexetil was identified as a novel and potent inhibitor of ZIKV infection. • Mechanistic studies have indicated that candesartan cilexetil targets a post-entry stage(s) of the ZIKV replication cycle. • Candesartan cilexetil also expressed potential broad-spectrum antiviral properties against DENV2, KUNV and CHIKV. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne virus that has garnered a lot of attention in recent years, due to the explosive epidemic from 2014 to 2016. Since its introduction in the Americas in late 2014, ZIKV has spread at an unprecedented rate and scale throughout the world and infected millions of people. Its infection has also been associated with severe neurological disorders like Guillain-Barré syndrome and microcephaly in fetuses. Despite these, there is currently no approved antiviral against ZIKV. In this study, an immunofluorescence-based high throughput screen was conducted on a library of 483 flavonoid derivatives to identify potential anti-ZIKV compounds. Flavonoids, which are natural polyphenolic compounds found in plants, represent an attractive source of antivirals due to their abundance in food and expected low toxicity. From the primary screen, three hits were selected for validation by cell viability and viral plaque reduction assays. Pinocembrin, a flavanone found in honey, tea and red wine, was chosen for downstream studies as it exhibited the strongest inhibition of ZIKV infection in human placental JEG-3 cells (IC 50 = 17.4 μM). Time-course studies revealed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. Furthermore, pinocembrin inhibits viral RNA production and envelope protein synthesis based on quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses. This study has demonstrated for the first time the in vitro anti-ZIKV activity of pinocembrin. Image 1 • 483 flavonoid derivatives were screened for anti-Zika virus (ZIKV) activity in human placental JEG-3 cells. • Three hits were validated using dose response and cell viability assays. • Pinocembrin, a flavanone found in honey and tea, exhibited the strongest inhibition of ZIKV replication (IC 50 = 17.4 μM). • Mechanism studies showed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. • Pinocembrin was also shown to have broad-spectrum antiviral effects against DENV2 and CHIKV. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) infection, a febrile illness caused by a mosquito-transmitted alphavirus, has afflicted millions of people worldwide. There is currently no approved effective antiviral treatment for CHIKV infection. In this study, we report a new class of small-molecule CHIKV inhibitors, the oxindole-labdanes, that potently block the replication of CHIKV with good selectivity. Andrographolide, a previously reported inhibitor of CHIKV infection, was used as the lead compound for our initial structure-activity relationship (SAR) study. From a focused library of 72 andrographolide analogues, we identified the lead compound (E) - 2 with improved antiviral activities. Further optimization of (E) - 2 led to the discovery of the normal -labdane 7-chloro-oxindole (E) - 42 as potent inhibitor against two low-passage CHIKV isolates from human patients with an EC 50 of 1.55 μM against CHIKV-122508 and 0.14 μM against CHIKV-6708. Compound (E) - 42 displayed minimal cytotoxic liability (CC 50 > 100 μM), thus furnishing good selectivity relative to the host cells. Mechanistically, (E) - 42 does not inactivate the viral particles but rather acts on the host cells to interfere with the viral replication, demonstrating both prophylactic and therapeutic effects. Our findings open a new avenue for the development of oxindole-labdane compounds as promising antiviral drugs against CHIKV infection. [Display omitted] • A series of novel oxindole-labdane hybrids were developed as small-molecule inhibitors against Chikungunya virus infection. • Compound (E) - 42 displays potent inhibitory effects against two CHIKV variants isolated from infected patients. • (E) - 42 acts on the host cells to interfere with the viral replication, demonstrating prophylactic and therapeutic effects. [ABSTRACT FROM AUTHOR]