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The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932 . However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932 , represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Influenza virus infection causes substantial morbidity and mortality worldwide. The limited efficacy of oseltamivir in delayed treatment, coupled with the increasing incidences of oseltamivir-resistant strains, calls for next-generation of antiviral drugs. In this study, we discovered NMS-873, an allosteric and specific p97 inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. NMS-873 shows potent antiviral activity with low-nanomolar EC 50 s against multiple human influenza A and B viruses, including adamantine-, oseltamivir-, or double resistant strains. Our data further showed that silencing of p97 via siRNA or inhibiting p97 by NMS-873 both inhibited virus replication and retained viral ribonucleoproteins (vRNPs) in the nucleus, confirming p97 is the drug target. Mechanistic studies have shown that the nuclear retention of vRNP with NMS-873 treatment is a combined result of two effects: the reduced viral M1 protein level (indirect effect), and the disruption of p97-NP interactions (direct effect). Taken together, our results suggest that p97 could be a novel antiviral target and its inhibitor, NMS-873, is a promising antiviral drug candidate., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC 50 < 1 μM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2A pro ) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2A pro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2A pro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2A pro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2A pro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2A pro and the identification of telaprevir as a potent EV-D68 2A pro inhibitor. These findings implicate EV-D68 2A pro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection. IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2A pro , and identify telaprevir-an FDA-approved drug used to treat hepatitis C virus (HCV) infections-as a potent antiviral with a novel mechanism of action toward 2A pro 2A pro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2A pro inhibition. This is the first inhibitor to selectively target the 2A pro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68., (Copyright © 2019 American Society for Microbiology.)

Influenza viruses are severe human pathogens that pose persistent threat to public health. Each year more people die of influenza virus infection than that of breast cancer. Due to the limited efficacy associated with current influenza vaccines, as well as emerging drug resistance from small molecule antiviral drugs, there is a clear need to develop new antivirals with novel mechanisms of action. The influenza virus polymerase complex has become a promising target for the development of the next-generation of antivirals for several reasons. Firstly, the influenza virus polymerase, which forms a heterotrimeric complex that consists of PA, PB1, and PB2 subunits, is highly conserved. Secondly, both individual polymerase subunit (PA, PB1, and PB2) and inter-subunit interactions (PA-PB1, PB1- PB2) represent promising drug targets. Lastly, growing insight into the structure and function of the polymerase complex has spearheaded the structure-guided design of new polymerase inhibitors. In this review, we highlight recent progress in drug discovery and assay development targeting the influenza virus polymerase complex and discuss their therapeutic potentials., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Influenza viruses are respiratory pathogens that are responsible for seasonal influenza and sporadic influenza pandemic. The therapeutic efficacy of current influenza vaccines and small molecule antiviral drugs is limited due to the emergence of multidrug-resistant influenza viruses. In response to the urgent need for the next generation of influenza antivirals, we utilized a fast-track drug discovery platform by exploring multi-component reaction products for antiviral drug candidates. Specifically, molecular docking was applied to screen a small molecule library derived from the Ugi-azide four-component reaction methodology for inhibitors that target the influenza polymerase PA C -PB1 N interactions. One hit compound 5 was confirmed to inhibit PA C -PB1 N interactions in an ELISA assay and had potent antiviral activity in an antiviral plaque assay. Subsequent structure-activity relationship studies led to the discovery of compound 12a, which had broad-spectrum antiviral activity and a higher in vitro genetic barrier to drug resistance than oseltamivir. Overall, the discovery of compound 12a as a broad-spectrum influenza antiviral with a high in vitro genetic barrier to drug resistance is significant, as it offers a second line of defense to combat the next influenza epidemics and pandemics if vaccines and oseltamivir fail to confine the disease outbreak.

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu ® ) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics., Competing Interests: The authors declare no conflict of interest.

The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adamantanes such as amantadine (1) and rimantadine (2) are FDA-approved anti-influenza drugs that act by inhibiting the wild-type M2 proton channel from influenza A viruses, thereby inhibiting the uncoating of the virus. Although adamantanes have been successfully used for more than four decades, their efficacy was curtailed by emerging drug resistance. Among the limited number of M2 mutants that confer amantadine resistance, the M2-V27A mutant was found to be the predominant mutant under drug selection pressure, thereby representing a high profile antiviral drug target. Guided by molecular dynamics simulations, we previously designed first-in-class M2-V27A inhibitors. One of the potent lead compounds, spiroadamantane amine (3), inhibits both the M2-WT and M2-V27A mutant with IC 50 values of 18.7 and 0.3 μM, respectively, in in vitro electrophysiological assays. Encouraged by these findings, in this study we further examine the in vitro and in vivo antiviral activity of compound 3 in inhibiting both amantadine-sensitive and -resistant influenza A viruses. Compound 3 not only had single to sub-micromolar EC 50 values against M2-WT- and M2-V27A-containing influenza A viruses in antiviral assays, but also rescued mice from lethal viral infection by either M2-WT- or M2-V27A-containing influenza A viruses. In addition, we report the design of two analogs of compound 3, and one was found to have improved in vitro antiviral activity over compound 3. Collectively, this study represents the first report demonstrating the in vivo antiviral efficacy of inhibitors targeting M2 mutants. The results suggest that inhibitors targeting drug-resistant M2 mutants are promising antiviral drug candidates worthy of further development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adamantanes (amantadine and rimantadine) are one of the two classes of Food and Drug Administration-approved antiviral drugs used for the prevention and treatment of influenza A virus infections. They inhibit viral replication by blocking the wild-type (WT) M2 proton channel, thus preventing viral uncoating. However, their use was discontinued due to widespread drug resistance. Among a handful of drug-resistant mutants, M2-S31N is the predominant mutation and persists in more than 95% of currently circulating influenza A strains. We recently designed two classes of M2-S31N inhibitors, S31N-specific inhibitors and S31N/WT dual inhibitors, which are represented by N-[(5-cyclopropyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine (WJ379) and N-[(5-bromothiophen-2-yl)methyl]adamantan-1-amine (BC035), respectively. However, their antiviral activities against currently circulating influenza A viruses and their genetic barrier to drug resistance are unknown. In this report, we evaluated the therapeutic potential of these two classes of M2-S31N inhibitors (WJ379 and BC035) by profiling their antiviral efficacy against multidrug-resistant influenza A viruses, in vitro drug resistance barrier, and synergistic effect with oseltamivir. We found that M2-S31N inhibitors were active against several influenza A viruses that are resistant to one or both classes of Food and Drug Administration-approved anti-influenza drugs. In addition, M2-S31N inhibitors display a higher in vitro genetic barrier to drug resistance than amantadine. The antiviral effect of WJ379 was also synergistic with oseltamivir carboxylate. Overall, these results reaffirm that M2-S31N inhibitors are promising antiviral drug candidates that warrant further development., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Adamantanes such as amantadine (1) and rimantadine (2) are FDA-approved anti-influenza drugs that act by inhibiting the wild-type M2 proton channel from influenza A viruses, thereby inhibiting the uncoating of the virus. Although adamantanes have been successfully used for more than four decades, their efficacy was curtailed by emerging drug resistance. Among the limited number of M2 mutants that confer amantadine resistance, the M2-V27A mutant was found to be the predominant mutant under drug selection pressure, thereby representing a high profile antiviral drug target. Guided by molecular dynamics simulations, we previously designed first-in-class M2-V27A inhibitors. One of the potent lead compounds, spiroadamantane amine (3), inhibits both the M2-WT and M2-V27A mutant with IC50 values of 18.7 and 0.3 μM, respectively, in in vitro electrophysiological assays. Encouraged by these findings, in this study we further examine the in vitro and in vivo antiviral activity of compound 3 in inhibiting both amantadine-sensitive and -resistant influenza A viruses. Compound 3 not only had single to sub-micromolar EC50 values against M2-WT- and M2-V27A-containing influenza A viruses in antiviral assays, but also rescued mice from lethal viral infection by either M2-WT- or M2-V27A-containing influenza A viruses. In addition, we report the design of two analogs of compound 3, and one was found to have improved in vitro antiviral activity over compound 3. Collectively, this study represents the first report demonstrating the in vivo antiviral efficacy of inhibitors targeting M2 mutants. The results suggest that inhibitors targeting drug-resistant M2 mutants are promising antiviral drug candidates worthy of further development.

The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications. [ABSTRACT FROM AUTHOR]

The present study aimed to analyze the contribution of Nicotiana benthamiana ARGONAUTE2 (NbAGO2) to its antiviral response against different viruses. For this purpose, dsRNA hairpin technology was used to reduce NbAGO2 expression in transgenic plants as verified with RT-PCR. This reduction was specific because the expression of other NbAGOs was not affected, and did not cause obvious developmental defects under normal growth conditions. Inoculation of transgenic plants with an otherwise silencing-sensitive GFP-expressing Tomato bushy stunt virus (TBSV) variant resulted in high GFP accumulation because antiviral silencing was compromised. These transgenic plants also exhibited accelerated spread and/or enhanced susceptibility and symptoms for TBSV mutants defective for P19 or coat protein expression, other tombusviruses, Tobacco mosaic virus , and Potato virus X ; but not noticeably for Foxtail mosaic virus . These findings support the notion that NbAGO2 in N. benthamiana can contribute to antiviral defense at different levels. [ABSTRACT FROM AUTHOR]