► Favipiravir (T-705) is an influenza drug in clinical development. ► Favipiravir inhibits (murine) norovirus-induced CPE and viral RNA synthesis. ► Favipiravir inhibits viral replication at a time ...point that coincides with the onset of viral RNA synthesis.
Human noroviruses are the primary cause of foodborne gastroenteritis. Potent and safe inhibitors are needed for the treatment/prophylaxis of norovirus infections. We demonstrate that Favipiravir T-705, a drug in advanced clinical development for the treatment of infections with the influenza virus inhibits in vitro murine norovirus replication. Time-of-drug addition studies reveal that T-705 exerts its activity at a time-point that coincides with onset of viral RNA synthesis, which is in line with the viral polymerase as the presumed target.
Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease ...inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II GII) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.
Norovirus outbreaks of acute gastroenteritis are highly prevalent, extensive and can disturb the functioning of health institutions, leading to the closure of hospital wards and causing ...life-threatening infections in long-term care facilities. There is no vaccine available; hence there is a pressing need for antivirals for the treatment (in immunodeficient patients) and prophylaxis of norovirus infections. We explored in a mouse model whether an inhibitor of norovirus replication can prevent/reduce transmission of the virus.
We reported recently that the viral polymerase inhibitor 2'-C-methylcytidine (2CMC) efficiently protects against murine norovirus (MNV)-induced diarrhoea and mortality in mice. Here, we established an MNV-transmission model, determined the 50% infectious dose and assessed the ability of an antiviral molecule to prevent or reduce transmission of (murine) norovirus when given either to the infected (seeder) mice or to the uninfected (sentinel) mice.
A robust norovirus transmission model was established using genogroup V (murine) norovirus in AG129 mice. The 50% infectious dose was determined to be ∼ 270 CCID50 (50% cell culture infectious dose). Treatment of infected mice with 2CMC reduced viral shedding and markedly reduced transmission to uninfected sentinels. Also, prophylactic treatment of sentinels with 2CMC resulted in protection against infection with MNV.
These findings constitute an important first step towards developing an efficient prophylaxis for the control of norovirus outbreaks.
The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a ...novel class of small molecules (1,2,3triazolo4,5-dpyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) ...has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.
► A panel of reference antiviral molecules was screened against norovirus. ► 2′-C-methylcytidine (2CMC) was identified as an inhibitor of the replication of (murine) norovirus. ► 2CMC inhibits ...virus-induced CPE formation, the production of infectious viruses and viral RNA synthesis. ► The antiviral effect coincides with the onset of viral RNA synthesis, suggesting the viral polymerase as target. ► When combined with ribavirin a marked antagonistic activity is observed.
We here report on the activity of 2′-C-methylcytidine (2CMC) a nucleoside polymerase inhibitor of the hepatitis C virus (HCV) on the in vitro replication of (murine) norovirus (MNV). 2CMC inhibits (i) virus-induced CPE formation, (ii) viral RNA synthesis and (iii) infectious progeny formation with EC50 values of ∼2μM. 2CMC acts at a time-point that coincides with the onset of viral RNA synthesis. Even following 30 passages of selective pressure no MNV-resistant virus was selected, which is in line with the high barrier to resistance of the nucleoside analogue for HCV. When combined with the broad-spectrum RNA virus inhibitor ribavirin, a marked antagonistic activity was observed indicating that these molecules should not be combined for the treatment of norovirus infections. Our results suggest that 2′-C-methyl nucleoside analogues should be further explored for the treatment and prophylaxis of norovirus infections.
HIV-1 reverse transcriptase (RT) was the first viral enzyme to be targeted by anti-HIV drugs. Despite 20 years of experience with RT inhibitors, new ways to inhibit this target and address viral ...resistance continue to emerge. In both licensed RT inhibitor classes, nucleosides (NRTIs) and non-nucleosides (NNRTIs), compounds with better resistance, pharmacokinetic and toxicity profiles are being developed. Second-generation NNRTIs active against HIV-1 strains resistant to current NNRTIs are being clinically evaluated. Beyond the classical NRTIs, nucleoside analogs that are no longer obligate chain terminators but nevertheless impede reverse transcription or even lead to viral ablation after several replication cycles, are being studied. RT inhibitor research has also yielded additional mechanisms to block RT. Driven by new insights the RNase H field remains in evolution. In addition, the binding of both substrates (deoxynucleotide and primer/template) to RT is now subject to competition by novel inhibitors. Further development of aptamers bears promise for gene therapy but perhaps more importantly, reveals additional new platforms for the development of small-molecule RT inhibitors. This promising research provides much optimism that RT inhibitors will continue to evolve with subsequent clinical benefit.
Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. ...We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target–inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.
► Development of a novel cellular screening assay focused on HIV-1 integrase activity. ► Nevirapine induced synchronization of HIV-1 vectors by temporary arrest at RT step. ► Validation of integrase ...focused assay by testing known integrase and RT inhibitors.
The discovery of HIV-1 integrase inhibitors has been enabled by high-throughput screening and rational design of novel chemotypes. Traditionally, biochemical assays focusing on the strand transfer activity of integrase have been used to screen compound libraries for identification of novel inhibitors. In contrast, cellular screening assays enable a phenotypic or multi-target approach, and may result in identification of compounds inhibiting integrase in its natural context, the pre-integration complex. Furthermore, a cellular assay encompassing 3′ processing, strand transfer and nuclear import may lead to the identification of compounds with novel mechanisms of action targeting cellular and viral factors. Therefore, a cellular screening assay was developed, which focused on integrase activity, where infection of MT4 cells with an HIV-1 based lentiviral vector was synchronized by temporary arrest at the reverse transcriptase step and subsequent release to enable integration. The assay was validated using a panel of antivirals and proved to be a robust cellular screening assay for the identification of novel integrase inhibitors.
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