Viral Polymerase Inhibitors Research Articles

Combinations of Oseltamivir and T-705 Extend the Treatment Window for Highly Pathogenic Influenza A(H5N1) Virus Infection in Mice.

Current anti-influenza therapy depends on administering drugs soon after infection, which is often impractical. We assessed whether combinations of oseltamivir (a neuraminidase inhibitor) and T-705 (a nonspecific inhibitor of viral polymerases) could extend the window for treating lethal infection with highly pathogenic A(H5N1) influenza virus in mice. Combination therapy protected 100% of mice, even when delayed until 96 h postinoculation. Compared to animals receiving monotherapy, mice receiving combination therapy had reduced viral loads and restricted viral spread in lung tissues, limited lung damage, and decreased inflammatory cytokine production. Next-generation sequencing showed that virus populations in T-705–treated mice had greater genetic variability, with more frequent transversion events, than did populations in control and oseltamivir-treated mice, but no substitutions associated with resistance to oseltamivir or T-705 were detected. Thus, combination therapy extended the treatment window for A(H5N1) influenza infection in mice and should be considered for evaluation in a clinical setting.

Inhibition of human norovirus by a viral polymerase inhibitor in the B cell culture system and in the mouse model

The recently developed human norovirus (HuNoV) B cell culture and mouse models hold promise for drug discovery and development but their suitability for antiviral studies has not been assessed. We demonstrate the inhibitory effect of the nucleoside analogue 2′-C-methylcytidine (2CMC) on HuNoV replication in the human B cell BJAB cell line and in Balb/c Rag/gamma chain-deficient (Rag-γc−/−) mice. These data suggest the applicability of both models for future study and development of antiviral drugs for the treatment of HuNoV infections.

The Viral Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model.

Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2’-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/β and IFN-γ receptor knock-out) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-γ and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its in vitro anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the in vivo efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines.

Direct-acting antiviral agents for hepatitis C: structural and mechanistic insights.

The treatment of HCV infection has evolved at an extremely rapid pace over the past few years. The development of direct-acting antiviral agents, which potently inhibit different stages in the viral life cycle, has led to the replacement of interferon with well-tolerated oral therapies with cure rates of >90% in most patient populations. Understanding the mechanisms of action of the various agents as well as related issues, including the molecular basis for resistance, helps to guide drug development and clinical use. In this Review, we provide a mechanistic description of NS3/4A protease inhibitors, nucleotide and non-nucleotide inhibitors of the NS5B viral polymerase and inhibitors of the NS5A protein, followed by a summary of clinical data from studies of each drug class alone and in combination. Remaining challenges in drug development efforts are also discussed.

Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors

Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5′ terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted.

Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design.

The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes.

A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus.

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038μM and 0.016μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC50 (0.98–6.2μM) compared with FPV (36.8μM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25–25μM). When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.

Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by this method were shown to possess in vitro activity against the polymerase of dengue virus. The most potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit submicromolar activity on the four dengue virus serotypes.

Treatment with a Nucleoside Polymerase Inhibitor Reduces Shedding of Murine Norovirus in Stool to Undetectable Levels without Emergence of Drug-Resistant Variants.

Prolonged norovirus shedding may occur in certain patients, such as organ transplant recipients. We established a mouse model for persistent norovirus infection (using the mouse norovirus MNV.CR6 strain). The nucleoside viral polymerase inhibitor 2'-C-methylcytidine (2CMC), but not favipiravir (T-705), reduced viral shedding to undetectable levels. Viral rebound was observed after stopping treatment, which was again effectively controlled by treatment with 2CMC. No drug-resistant variants emerged.

Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes.

The drug-drug interaction (DDI) potential of deleobuvir, an hepatitis C virus (HCV) polymerase inhibitor, and its two major metabolites, CD 6168 (formed via reduction by gut bacteria) and deleobuvir-acyl glucuronide (AG), was assessed in vitro. Area-under-the-curve (AUC) ratios (AUCi/AUC) were predicted using a static model and compared with actual AUC ratios for probe substrates in a P450 cocktail of caffeine (CYP1A2), tolbutamide (CYP2C9), and midazolam (CYP3A4), administered before and after 8 days of deleobuvir administration to HCV-infected patients. In vitro studies assessed inhibition, inactivation and induction of P450s. Induction was assessed in a short-incubation (10 hours) hepatocyte assay, validated using positive controls, to circumvent cytotoxicity seen with deleobuvir and its metabolites. Overall, P450 isoforms were differentially affected by deleobuvir and its two metabolites. Of note was more potent CYP2C8 inactivation by deleobuvir-AG than deleobuvir and P450 induction by CD 6168 but not by deleobuvir. The predicted net AUC ratios for probe substrates were 2.92 (CYP1A2), 0.45 (CYP2C9), and 0.97 (CYP3A4) compared with clinically observed ratios of 1.64 (CYP1A2), 0.86 (CYP2C9), and 1.23 (CYP3A4). Predictions of DDI using deleobuvir alone would have significantly over-predicted the DDI potential for CYP3A4 inhibition (AUC ratio of 6.15). Including metabolite data brought the predicted net effect close to the observed DDI. However, the static model over-predicted the induction of CYP2C9 and inhibition/inactivation of CYP1A2. This multiple-perpetrator DDI scenario highlights the application of the static model for predicting complex DDI for CYP3A4 and exemplifies the importance of including key metabolites in an overall DDI assessment.

Identification of dengue viral RNA-dependent RNA polymerase inhibitor using computational fragment-based approaches and molecular dynamics study

Dengue is a major public health concern in tropical and subtropical countries of the world. There are no specific drugs available to treat dengue. Even though several candidates targeted both viral and host proteins to overcome dengue infection, they have not yet entered into the later stages of clinical trials. In order to design a drug for dengue fever, newly emerged fragment-based drug designing technique was applied. RNA-dependent RNA polymerase, which is essential for dengue viral replication is chosen as a drug target for dengue drug discovery. A cascade of methods, fragment screening, fragment growing, and fragment linking revealed the compound [2-(4-carbamoylpiperidin-1-yl)-2-oxoethyl]8-(1,3-benzothiazol-2-yl)naphthalene-1-carboxylate as a potent dengue viral polymerase inhibitor. Both strain energy and binding free energy calculations predicted that this could be a better inhibitor than the existing ones. Molecular dynamics simulation studies showed that the dengue polymerase–lead complex is stable and their interactions are consistent throughout the simulation. The hydrogen-bonded interactions formed by the residues Arg792, Thr794, Ser796, and Asn405 are the primary contributors for the stability and the rigidity of the polymerase–lead complex. This might keep the polymerase in closed conformation and thus inhibits viral replication. Hence, this might be a promising lead molecule for dengue drug designing. Further optimization of this lead molecule would result in a potent drug for dengue.

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates.

Alpha-carboxynucleoside phosphonates (α-CNPs) are novel viral DNA polymerase inhibitors that do not need metabolic conversion for enzyme inhibition. The prototype contains a cyclopentyl linker between nucleobase and α-carboxyphosphonate and preferentially (50- to 100-fold) inhibits HIV-1 RT compared with herpetic DNA polymerases. A synthesis methodology involving three steps has been developed for the synthesis of a series of novel α-CNPs, including a Rh(II)-catalyzed O-H insertion that connects the carboxyphosphonate group to a linker moiety and an attachment of a nucleobase to the other end of the linker by a Mitsunobu reaction followed by final deprotection. Replacing the cyclopentyl moiety in the prototype α-CNPs by a more flexible entity results in a selectivity shift of ∼ 100-fold in favor of the herpetic DNA polymerases when compared to selectivity for HIV-1 RT. The nature of the kinetic interaction of the acyclic α-CNPs against the herpetic DNA polymerases differs from the nature of the nucleobase-specific kinetic interaction of the cyclopentyl α-CNPs against HIV RT.

Multi-scale model for hepatitis C viral load kinetics under treatment with direct acting antivirals

Hepatitis C virus (HCV) infections are a global health problem, and extensive research over the last decades has been targeted at understanding its molecular biology and developing effective antiviral treatments. Recently, a number of potent direct acting antiviral drugs have been developed targeting specific processes in the viral life cycle. Here, we developed a mathematical multi-scale model of the within-host dynamics of HCV infection by integrating a standard model for viral infection with a detailed model of the viral replication cycle inside infected cells. We use this model to study patient time courses of viral load under treatment with daclatasvir, an inhibitor of the viral non-structural protein NS5A. Model analysis predicts that treatment efficacy can be increased by combining daclatasvir with dedicated viral polymerase inhibitors, corresponding to promising current strategies in drug development. Hence, our model presents a predictive tool for in silico simulations, which can be used to study and optimize direct acting antiviral drug treatment.

ID: 68: IFN-β treatment for Ebola virus disease: Bench to bedside

To date there are no approved vaccines or antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in preclinical studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles, we compared the antiviral activities of 8 different candidate antivirals from 3 drug classes: IFN-α and IFN-s, viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF), and the estrogen receptor modulator, toremifene (TOR). We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-β inhibited viral replication 24 h post-infection (IC50 0.038 μM and 0.016 μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC50 (0.98–6.2 μM) compared with FPV (36.8 μM), when administered 24 h post-infection. CDF had a narrow therapeutic window (6.25–25 μM). When dosed >50 μM, CDF treatment enhanced viral infection. IFN- β exhibited strong synergy with 3TC (97.9% inhibition) or in triple combination with 3TC and AZT (98.5% inhibition). Our data indicate that IFN-β is a potent inhibitor of Ebola virus, superior to the antivirals we tested, contributing to the decision to conduct a clinical trial of IFN-β treatment for Ebola virus disease in Coyah, Guinea. Results from the trial, including effects of IFN treatment on viremia, disease symptoms and survival will be presented.

Novel methods of hepatitis C treatment and prevention

Despite available treatment, Hepatitis C remains one of most serious burdens to public health. Current therapy based on pegylated interferon-alpha and ribavirin has significant side effects and its effectiveness varies for different genotypes of the virus. Four novel drugs - viral protease inhibitors (telaprevir, boceprevir, simeprevir) and polymerase inhibitor - sofosbuvir have been introduced in last years for use in combination with standard-of-care treatment. For the first time interferon free therapies were approved with the use of combination of sofosbuvir+ribavirin. New therapies improve virological response rates but also increase the cost, side effects and raise the issue of drug resistance. Numerous novel anti-HCV compounds have been evaluated in advanced clinical trials including inhibitors of viral proteins (protease, polymerase and NS5A) and inhibitors of host factors involved in HCV replication (cyclophilin A, microRNA - miR-122). New interferon-free therapies reducing severe side effects are expected to enter the market within few months. At the same time efforts are undertaken to determine the host and viral factors with predictive value for HCV treatment response, enabling personalized therapy approach. The main success in this field was the discovery of interleukin IL28B polymorphism, which correlates with positive standard-of-care treatment response. An effective vaccination may be an alternative for antiviral drugs, but no anti-HCV vaccine is available currently. It is well proved that successful vaccination should induce antibody and T-cell responses specific against a range of HCV genotypes. With this aim, new subunit and genetic candidate vaccines have been evaluated in I and II phase clinical trials. This review summarizes the recent developments in the field of new drug development and vaccine studies against hepatitis C virus.

Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon‐13 and carbon‐14

Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide derivatives of (1) and (2), is also reported. Aniline-(13) C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-nitrophenyl-(13) C6 )acrylate [(13) C6 ]-(11) in six steps. This intermediate was then used to obtain [(13) C6 ]-(1) and [(13) C6 ]-(2) in five and four more steps, respectively. For the radioactive synthesis, potassium cyanide-(14) C was used to prepare 1-cylobutylaminoacid [(14) C]-(23) via Buchrer-Bergs reaction. The carbonyl chloride of this acid was then used to access both [(14) C]-(1) and [(14) C]-(2) in four steps. The acyl glucuronide derivatives [(13) C6 ]-(3), [(13) C6 ]-(4) and [(14) C]-(3) were synthesized in three steps from the acids [(13) C6 ]-(1), [(13) C6 ]-(2) and [(14) C]-(1) using known procedures.

Vesicular stomatitis virus (VSV) as a paradigm for predicting antiviral activity against Ebola virus (EBOV)

There are at present no antivirals available which have been formally licensed for clinical use for the treatment of Ebola virus (EBOV) infections in humans. The most advanced to be approved for this purpose is favipiravir (T-705), a viral RNA polymerase inhibitor. Under consideration are BCX4430, also a viral RNA polymerase inhibitor, and 3-deazaneplanocin A and various other S-adenosylhomocysteine (SAH) hydrolase inhibitors. A number of compounds which have been approved for other purposes seem to interact with the cell entry of EBOV. Some compounds like pyrazofurin have been found to be exquisitely potent inhibitors of vesicular stomatitis virus (VSV). VSV belongs to the rhabdoviridae, a family closely related to the family of the filoviridae to which EBOV and Marburg virus belong. VSV, unlike EBOV and Marburg virus which require biosafety level 4, can be handled in conventional safety conditions. Key words: Ebola virus (EBOV); vesicular stomatitis virus (VSV); rhabdoviridae; filoviridae; favipiravir; BCX4430; pyrazofurin; SAH hydrolase

Design and synthesis of a novel 5-(aminomethylene)thiazolidine-2,4-dione derivatives as potent hepatitis-B virus polymerase inhibitors

<p>A series of novel thiazolidinedione analogues (TZD) were designed and synthesized potent inhibitors of HBV capsid assembly. The synthesis of thiazolidine-2,4-dione derivatives (4a–4o), starting from the condensation of 5-(ethoxymethylene)thiazolidine-2,4-dione (1) with various secondary amines (3) derived from biologically active compounds. The newly synthesized TZD analogues 4a-4o were characterized by <sup>1</sup>H NMR, <sup>13</sup>C NMR, and MS and evaluated for their anti-HBV activity. Most of the compounds inhibited the expression of viral antigens at low concentration. Six compounds, 4g, 4h, 4l, 4m, 4n, and 4o, demonstrated potent inhibition of HBV DNA replication at submicromolar range. Of these five initial hits, compound 4o was the most active when compared with lamivudine.</p><p> </p><p> </p>

Towards interferon-free treatment for all HCV genotypes

It is surely unique in modern medicine that regulatory agencies should approve seven new compounds to treat a specific disease within just 12 months. The European Commission granted marketing authorisation for sofosbuvir, a nucleotide hepatitis C virus (HCV) polymerase inhibitor, in January, 2014. Since then, the HCV protease inhibitor simeprevir (May, 2014), the NS5A inhibitor daclatasvir (August, 2014), and the single-tablet combination of the HCV NS5 inhibitor ledipasvir with sofosbuvir (November, 2014) have reached the market; followed most recently by the three-drug combination of ombitasvir (NS5A inhibitor), the ritonavir-boosted protease inhibitor paritaprevir, and the first non-nucleosidic polymerase inhibitor dasabuvir (January, 2015).

Hepatitis C Virus of Subtype 2l in Marseille, Southeastern France

The rate of eradication of chronic hepatitis C considerably increases with direct-acting antiviral agents, particularly hepatitis C virus (HCV) polymerase inhibitors. While implementing full-length HCV NS5B polymerase sequencing in our clinical microbiology laboratory, we identified atypical HCV sequences, classified as subtype 2l, from 2 patients. HCV-2l NS5B polymerase sequences were detected from 5 and 14 additional patients by screening our laboratory hepatitis virus sequence database and the NCBI GenBank sequence database. Phylogenetic analyses show unambiguously that all HCV-2l sequences are clustered apart from HCV 2 non-l sequences, which compose a second cluster. Mean (±SD) nucleotide identity between near full-length NS5B fragments of subtype 2l was 93.4 ± 0.8% (range: 92.4-95.1). Of note, all HCV-2l sequences obtained in our laboratory and in other centers were from serum samples collected in France. Analysis of the HCV-2l NS5B polymerase amino acid sequences at 30 positions critical for interaction with or resistance to HCV polymerase inhibitors showed specific patterns.