bluuebunny/crossref_metadata_2025 huggingface dataset:
Helicase Primase: Targeting the Achilles Heel of Herpes Simplex Viruses
Authors: Kleymann Gerald
Published: March 2015

The majority of the population is infected by several herpesviruses. Once these infections are established the viruses persist for life. Therefore, current therapy may at best reduce symptoms but does not cure the infection. Moreover, the only classes of compounds licensed for systemic treatment of disease are nucleoside, nucleotide and pyrophosphate analogues; all of these ultimately target the herpesvirus DNA polymerase. A vaccine against varicella zoster virus (VZV) is available, but so far no effective vaccines against other human herpesviruses have been launched. At the same time, rising resistance to current medication, especially in the immunocompromised patient population, is a concern. For these reasons, there is an urgent need for new treatment options. Recently, some promising new drugs have been discovered; one of these compounds, developed at Bayer HealthCare under the name BAY 57–1293, is a potent HSV helicase primase inhibitor.
Variability in the PR and RT Genes of HIV-1 Isolated from Recently Infected Subjects
Authors: Turner Dan , Roldan Ariel , Brenner Bluma , Moisi Daniela , Routy Jean-Pierre , Wainberg Mark A
Published: March 2015

To study variability in reverse transcriptase (RT) and protease (PR) genes of HIV-1 isolated from recently infected patients identified between 1997 and 2003, sequences were obtained on the RT and PR genes of viruses harvested from plasma of 121 non-treated subjects who had undergone primary HIV infection. The degree of dissimilarity between the viruses studied and a reference HIV-1 subtype B strain (LAV-1) was calculated for each of RT and PR by counting all of the nucleotide substitutions that could be identified. Mutations associated with drug resistance were excluded from analysis. We observed a mean percentage of variation in the RT and PR genes of the viruses analysed of 0.42% between the years 1997–2003 ( P<0.01). In PR, the mean variation was 0.71% ( P<0.05), while that in RT was 0.3% ( P<0.05). Increased diversity was also observed among nucleotides conferring amino-acid changes, although no significant differences in patterns of nucleotide substitutions were apparent over the period of analysis. In conclusion, variability in the RT and PR genes of viruses from recently infected patients has increased over time in a manner that is independent of variability attributable to HIV drug resistance.
Remofovir Mesylate: A Prodrug of PMEA with Improved Liver-Targeting and Safety in Rats and Monkeys
Authors: Lin Chin-Chung , Yeh Li-Tain , Vitarella Domenico , Hong Zhi , Erion Mark D
Published: March 2015

Adefovir dipivoxil (Hepsera™), a first-line therapy for chronic hepatitis B, is an esterase-activated prodrug of PMEA. Dose-limiting nephrotoxicity necessitates suboptimal dosing at 10 mg/day. Remofovir mesylate (MB06866Q) (Hepavir B) is a CYP3A4-activated prodrug of PMEA based on the HepDirect™ technology that targets PMEA to the liver. In a whole body autoradiography study in rats after oral dosing (30 mg/kg) of [14C]adefovir dipivoxil or [14C]remofovir mesylate, remofovir yielded 15 times higher concentrations of radioactivity in the liver than adefovir dipivoxil, but only one-third of the concentrations in the kidney. After oral dosing (4 mg/kg) of the same radiolabelled agents in cynomolgus monkeys, remofovir mesylate yielded 60 times higher levels of total radioactivity in the liver, but only two-thirds of total radioactivity levels in the kidney. Thus, remofovir mesylate may provide better efficacy and reduced nephrotoxicity. In portal vein-cannulated rats (30 mg/kg) after a single oral dose of [14C]adefovir dipivoxil or [14C]remofovir mesylate, no PMEA was detectable in rat portal plasma early after dosing, indicating that intestinal CYP3A4 does not play a role in conversion of remofovir mesylate to PMEA. The portal/systemic extraction ratio was quite high in both models, suggesting good liver-targeting properties. Portal and systemic remofovir/PMEA ratio indicates that the liver is the site of conversion of remofovir to PMEA. 28-Day toxicity studies demonstrated renal toxicity in rats at doses of 100 mg/kg or higher with no safety concerns at 30 mg/kg and acceptable safety in monkeys at doses up to 60 mg/kg. Thus, in rats and non-human primates, remofovir mesylate has liver-targeting properties and is safer than adefovir dipivoxil.
Bicyclic Nucleoside Inhibitors of Varicella-Zoster Virus Modified on the Sugar Moiety: 3′ and 5′ Derivatives
Authors: Luoni Giovanna M , McGuigan Christopher , Andrei Graciela , Snoeck Robert , De Clercq Erik , Balzarini Jan
Published: March 2015

Bicyclic furanopyrimidine nucleoside analogues (BCNAs) have been previously reported as potent and selective anti-varicella-zoster virus (VZV) agents. Few modifications on the sugar moiety have been considered so far but some of them have shown interesting activity against human cytomegalovirus (HCMV) while losing activity against VZV. In addition, recent work has led to an entirely new series of anti-HCMV bicyclic furopyrimidine agents, acting through a non-nucleoside mechanism. In order to further investigate structure-activity relationship studies on the sugar moiety, some 3′- and 5′-chloro derivatives and 5′-deoxygenated derivatives have been synthesized. The lack of anti-VZV activity of the 5′-modified derivatives is further proof of a mechanism of action involving VZV thymidine kinase (TK)-mediated phosphorylation. Similarly, the replacement of the 3′-OH with chlorine showed a decrease of antiviral activity, which can be correlated to the lack of interaction with VZV TK as demonstrated by enzyme assays. These results confirm free 5′-OH and 3′-OH as necessary requirements for efficient recognition by VZV TK and for potent anti-VZV activity in cell culture.
Colorimetric Lactate Dehydrogenase (LDH) Assay for Evaluation of Antiviral Activity against Bovine Viral Diarrhoea Virus (BVDV) in Vitro
Authors: Baba Chiaki , Yanagida Koichiro , Kanzaki Tamotsu , Baba M
Published: March 2015

A rapid and sensitive screening assay has been established for in vitro evaluation of antiviral compounds against bovine viral diarrhoea virus (BVDV), which is widely used as a surrogate for hepatitis C virus (HCV). The procedure is based on photospectrometrical assessment for the viability of virus-infected cells via extracellular leakage of lactic dehydrogenase (LDH). The level of LDH in culture supernatants of BVDV-infected Madin-Darby bovine kidney (MDBK) cells was significantly higher than those of mock-infected MDBK cells. Under optimized assay conditions, the LDH level was found to correlate well with the degree of viral replication. When the 50% effective concentrations (EC50s) of ribavirin, cyclosporine A and human interferon-α for BVDV replication were determined by the established LDH method and compared with those obtained by a conventional tetrazolium colorimetric (MTT) method, there was a complete correlation in EC50s between the two methods. Furthermore, a much higher ratio of background activity (noise) to sample activity (signal) could be achieved with the LDH method than with the MTT method, indicating that the present LDH assay permits a sensitive, rapid and reliable screening of compounds for their anti-BVDV activity and may be useful for the discovery of novel anti-HCV agents.
Risk Factors for Hyperlactataemia in HIV-Infected Patients, Aquitaine Cohort, 1999–2003
Authors: Bonnet Fabrice , Balestre Eric , Bernardin Elise , Pellegrin Jean-Luc , Neau Didier ,   for the Groupe d'Epidémiologie Clinique du SIDA en Aquitaine25
Published: March 2015

The objectives of our study were to describe the characteristics of a subset of patients who had been prescribed serum lactate in clinical practice within a large cohort of HIV-infected patients and to determine the factors associated with hyperlactataemia. Hyperlactataemia (⩾T2 mmol/l) was found in 219 [29% (95% confidence interval: 25.3–31.7)] of the 768 HIV-infected participants. In multivariate analysis (logistical regression), an increased risk of hyperlactataemia was associated with increasing age, CD4 count <500/mm3, triglycerides >2.2 mmol/L, lipoatrophy and stavudine use. In a second model coding for the NRTI-based drug combinations, only those including stavudine were associated with an increased risk of hyperlactataemia. In a third model including exposure duration to NRTIs, we estimated a 20% increased risk of hyperlactataemia per year of exposure to didanosine or stavudine. The risk of hyperlactataemia could increase over time in patients treated with these drugs and is also closely associated with increased age, decreased CD4 count, lipodystrophy and increased plasma triglycerides. It could be proposed that patients having one or more of these risk factors undergo regular monitoring of plasma lactate and renal function to prevent lactic acidosis.
Oligonucleotides and Polyribonucleotides: A Review of Antiviral Activity
Authors: Markosian Michael , Hyde Robyn M
Published: March 2015

Current antiviral therapies are insufficient for treating emerging, re-emerging and established viral diseases. In an effort to find new therapeutics, oligo- and polyribonucleotides are being studied for their antiviral capabilities. Studies have shown that uniquely modified single- and double-stranded nucleic acid constructs are effective in inhibiting viral proliferation by various mechanisms. This review gives a brief history and highlights the development of oligo- and polyribonucleotides as antiviral agents primarily in the fields of interferon induction, mRNA complementation and reverse transcriptase inhibition.
Inhibition of Hepatitis B Virus Gene Expression and Replication by Helioxanthin and its Derivative
Authors: Li Ying , Fu Lei , Yeo Hosup , Zhu Ju-Liang , Chou Chen-Kung , Kou Yueh-Hsiung , Yeh Sheau-Farn , Gullen Elizabeth , Austin David , Cheng Yung-Chi
Published: March 2015

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-α (IFN-α) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 μM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 μM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.
Pointer 4'-Ethynylstavudine (4'-Ed4T) Has Potent Anti-HIV-1 Activity with Reduced Toxicity and Shows a Unique Activity Profile against Drug-Resistant Mutants
Authors: Tanaka Hiromichi , Haraguchi Kazuhiro , Kumamoto Hiroki , Baba Masanori , Cheng Yung-Chi
Published: March 2015

A nucleoside analogue 4'-ethynylstavudine (4'-Ed4T) was recently synthesized during chemical studies directed towards the development of a new route to 4' -carbon-substituted nucleosides. This compound was found to be more anti-HIV-1 active than the parent compound stavudine (d4T) and much less toxic to various cells and also to mitochondrial DNA synthesis. It became apparent that 4' -Ed4T is a better substrate for human thymidine kinase than d4T, and very much more resistant to catabolism by thymidine phosphorylase. The study of 4' -Ed4T against various drug-resistant HIV-1 mutants has disclosed its unique activity profile.
Mutations in Both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology
Authors: Hantson Anke , Fikkert Valery , Van Remoortel Barbara , Pannecouque Chistophe , Cherepanov Peter , Matthews Barry , Holan George , De Clercq Erik , Vandamme Anne-Mieke , Debyser Zeger , Witvrouw Myriam
Published: March 2015

A drug-resistant NL4.3.SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3.SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3.SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3.SPL2923 towards DS was fully reproduced following gp160recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3.SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17.gp160-CVT. Phenotypic analysis of the NL4.3.SPL2923 p17- and p17.gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3.SPL2923 towards SPL2923.
Synthesis and Anti-Rhinovirus Properties of Fluoro-Substituted Flavonoids
Authors: Conti Cinzia , Mastromarino Paola , Goldoni Paola , Portalone Gustavo , Desideri Nicoletta
Published: March 2015

Fluoro-substituted flavones and 2-styrykhromones, related to natural and synthetic flavonoids previously described, were prepared, characterized and tested for anti-rhinovirus activity. Structural elucidation of the new compounds was performed by IR, NMR spectra and X-ray crystal structure analysis for 6-fluoro-3-hydroxy-2-styrylchromone. The antiviral potency was evaluated by a plaque reduction assay in HeLa cell cultures infected with rhinoviruses 1B and 14, selected as representative serotypes for viral groups B and A of human rhinoviruses, respectively. In comparison with results previously obtained, the introduction of the fluorine atom seems to exert a positive influence on the activity against serotype 14 while counteracting the effect against serotype 1B.
A Novel Small Molecular Weight Compound with a Carbazole Structure That Demonstrates Potent Human Immunodeficiency Virus Type-1 Integrase Inhibitory Activity
Authors: Yan Hua , Mizutani Tomoko Chiba , Nomura Nobuhiko , Takakura Tadakazu , Kitamura Yoshihiro , Miura Hideka , Nishizawa Masako , Tatsumi Masashi , Yamamoto Naoki , Sugiura Wataru
Published: March 2015

The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00 ±3.31 μM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48–1.52 μM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.
Synthesis and Antiviral Activities of 1,2,3-triazole Functionalized Thymidines: 1,3-dipolar Cycloaddition for Efficient Regioselective Diversity Generation
Authors: Zhou Longhu , Amer Adel , Korn Michael , Burda Robert , Balzarini Jan , De Clercq Erik , Kern Earl R. , Torrence Paul F.
Published: March 2015

Efficient regioselective synthesis of nucleoside conjugates was achieved by cycloaddition reaction of azides and alkynes using sodium ascorbate/CuSO4 system as a catalyst. These 16 novel thymidine analogues were obtained in excellent yields (75–100%), employing mild reaction conditions with a broad scope of structural modification. For the compounds tested, no specific antiviral effects could be witnessed against a broad range of viruses.
In Vitro Anti-HCV Activities of Saxifraga Melanocentra and its Related Polyphenolic Compounds
Authors: Zuo Guo-Ying , Li Zheng-Quan , Chen Li-Rong , Xu Xiao-Jie
Published: March 2015

The aim of this study was to search for new natural anti-HCV agents from Chinese herbal medicine. Bioactivity-guided extraction and isolation methods were used. Active part and pure compounds were obtained from ethanolic extract of Saxifraga melanocentra Franch. and their in vitro inhibitory activities (IC50) against HCV NS3 serine protease were tested by enzyme-linked immunosorbent assay. Results showed that the polyphenolic ethyl acetate part of the herbal extract was the most active, and from this 18 polyphenols representing active compounds were isolated and identified. IC50 values of these compounds and five related ones were obtained. A broad-degree of anti-HCV activity was observed among them in the following order: gallated esters of D-glucose and rutin (0.68–4.86 μM)> flavonoids (33.11–370.37 μM)> gallic acid and its methyl and ethyl esters, Bergenin and others (over 1000 μM). The most active compound was 1,2,3,4,6-penta- O-galloyl-β-D-glucoside (0.68 μM). In conclusion, polyphenols were responsible for the anti-HCV constitution of S. melanocentra, and multigallated esters of D-glucose possessed the strongest inhibition against HCV NS3 serine protease and little cytotoxic effect, suggesting the potential use of these compounds for designing and developing drugs for treatment of the viral infection.
Synergistic Combination Effect of Cidofovir and Idoxuridine on Vaccinia Virus Replication
Authors: Remichkova Mimi , Petrov Nikolay , Galabov Angel S
Published: March 2015

In view of the potential menace of a terrorism attack with smallpox virus, an intensive search of chemotherapeutic agents active against orthopoxviruses is underway. We comparatively studied the antiviral activity of cidofovir (CDV) and idoxuridine (IUdR) against two vaccinia virus (VV) strains, Bratislava and RIIPD, in cell cultures of chick embryo fibroblasts (CEF). The investigations were carried out according to cytopathic effect (CPE) inhibition assay protocols. To determine the cytotoxicity of the compounds, maximal tolerated concentration (MTC) was calculated in CEF cell monolayers and 50% cell growth inhibitory concentration (CGIC50) was calculated in growing cell cultures. It was found that the antiviral effects were strongly dependent on virus inoculum size. There were no marked differences in the susceptibility to CDV and IUdR between the two VV strains. The individual half maximal inhibitory concentration (IC50) for CDV varied from 7.1–8.5 µM at 10/100 virus 50% infectious dose (ID50) to 13.6–26.5 µM at 10,000 ID50. The CDV selectivity index was also virus dose-dependent with MTC/IC50 and CGIC50/IC50 values ranging between 37.8–141.4 and 33.3–124.6, respectively. For IUdR, IC50 ranged from 0.58 to 0.85 µM, but the selectivity index for monolayer CEF and growing cell cultures produced substantial different results with MTC/IC50 and CGIC50/IC50 values between 117.7–172.4 and 20.4–33.3, respectively. The combination effects of CDV and IUdR against VV Bratislava strain in the CPE inhibition test were also determined. The test design of both combination antiviral effect and combined cytotoxicity followed a three-dimensional model. The combined effect of CDV and IUdR on VV replication in monolayer CEF cultures was characterized as a markedly synergistic one. In contrast, CDV and IUdR together reduced cytotoxicity in both monolayer and growing CEF cells.
Anti-Herpes Simplex Virus Activities of Two Novel Disulphated Cyclitols
Authors: Ekblad Maria , Bergström Tomas , Banwell Martin G , Bonnet Muriel , Renner Jens , Ferro Vito , Trybala Edward
Published: March 2015

By screening a library of sulphated compounds of low molecular weight, we have found that several cyclitol derivatives, each modified with two sulphate groups in addition to pyrrole and various aromatic moieties, inhibited infectivity of herpes simplex virus (HSV) at concentrations approximately 100 times lower than those toxic for cultured cells. These disulphated cyclitols interfered with HSV-1 attachment to cells, and efficiently reduced the cell-to-cell spread of the virus. This effect is most likely due to their low molecular weight and associated with the compounds' capability to access the narrow intercellular spaces. Furthermore, these disulphated cyclitols also inactivated infectivity of HSV. However, the virus-inactivating activities of these compounds were to some extent diminished in the presence of human cervical secretions or other protein-rich solutions suggesting that disulphated cyclitols may have some features of surfactant-type virucides. In conclusion, this new class of anti-HSV compounds offers potential for further development.
Inhibitory Activity of 4-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethylpyrimidin-2-yl) Benzenesulphonamide and its Derivatives against Orthopoxvirus Replication in vitro
Authors: Selvam Periyasamy , Murugesh Narayanan , Chandramohan Markandavel , Keith Kathy A , Kern Earl R
Published: March 2015

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethylpyrimidin-2-yl) benzenesulphonamide and its derivatives were tested in vitro for antiviral activity against vaccinia and cowpox virus replication in human foreskin fibroblast (HFF) cells, and their activity was compared with cidofovir (CDV). Among the tested compounds, 4-[(5-methyl-1,2-dihydro-2-oxo-3-H-indol-3-ylidene)amino]- N-(4,6-dimethylpyrimidin-2-yl)benzene-sulphonamide was the most active against vaccinia virus, with a 50% effective concentration (EC50) value of 18 µM and 4-[(N-acetyl-1,2–dihydro-2-oxo-3-H-indol-3-ylidene)amino]- N-(4,6-dimethylpyrimidin-2-yl) benzenesulphonamide was the most active against cowpox virus (EC50=33 µM). Cidofovir was found to have an EC50 of 20 µM and 32 µM against vaccinia and cowpox virus, respectively. Most of the tested compounds were non-cytotoxic (>300 µM) in HFF cells as determined by a neutral red uptake assay. The substitution of a halogen atom at the 5-position of isatin abolished the antiviral activity.
Rapid Propagation of Low-Fitness Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 by a Streptococcal Metabolite Sparsomycin
Authors: Miyauchi Kosuke , Komano Jun , Myint Lay , Futahashi Yuko , Urano Emiko , Matsuda Zene , Chiba Tomoko , Miura Hideka , Sugiura Wataru , Yamamoto Naoki
Published: March 2015

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the –1 frameshift efficiency required to produce the Gag—Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.
Combinatorial Ribavirin and Interferon Alfacon-1 Therapy of Acute Arenaviral Disease in Hamsters
Authors: Gowen Brian B , Smee Donald F , Wong Min-Hui , Pace Anne M , Jung Kie-Hoon , Bailey Kevin W , Blatt Lawrence M , Sidwell Robert W
Published: March 2015

Several arenaviruses endemic to South America (Junin, Machupo, and Guanarito) and Africa (Lassa) are known to cause frequently fatal haemorrhagic fever. With the exception of ribavirin, which has demonstrated efficacy in cases of Lassa fever, there is no other effective therapeutic for the treatment of arenaviral haemorrhagic fever. We have recently reported that consensus interferon-α (IFN alfacon-1) can protect hamsters from lethal Pichinde virus (PCV) infection, which serves as a model for acute arenaviral disease in humans. Here we demonstrate highly effective therapy through the combined use of ribavirin with IFN alfacon-1 for the treatment of PCV infection in hamsters. Ribavirin was given orally, twice per day for 7 days, and IFN alfacon-1 was administered intraperitoneally once per day for 10 days. Treatments were initiated 1–5 days post-virus challenge using various dose combinations, many of which were less than optimal when the drugs were given independently. Combining suboptimal doses of ribavirin (5–10mg/kg/day) with IFN alfacon-1 (5–10 µg/kg/day), we were able to demonstrate increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. Our data indicate that combination therapy results in synergistic activity that may slow down the progression of the disease and decrease fatality rates associated with severe arenaviral infections in humans. Further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity.
Activities of Oseltamivir and Ribavirin Used Alone and in Combination against Infections in Mice with Recent Isolates of Influenza a (H1N1) and B Viruses
Authors: Smee Donald F , Wong Min-Hui , Bailey Kevin W , Sidwell Robert W
Published: March 2015

Mouse models have been widely used for evaluating potential influenza virus inhibitors. However, the viral strains traditionally used in these models are fairly old and do not represent currently circulating viruses in nature. We developed two new lethal infection models in mice using mouse-adapted influenza A/New Caledonia/20/99 (H1N1) and influenza B/Sichuan/379/99 viruses. Both virus infections were used to study oral treatment with oseltamivir and ribavirin, both alone and in combination. Oral treatments were given twice daily for 5 days starting 4 h before infection in initial studies. Against influenza A, oseltamivir was active at 10, 20, and 40 mg/kg/day, protected 80–100% of mice from death and reduced lung consolidation — ribavirin was similarly effective at 20, 40, and 80 mg/kg/day. When treatments were initiated after virus challenge, delaying treatment with oseltamivir even 1 day caused it to be ineffective. Ribavirin prevented mortality by 50–80% when treatments were delayed 1–4 days after infection. The combination of the two drugs (oseltamivir at 20 mg/kg/day and ribavirin at 40 mg/kg/day) was no better than ribavirin alone. In contrast to what we observed with influenza A virus infections, oseltamivir and ribavirin showed similar dose-related antiviral activities against influenza B virus infections. The compounds both significantly increased survival when treatments started up to 4 days after infection, but ribavirin was more active than oseltamivir (50–80% survival compared to 30–40% survival, respectively, when starting treatments on days 2–4 after infection). By varying the doses of each drug that were used in combination (oseltamivir at 1.25, 2.5 and 5 mg/kg/day; ribavirin at 5, 10 and 20 mg/kg/day) certain dosage combinations were superior to either compound used alone as assessed by decreased mortality, lung virus titre, lung score and lung weight parameters. These activities differed from published results with older, more established virus strains as oseltamivir was less effective and ribavirin was more active than previously reported.
AZT and AZT-monophosphate Prodrugs Incorporating HIV-protease Substrate Fragment: Synthesis and Evaluation as Specific Drug Delivery Systems
Authors: Liotard Jean-François , Mehiri Mohamed , Di Giorgio Audrey , Boggetto Nicole , Reboud-Ravaux Michèle , Aubertin Anne-Marie , Condom Roger , Patino Nadia
Published: March 2015

With the view to deliver anti-HIV nucleoside and nucleoside-monophosphate (MP) analogues specifically into HIV-infected cells, we synthesized a series of ester and phosphoramidate peptide conjugates of zidovudine (AZT) and of AZT-MP, respectively, wherein the peptide sequences derive from a HIV-protease (PR) hydrolysable substrate. Their in vitro stability with respect to hydrolysis, anti-HIV activity and cytotoxicity, and ability to inhibit the HIV-PR activity were investigated. Concerning the ester AZT-peptide conjugates, their antiviral activity level in thymidine kinase-expressing (TK+) CEM-SS and MT-4 cells was in most cases closely correlated to their hydrolysis rate: the faster the hydrolysis, the closer the anti-HIV activity to that of AZT. None of them was a HIV-PR substrate, indicating that their antiviral activity was not related to their intracellular hydrolysis by this enzyme. None of them inhibited HIV in TK-deficient (TK−) CEM cells, demonstrating that they probably act as prodrugs of AZT. Most of the phosphoramidate peptide conjugates of AZT-MP were rapidly degraded in a physiological buffer into several metabolites including AZT. Their anti-HIV activity in TK+ CEM-SS and MT-4 cells was much lower than that of AZT, indicating that only low amounts of AZT or AZT-MP were released into cells during incubation. Antiviral activities measured on TK− CEM cells for some phosphoramidates suggest that low amounts of AZT-MP could be released intracellularly. However, this AZT-MP release was not initiated by a HIV-PR hydrolysis, as no evidence for peptide cleavage was obtained by HPLC analysis of one representative compound after incubation with HIV-PR.
A Novel Colorimetric Assay for CXCR4 and CCR5 Tropic Human Immunodeficiency Viruses
Authors: Kajiwara Keiko , Kodama Eiichi , Matsuoka Masao
Published: March 2015

The majority of HIV isolated from infected patients uses CCR5 as a coreceptor (R5-HIV). Although R5-HIV fails to replicate efficiently in human transformed T-cell lines, HIV using CXCR4 (X4-HIV) can replicate well in such cell lines. Therefore, most of screening systems using the T-cell lines detect only X4-HIV replication. Here we report a new assay to monitor the replication of R5- as well as X4-HIV. An MTT assay using CD4-, CXCR4-, and CCR5-transduced human glioma NP-2 cells (NCK45 cells) was established and then compared with the representative assays including multinuclear activation of a galactosidase indicator assay (MAGI assay). The antiviral activities of not only an adsorption inhibitor and reverse transcriptase inhibitors but also a Tat antagonist in the NCK45 cells, were comparable to those obtained from the MTT assay using MT-4 cells or the MAGI assay. However, the activity of protease inhibitors (PIs) was underestimated, even though expressions of major multidrug resistant genes involved in efflux of PIs were comparable in MT-2, NP-2, and NCK45 cells. After cultivation of more than 6 months, NCK45 cells remained susceptible to HIV infection since NCK45 cells consistently expressed CD4, CXCR4, and CCR5. On the other hand, MAGI cells lost the CD4 expression during culture. Thus, this assay system can stably detect the replication of both X4- and R5-HIV, indicating that it should be useful for the evaluation of HIV replication and drug susceptibility.
3'-Carbon-Substituted Pyrimidine Nucleosides Having a 2',3'-dideoxy and 2',3'-didehydro-2',3'-dideoxy Structure: Synthesis and Antiviral Evaluation
Authors: Kumamoto Hiroki , Onuma Sayoko , Tanaka Hiromichi , Dutschman Ginger E , Cheng Yung-Chi
Published: March 2015

The bis(tributylstannyl) derivative of 2',3'-didehydro-2',3'-dideoxyuridine (d4U) underwent an anionic 5'-O→3'-C stannyl migration to yield the 3'-tributylstannyl-d4U. This compound, with its vinylstannane structure, allowed ready access to the preparation of 3'-carbon-substituted analogues through the Stille reaction. A conventional transformation of the uracil moiety of these d4U analogues led to the corresponding 2',3'-didehydro-2',3'-dideoxycytidine (d4C) counterparts. Some 2',3'-dideoxycytidine (ddC) analogues were also synthesized. Antiviral evaluation revealed that none of these analogues showed activity against HIV, hepatitis B virus, herpes simplex virus-1 (HSV-1) and HSV-2.
High Frequency of Spontaneous Helicaseprimase Inhibitor (BAY 57–1293) Drug-Resistant Variants in Certain Laboratory Isolates of HSV-1
Authors: Biswas Subhajit , Swift Mihaiela , Field Hugh J
Published: March 2015

Herpes simplex virus (HSV) helicase-primase is the target for a new group of potent antivirals that show great promise in vivo. A claimed advantage of this class of compounds is the low rate of drug resistance, which is reported to occur at a lesser rate than acyclovir (ACV)-resistance in cell culture. We confirmed that BAY 57–1293 is highly active against HSV-1 and superior to ACV when tested in Vero cells. Notably, drug resistance was detected in laboratory working stocks in two different strains of HSV at 10−4to 10−5and there was evidence that the resistant variants were present in the virus population before the selection was applied. Plaque-purified clones obtained from the parental viruses showed a lower level of resistance selection in the presence of drug (10−6) and this value is similar to published reports. In the case of HSV-1 SC16, no difference was observed between a working stock and a plaque-pure clone in the rate of resistance to the nucleoside analogue ACV. The working stocks were found to contain variants with resistance to BAY 57–1293 ranging from ∼15-fold to 4,000-fold suggesting that these viruses have the potential to subvert effective therapy. Sequence analysis of HSV-1 helicase protein showed that most of the amino acid substitutions in the variants described in this study tallied with published results, with some interesting exceptions in the case of HSV-1 strain PDK. Resistant variants did not readily revert to a sensitive phenotype in the absence of the inhibitor and representative BAY 57–1293-resistant variants were cross-resistant to an alternative helicase-primase inhibitor, BILS 22 BS. Variants resistant to BAY 57–1293 retained sensitivity to the nucleoside analogue, ACV.
Interaction of 2′-deoxyguanosine Triphosphate Analogue Inhibitors of HIV Reverse Transcriptase with Human Mitochondrial DNA Polymerase γ
Authors: Ray Adrian S , Feng Joy Y , Murakami Eisuke , Chu Chung K , Schinazi Raymond F , Anderson Karen S
Published: March 2015

Mitochondrial toxicity is a limiting factor in the use of some nucleoside reverse transcriptase inhibitors of HIV. To further understand the impact of structural features on the incorporation and exonuclease removal of nucleoside monophosphate (MP) analogues by human mitochondrial DNA polymerase (pol γ), transient kinetic studies were done with analogues of 2′-deoxyguanosine triphosphate. The kinetic parameters for the incorporation and removal of carbovir (CBV)-MP, dioxolane guanosine (DXG)-MP and 2′,3′-dideoxy-2′,3′-didehydroguanosine (d4G)-MP were studied with pol γ holoenzyme. The importance of the ribose oxygen in incorporation by pol γ was illustrated by an approximate 3,000-fold decrease in the incorporation efficiency of an analogue lacking the ribose oxygen (CBV-TP) relative to those containing a ribose oxygen (DXG-TP and d4G-TP). As a result, a comparison with previous data for the incorporation by HIV reverse transcriptase showed CBV-TP to be approximately 800–8,000-fold more selective for its antiviral target over pol γ relative to the other guanosine analogues. However, DXG-TP and d4G-TP were found to be much more selective than previously reported values for mitochondrial toxic nucleoside analogues. Structural modelling based on sequence homology with other polymerase A family members suggests that an interaction between the ribose oxygen and arginine 853 in pol γ may play a critical role in causing this differential incorporation. Exonuclease removal of a chain-terminating CBV-MP was also found to be more efficient by pol γ. These results help to further elucidate the structure activity relationships for pol γ and should aid in the design of more selective antiviral agents.
Superior Efficacy of Helicase-Primase Inhibitor BAY 57–1293 for Herpes Infection and Latency in the Guinea Pig Model of Human Genital Herpes Disease
Authors: Baumeister Judith , Fischer Ruediger , Eckenberg Peter , Henninger Kerstin , Ruebsamen-Waigmann Helga , Kleymann Gerald
Published: March 2015

The efficacy of BAY 57–1293, a novel non-nucleosidic inhibitor of herpes simplex virus 1 and 2 (HSV-1 and HSV-2), bovine herpesvirus and pseudorabies virus, was studied in the guinea pig model of genital herpes in comparison with the licensed drug valaciclovir (Valtrex™). Early therapy with BAY 57–1293 almost completely suppressed the symptoms of acute HSV-2 infection, and reduced virus shedding and viral load in the sacral dorsal root ganglia by up to three orders of magnitude, resulting in decreased latency and a greatly diminished frequency of subsequent recurrent episodes. In contrast, valaciclovir showed only moderate effects in this set of experiments. When treatment was initiated late during the course of disease after symptoms were apparent, that is, a setting closer to most clinical situations, the efficacy of therapy with BAY 57–1293 was even more pronounced. Compared with valaciclovir, BAY 57–1293 halved the time necessary for complete healing. Moreover, the onset of action was fast, so that only very few animals developed new lesions after treatment commenced. Finally, in a study addressing the treatment of recurrent disease in animals whose primary infection had remained untreated BAY 57–1293 was efficient in suppressing the episodes. In summary, superior potency and efficacy of BAY 57–1293 over standard treatment with valaciclovir was demonstrated in relevant animal models of human genital herpes disease in terms of abrogating an HSV infection, reducing latency and the frequency of subsequent recurrences. Furthermore, BAY 57–1293 shortens the time to healing even if initiation of therapy is delayed.
Antiviral Profiles of Novel Iminocyclitol Compounds against Bovine Viral Diarrhea Virus, West Nile Virus, Dengue Virus and Hepatitis B Virus
Authors: Gu Baohua , Mason Peter , Wang Lijuan , Norton Pamela , Bourne Nigel , Moriarty Robert , Mehta Anand , Despande Mehendra , Shah Rajendra , Block Timothy
Published: March 2015

The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family ( Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-95II), led to a structure that was as well tolerated as DNJ (CC50>500 µM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.
Apricitabine: A Novel Deoxycytidine Analogue Nucleoside Reverse Transcriptase Inhibitor for the Treatment of Nucleoside-Resistant HIV Infection
Authors: Wainberg Mark A , Cahn Pedro , Bethell Richard C , Sawyer James , Cox Susan
Published: March 2015

Existing nucleoside reverse transcriptase inhibitors for HIV disease are limited by problems of resistance and, in some cases, long-term toxicity. Apricitabine (ATC; formerly BCH10618, SPD754 and AVX754) is a deoxycytidine analogue nucleoside reverse transcriptase inhibitor in clinical development. ATC retains substantial in vitro activity against HIV-1 containing many mutations associated with nucleoside reverse transcriptase inhibitor resistance, showing a less than twofold reduction in susceptibility in the presence of either up to five thymidine analogue mutations or the M184V mutation. ATC showed a low potential for cellular or mitochondrial toxicity in vitro. ATC is well absorbed orally, with a bioavailability of 65–80%. Its plasma elimination half-life (approximately 3 h), and the intracellular half-life of its triphosphate (TP) metabolite (6–7 h) support twice-daily dosing. Intracellular ATC-TP levels are markedly reduced in the presence of lamivudine or emtricitabine, indicating that clinical co-administration of ATC together with these agents will not be possible. The drug is renally eliminated, giving a low potential for hepatic drug interactions. In a double-blind, randomized, placebo-controlled Phase II monotherapy trial in antiretroviral-naive patients, ATC doses of 1,200 and 1,600 mg/day reduced plasma viral load levels by 1.65 and 1.58 log10 HIV RNA copies/ml, respectively, after 10 days of treatment ( P<0.0001 versus placebo). ATC showed a low propensity to select for resistance mutants in vitro and during clinical monotherapy. ATC was well tolerated in volunteers and in HIV-infected patients. This promising profile suggests that ATC may be useful in treating patients who have failed previous lamivudine- or emtricitabine-containing regimens. Further studies to evaluate the long-term efficacy and tolerability of ATC are underway.
Effect of a Plant Polyphenol-Rich Extract on the Lung Protease Activities of Influenza-Virus-Infected Mice
Authors: Serkedjieva Julia , Toshkova Reneta , Antonova-Nikolova Stefka , Stefanova Tsvetanka , Teodosieva Ani , Ivanova Iskra
Published: March 2015

Influenza infection was induced in white mice by intranasal inoculation of the virus A/Aichi/2/68 (H3N2). The lung protease and the protease-inhibitory activities were followed for 9 days after infection. The intranasal application of a polyphenol-rich extract (PC) isolated from Geranium sanguineum L. induced a continuous rise in the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. Influenza virus infection triggered a slight reduction in protease activity in the lungs at 5 and 48 h post infection (p.i.) and a marked increase at 24 h and 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h and 6 and 9 days p.i. PC treatment brought both activities to normal levels. The restoration of the examined parameters was consistent with a prolongation of mean survival time and reduction of mortality rate, infectious virus titre and lung consolidation. PC reinstated superoxide production by alveolar macrophages and increased their number in virus-infected mice. The favourable effect on the protease and the protease-inhibitory activities in the lungs of influenza-virus-infected mice apparently contributes to the overall protective effect of PC in the murine experimental influenza A/Aichi infection. The antiviral effect of the individual constituents was evaluated.
Tropolone and its Derivatives as Inhibitors of the Helicase Activity of Hepatitis C Virus Nucleotide Triphosphatase/helicase
Authors: Borowski Peter , Lang Melanie , Haag Annemarie , Baier Andrea
Published: March 2015

In this report, we demonstrate the interaction of the non-structural protein 3 (NS3) of hepatitis C virus (HCV) with alkaloide tropolone (2-hydroxy-2,4,6-heptatriene-1-one) and its derivatives. The compounds were biochemically screened separately against the ATPase and helicase activities of HCV NS3. In the investigations presented, alkaloide tropolone and its derivatives significantly inhibited the helicase activity of the viral protein when using a DNA substrate, with 50% inhibitory concentration values within a low micromolar range. The results using the RNA substrate were unexpected — none of the tropolone derivatives excerted any modulating influence towards the unwinding activity. Surprisingly, no influence of the nucleoside triphosphatase (NTPase) turnover was observed. Evidence is presented confirming that these compounds do not act by blocking the NTP-binding site, but by occupying an additional allosteric regulatory site. Further mechanisms of action, particularly of some of the derivatives, are discussed.