bluuebunny/crossref_metadata_2025 huggingface dataset:
Ganciclovir and Penciclovir, but Not Acyclovir, Induce Apoptosis in Herpes Simplex Virus Thymidine Kinasetransformed Baby Hamster Kidney Cells
Authors: Shaw MM , Gurr WK , Watts PA , Littler E , Field HJ
Published: March 2015

The efficacies of ganciclovir (GCV), penciclovir (PCV) and acyclovir (ACV) in inducing cell death in the herpes simplex virus thymidine kinase (HSVTK) system were compared. HSVTK-transformed baby hamster kidney cells treated with GCV, PCV or ACV were monitored for growth by viable count, and for death by TUNEL assay, propidium iodide staining, detection of phosphatidyl serine translocation and detection of DNA laddering. All compounds delayed growth or reduced viability of HSVTK-transformed cells. Drug treatment reduced levels of cyclin B1 message (which normally peaks in G2/M-phase of the cell cycle) and induced a four- to fivefold upregulation of GADD45 message. Treatment with GCV or PCV induced rapid accumulation of cells in S-phase and apoptotic death. Treatment with ACV, however, was associated with sustained S-phase arrest. GCV (and to a lesser extent PCV) increased phosphatidyl serine translocation, induced positive TUNEL results with alterations in cell morphology, caused marked propidium iodide staining and induced DNA laddering. By contrast, up to 7 days' exposure to ACV did not induce DNA laddering, with very little TUNEL staining. ACV treatment had little effect on phosphatidyl serine translocation and propidium iodide staining was markedly reduced compared with treatment with the other compounds. Thus, by all criteria, GCV was the most potent inducer of cell death. The current theories regarding apoptosis or necrosis as the preferred form of cell death in prodrug gene therapy are considered and the suitability of PCV or ACV as potential alternatives to GCV in the HSVTK system is discussed.
Influence of a Cα-Substitution on the S-Pivaloyl-2-Thioethyl Chain on the Anti-HIV Activity and Stability of the Resulting Zidovudine Mononucleoside Phosphotriester
Authors: Peyrottes S , Périgaud C , Aubertin A-M , Gosselin G , Imbach J-L
Published: March 2015

We report the synthesis, in vitro anti-HIV-1 activity and stability study of a mononucleoside phosphotriester derivative of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a new biolabile phosphate-protection, namely S-pivaloyl-2-thioisopropyl ( tBuSATP). This transient protection was characterized by the presence of a methyl substituent at the Cα-position of the previously described S-pivaloyl-2-thioethyl ( tBuSATE) group. Results demonstrated that the new phosphotriester entity was able to deliver selectively the corresponding 5′-mononucleotide within the infected cells. The introduction of a methyl group at the Cα-position of the tBuSATE chain decreased the rate of this delivery.
Effect of Long-Term, Low-Dose Acyclovir Suppressive Therapy on Susceptibility to Acyclovir and Frequency of Acyclovir Resistance of Herpes Simplex Virus Type 2
Authors: Honda Mariko , Okuda Tomoko , Hasegawa Tomomi , Kurokawa Masahiko , Shiraki Kimiyasu , Matsuo Koma , Komatsuzaki Makoto , Niimura Michihito
Published: March 2015

We have examined the susceptibility to acyclovir and frequency of acyclovir-resistant viruses in herpes simplex virus type (HSV) 2 clones isolated directly from genital lesions of 11 patients who had taken suppressive therapy (200 mg/day) for 1–9 years and 15 patients naive to acyclovir. Suppressive therapy significantly reduced the incidence of recurrence and the severity of the skin lesions. HSV samples from genital lesions were directly inoculated into Vero cells, and viral clones were isolated in the absence and presence of 10 pg/ml acyclovir. Five-hundred-and-ninety-two clones, isolated in the absence of acyclovir, were subjected to the acyclovir susceptibility test, and 155 clones isolated in the presence of acyclovir were analysed for the mechanisms of resistance to acyclovir. There were no significant differences in the susceptibility to acyclovir, the frequency of acyclovir-resistant virus and the ratio of thymidine kinase-deficient viruses in acyclovir-resistant viruses between the two groups. The frequency of acyclovir-resistant clones was about three per 10000 plaque forming units (PFU), and genital lesions contained up to 3times106 PFU of replicating virus in the specimens from the patients with genital herpes with or without acyclovir-suppressive therapy. Thus, the low dose of acyclovir suppressive therapy did not affect the susceptibility to acyclovir or increase the frequency of acyclovir-resistant viruses in the genital lesions.
Inhibition of Measles Virus Replication by 5′-Nor Carbocyclic Adenosine Analogues
Authors: Barnard Dale L , Stowell Valerie D , Seley Katherine L , Hegde Vishnumurthy R , Das Subha R , Rajappan Vasanthakumar P , Schneller Stewart W , Smee Donald F , Sidwell Robert W
Published: March 2015

Despite intense efforts to increase vaccine coverage, measles virus (MV) still causes significant morbidity and mortality in the world, sometimes as the result of severe, chronic, lethal disease. In an effort to develop therapies to supplement immunization strategies, a number of 5′-nor carbocyclic adenosine analogues were evaluated for anti-MV activity in CV-1 monkey kidney cells. Of those compounds tested, those either unsubstituted at C4 or possessing a hydroxyl, azido or amino substituent at that position were the most active, with particularly significant inhibition of MV, strain Chicago-1. The EC50 values against this strain ranged from <0.1 to 1 mg/ml, as determined by cytopathic effect reduction assay, and confirmed by neutral red uptake. By virus yield reduction assay (+)-(1 S,2S,3R,4S)-4-(6′-amino-9'H-purin-9′- yl)cyclopentane-1,2,3-triol (2) (-)-(1R,2S,3R)-1-(6′- amino-9'H-purin-9′-yl)-2,3-dihydoxycyclopent-4- ene (3) (-)-(1R,2S,3R)-1-(6-amino-9H-purin-9- yl)cyclopentane-2,3-dihydoxycyclopentane (5) and (-)-(1R,2R,3R,4S)-4-amino-1-(6-amino-9H-purin-9- yl)cyclopentane-2,3-diol (8) were the most potent compounds tested, all with EC90 values of <0.4 mg/ml. Compounds 3 and 5 were also tested against other MV strains, and similarly inhibited those strains except for four designated as Bil, Edmonston, SA and X-1108. Compound 8 did not potently inhibit these other MV strains. In addition, 3, 5 and 8 demonstrated synergistic (additive) inhibition of MV replication in combination with ribavirin at several concentrations. Compounds 3, 5 and 8 were also potent MV inhibitors even when added to infected cells 24 h after virus exposure. None of these three compounds was virucidal at concentrations that inhibited viral replication as determined by virus yield reduction assay. Most compounds tested were also not toxic at concentrations >100 mg/ml in actively growing and stationary-phase cells. Results suggest that these compounds may be clinically useful anti-MV virus agents.
Inhibition of the Bovine Viral Diarrhoea Virus NS3 Serine Protease by a Boron-Modified Peptidyl Mimetic of its Natural Substrate
Authors: Bukhtiyarova Marina , Rizzo Christopher J , Kettner Charles A , Korant Bruce D , Scarnati Helen T , King Robert W
Published: March 2015

Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1′. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144–00, inhibited approximately 75% of the NS3/4 activity at 10 μM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144–00 in BVDV-infected Madin—Darby bovine kidney cells at concentrations as great as 90 μM, suggesting permeability or that other cellular-derived limitations were present.
Novel DL-Galactan Hybrids from the Red Seaweed Gymnogongrus Torulosus are Potent Inhibitors of Herpes Simplex Virus and Dengue Virus
Authors: Pujol CA , Estevez JM , Carlucci MJ , Ciancia M , Cerezo AS , Damonte EB
Published: March 2015

A novel series of DL-galactan hybrids extracted from the red seaweed Gymnogongrus torulosus, was evaluated for its in vitro antiviral properties against herpes simplex virus type 2 (HSV-2) and dengue virus 2 (DEN-2). These compounds were very active against both viruses with inhibitory concentration 50% (IC50) values in the range 0.6–16 μg/ml for HSV-2 and 0.19–1.7 μg/ml for DEN-2, respectively, as determined in a virus plaque reduction assay in Vero cells. The DL-galactans lacked of cytotoxic effects, on stationary as well as on actively dividing cells, and anticoagulant properties. Some of the compounds showed a variable level of direct inactivating effect on both virions, with virucidal concentration 50% values exceeding the IC50s obtained by plaque reduction assay. Full inhibitory activity was achieved when the galactans were present during virus adsorption period, suggesting that the mode of action of these compounds is an interference in the binding of the surface envelope glycoprotein with the cell receptor.
Comparative Study of Bis(Benzyl)Phosphate Triesters of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine (d4T) and CycloSal-d4TMP — Hydrolysis, Mechanistic Insights and Anti-HIV Activity
Authors: Meier Chris , Muus Ulrike , Renze Jürgen , Naesens Lieve , De Clercq Erik , Balzarini Jan
Published: March 2015

A comparative study of three cycloSal-d4TMP 1, 2 and 3 and a variety of bis(benzyl) phosphate triester 4–8 of the antivirally active nucleoside analogue 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T) will be described. This study has been initiated by the observation that the introduction of a simple 7-methyl group in the cycloSal-structure (2) led to a completely different hydrolysis pattern as compared to the prototype cycloSal- d4TMP 1. Instead of the selective formation of d4TMP, a phenyl phosphate diester was formed in the case of the 7-methyl-substituted compound 2. The difference in degradation pathway was caused by a change of the reaction mechanism. The phenyl phosphate diester was chemically and enzymatically inert to further cleavage to yield d4TMP. For comparison bis(benzyl)-d4TMP 4, bis(α-methylbenzyl)-d4TMP 5, bis(α-methoxycarbonylmethyl [MCM]-benzyl)-d4TMP 6 as well as the enzyme-cleavable bis(4-acetoxybenzyl)- d4TMP [bis(AB)-d4TMP(7 and bis(α-methoxycarbonylmethyl-4-acetoxybenzyl)-d4TMP [bis(α-MCM-AB)-d4TMP] 8 were synthesized. Chemical hydrolysis studies proved that all bis(benzyl) triesters hydrolyze to give the intermediate benzyl phosphate diesters. Moreover, the latter two triesters 7,8 and cycloSal-d4TMPs 1 and 3 led finally to the delivery of d4TMP. The chemical hydrolysis studies allowed a detailed mechanistic interpretation of the degradation pathways of triesters 1–8. Cell extract studies of the bis(benzyl) triesters 4–8 confirmed that only triesters 7 and 8 released d4TMP although with a considerable increase of the reaction rate. Anti-HIV evaluation of the compounds showed that cycloSal-d4TMP 1 and the bis(AB) triesters 7,8 were entirely independent of the presence of cellular thymidine kinase (TK).
Aryl-Substituted and Benzo-Annulated CycloSal-Derivatives of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine Monophosphate — Correlation of Structure, Hydrolysis Properties and Anti-HIV Activity
Authors: Ducho Christian , Balzarini Jan , Naesens Lieve , De Clercq Erik , Meier Chris
Published: March 2015

The synthesis of phenyl-substituted and benzo-annulated cycloSal phosphate triesters of the nucleoside analogue 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T, Zerit™) as lipophilic, membrane-soluble pronucleotides is described. The cycloSal moiety was introduced by using cyclic chlorophosphite agents prepared from phenyl-substituted saligenin derivatives and orthohydroxymethylated naphthols, respectively. Hydrolysis studies (HPLC analysis) of the triesters 2, 3 showed a range of hydrolytic stability from 1.4 h up to 5.1 h and the stability could be correlated with the substitution pattern in the cycloSal moiety. A slight decrease of their stability was observed, if phenyl-substituted derivatives were hydrolyzed in human CEM/O cell extracts. D4T and thymine, possible products of enzymatic cleavage of the pronucleotides, were not detected in the cell extracts. A further investigation of the hydrolysis process was performed by 31P-NMR spectroscopy. This technique allowed a precise monitoring of the degradation products and the exact determination of the product ratio. Finally, the newly synthesized compounds were tested concerning their antiviral activity against HIV in vitro. A strong correlation of the hydrolysis properties and the antiviral activity was found. 3-phenyl- cycloSal-d4TMP showed a threefold increase in its anti-HIV-1 activity and retained full activity in thymidine kinase (TK) deficient cells, indicative of a successful TK-bypass.
Antiviral Properties of Prodelphinidin B-2 3′-O-Gallate from Green Tea Leaf
Authors: Cheng Hua-Yew , Lin Chun-Ching , Lin Ta-Chen
Published: March 2015

Prodelphinidin B-2 3′-O-gallate, a proanthocyanidin gallate isolated from green tea leaf, was investigated for its anti-herpes simplex virus type 2 properties in vitro. Prodelphinidin B-2 3′- O-gallate exhibited antiviral activity with IC50 of 5.0 ± 1.0 μM and 1.6 ± 0.3 μM for XTT and plaque reduction (PRA) assays, respectively. Cytotoxicity assay had shown that prodelphinidin B-2 3′- O-gallate possessed cytotoxic effect toward Vero cell at concentration higher than its IC50. The 50% cytotoxic concentration for cell growth (CC50) was 33.3 ± 3.7 μM. Thus, the selectivity index (SI) (ratio of IC50 to CC50) for XTT assay and PRA was 6.7 and 20.8, respectively. Prodelphinidin B-2 3′- O-gallate significantly reduced viral infectivity at concentrations 10 μM or more. Result of time-of-addition studies suggested that prodelphinidin B-2 3′- O-gallate affected the late stage of HSV-2 infection. In addition, it was also shown to inhibit the virus from attaching and penetrating into the cell. Thus, prodelphinidin B-2 3′- O-gallate was concluded to possess antiviral activity with mechanism of inhibiting viral attachment and penetration, and disturbing the late stage of viral infection.
Anti-HSV-1 Activity of Synthetic Humic Acid-Like Polymers Derived from p -Diphenolic Starting Compounds
Authors: Klöcking R , Helbig B , Schötz G , Schacke M , Wutzler P
Published: March 2015

A panel of ten humic-acid-like polymers was synthesized by oxidation of p -diphenolic compounds and characterized by relative molecular weights, FT-IR spectra and functional group analysis. Using the XTT-based tetrazolium reduction assay EZ4U, both the low-molecular starting compounds and the synthesized polymers were examined for antiviral and cytotoxic activities in HSV-1-infected Vero cells. With the exception of hydroquinone, 2,5-dihydroxytoluene and 2,5-dihydroxybenzoquinone, the starting compounds failed to inhibit herpesvirus replication. The polymeric oxidation products, however, developed anti-HSV-1 activity with EC50 values in the range of 0.65 (2,5-DHPOP) and 322 μg/ml (2,5-DHBQOP). The CC50 values of the polymers varied among 32.0 (TMHYDROP) and >512 μg/ml (2,5-DHBQOP, HYDSULFOP). The most effective polymers were found to be 2,5-DHPOP, 2,5-DHTOP and GENOP (EC50: 0.65, 1.6 and 2.2 μg/ml, respectively, and SI: ≥400, ≥80 and ≥58, respectively). Functional group analysis revealed that increasing numbers of carboxyl groups together with a high content of hydroxyl groups tend to enhance the antiviral activity of polymers derived from p -diphenolic compounds.
Synthesis and Anti-HIV Activity of Carbamates of Antiviral Agent Stavudine
Authors: Venkatachalam TK , Samuel P , Kourinov IV , Uckun FM
Published: March 2015

An efficient synthesis of carbamate analogues of the NRTI compound stavudine, has been achieved in five steps starting from commercially available thymidine. The synthesis involves conversion of thymidine into stavudine followed by condensation with carbaimidazole derivative obtained from various aromatic and heterocyclic amines in dimethylformamide solvent. The analogues thus obtained were further purified by crystallization to furnish analytically pure products. Examination of biological activity of these carbamate derivatives of stavudine showed that they inhibited HIV replication only at micro-molar concentrations.
Inhibitors of the IMPDH Enzyme as Potential Anti-Bovine Viral Diarrhoea Virus Agents
Authors: Stuyver Lieven J , Lostia Stefania , Patterson Steven E , Clark Jeremy L , Watanabe Kyoichi A , Otto Michael J , Pankiewicz Krzysztof W
Published: March 2015

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 μM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4–800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 μM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 μM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 μM].
Inhibition of the Replication of a Hepatitis C Virus-Like RNA Template by Interferon and 3′-Deoxycytidine
Authors: King Robert W , Zecher Marianne , Jefferies Matthew W
Published: March 2015

The development of low molecular weight inhibitors of hepatitis C virus (HCV) replication has been hindered by the lack of a good cell-based system that models the entire HCV replication cycle. To date the only two therapies approved for the treatment of HCV infection are interferon (IFN)-α and the nucleoside analogue, ribavirin. We have created a cell-based system that allows for the accurate quantification of the replication of an HCV-like RNA template by proteins that are encoded for by the HCV genome. The system consists of a cell line that constitutively produces luciferase in response to the production of functional HCV replicative proteins. The 293B4α cell line has been formatted into a semi-high throughput, cell-based screen for inhibitors of HCV replication. When these cells were treated with either IFN-α or -β, luciferase production decreased in a dose-responsive manner. Counterscreening these molecules in another cell line, 293SVLuc, in which luciferase production in not dependent the presence of functional HCV proteins, showed that the inhibition of luciferase in the 293B4α cell line was due to inhibition of the replication of the HCV-like RNA template and not anti-cellular or -luciferase activity. Moreover, when the 293B4α cell line was treated with the ribonucleoside analogue, 3′-deoxycytidine, luciferase decreased in a dose-responsive manner. 3′-deoxyguanosine and 3′-deoxyuridine did not inhibit luciferase production and 3′-deoxyadenosine was too cytotoxic to determine if it had any anti-HCV activity
Hepatitis C Virus Therapies: Current Treatments, Targets and Future Perspectives
Authors: Walker Michelle P , Appleby Todd C , Zhong Weidong , Lau Johnson Yn , Hong Zhi
Published: March 2015

Chronic hepatitis C virus (HCV) infection is the cause of an emerging global epidemic of chronic liver disease. Current combination therapies are at best 80% efficacious and are often poorly tolerated. Strategies to improve the therapeutic response include the development of novel interferons, nucleoside analogues with reduced haemolysis compared with ribavirin and inosine 5′-monophosphate dehydrogenase inhibitors. Compounds in preclinical or early clinical trials include small molecules that inhibit virus-specific enzymes (such as the serine proteases, RNA polymerase and helicase) or interfere with translation (including anti-sense molecules, iRNA and ribozymes). Advances in understanding HCV replication, obtaining a sub-genomic replicon and contriving potential small animal models, in addition to solving crystallographic structures for the replication enzymes, have improved prospects for developing novel therapies. This review summarizes current and evolving treatments for chronic hepatitis C infection. In addition, progress in HCV targets and drug discovery tools valuable in the search for novel anti-HCV agents is detailed.
Cellular Pharmacology of D-d4FC, a Nucleoside Analogue Active against Drug-Resistant HIV
Authors: Erickson-Viitanen Susan , Wu Jing-Tao , Shi Guoen , Unger Steven , King Robert W , Fish Barbara , Klabe Ronald , Geleziunas Romas , Gallagher Karen , Otto Michael J , Schinazi Raymond F
Published: March 2015

The backbone of effective highly active antiretroviral therapy regimens for the treatment of HIV infections currently contains at least two nucleosides. Among the features that influence the potency of each component of a regimen and the overall efficacy of the combination are the cellular uptake and bioconversion of nucleoside analogues to their active triphosphate form, and the extent of possible interactions in these steps that might occur when more than one nucleoside is used in a regimen. D-d4FC (Reverset™), a new cytidine analogue with the ability to inhibit many nucleoside-resistant viral variants, was examined for these parameters. In phytohemaglutinin-stimulated human peripheral blood mononuclear cells, D-d4FC was taken up in a rapid (8 h to 50% maximal value), saturable (plateau above 10 μM parent nucleoside concentration) process, resulting in levels of D-d4FC triphosphate that should provide potent antiviral activity against a variety of virus genotypes. Based on measurement of antiviral effects in cell culture, additive and in some cases, synergistic interactions were observed with protease inhibitors, non-nucleoside reverse transcriptase inhibitors or other nucleosides, including cytidine analogues.
Inhibition of Japanese Encephalitis Virus Infection by Diethyldithiocarbamate is Independent of its Antioxidant Potential
Authors: Saxena Shailendra Kumar , Mathur Asha , Srivastava Ramesh Chandra
Published: March 2015

Diethyldithiocarbamate (DDTC), a low molecular weight dithiol, has been described as an immunomodulator and modifier of diverse biological actions in human and animal models, and has also been shown to be effective in several disease conditions. Therefore, we studied the therapeutic aspect of DDTC in providing inhibition of Japanese encephalitis virus (JEV) infection. DDTC tested at various doses (10–100 μmol/kg) revealed that administration at low concentration (10 μmol/kg; i.p.) on alternate days prolonged the average survival time (AST) of mice infected with lethal dose of JEV (102 LD50, i.c.) and delayed progression of the disease. The low dose also provided >80% survival in sub-clinical (105 LD50, i.c.) JEV infection. Administration of DDTC to JEV-infected mice enhanced the inducible nitric oxide synthase (iNOS) activity in brain and level of serum tumour necrosis factor-α (TNF-α). We have recently demonstrated the production of nitric oxide (NO) via induction of iNOS activity is meditated by circulating macrophage-derived factor (MDF), which may be responsible for the delayed progression of the disease. DDTC-mediated inhibition of JEV is believed to involve the augmentation of protective role of MDF as evidenced by the observation that pretreatment with anti-MDF antibody significantly decreased the AST of mice and together with the inhibition of iNOS activity. Interestingly, DDTC alone did not stimulate iNOS and TNF-α in mock-infected normal mice. These results show that DDTC may have a possible therapeutic role during JEV infection.
Inhibitory Effect of Thiosemicarbazone Derivatives on Junin Virus Replication In Vitro
Authors: García Cybele C , Brousse Beatriz N , Carlucci Marla J , Moglioni Albertina G , Alho Miriam Martins , Moltrasio Graciela Y , D'Accorso Norma B , Damonte Elsa B
Published: March 2015

The inhibitory effect of several thiosemicarbazones (TSCs), synthesized from aromatic ketones and terpenones, and their heterocyclic thiadiazoline (TDZ) derivatives, was investigated against Junin virus (JUNV), an arenavirus agent of Argentine haemorrhagic fever. From the 25 compounds tested, six compounds belonging to the TSC group were found to be selective inhibitors of JUNV, with EC50 values determined by a virus yield inhibition assay in the range 3.4–12.5 μM, and selectivity indices greater than 10. By contrast, most of the TDZs obtained by heterocyclization of the TSCs were not active against JUNV. No conclusive structure-activity relationships could be established but systematically higher activity was associated to TSCs derived from aromatic ketones. The mode of action of one of the most active compound, the 3,4-dihydronaphtalen-1(2 H)one thiosemicarbazone (tetralone thiosemicarbazone), was studied further. This TSC lacked virucidal effects on JUNV virions. Results from time of addition experiments and viral protein expression assays suggest that tetralone thiosemicarbazone inhibited a late stage in the replicative cycle of JUNV.
Benzothiadiazine Dioxide Human Cytomegalovirus Inhibitors: Synthesis and Antiviral Evaluation of Main Heterocycle Modified Derivatives
Authors: Martinez Ana , Gil Carmen , Castro Ana , Bruno Ana M , Pérez Concepción , Prieto Columbiana , Otero Joaquin
Published: March 2015

The benzothiadiazine dioxide derivatives are potent non-nucleoside human cytomegalovirus (HCMV) inhibitors. As part of our comprehensive structure-activity relationship (SAR) study of these compounds, we have now proposed structural modifications on the heterocyclic moiety both on the number and the nature of the fused heterocycle and on the kind of heteroatoms pre-sent on it. Synthesis of these new compounds (benzyl derivatives of thiadiazines, thienothiadiazines, benzothienothiadiazines and quinazolines) and the antiviral evaluation against HCMV has been performed. SAR investigation on this class of compounds has defined the structural requirements for potency and toxicity. They have revealed two important clues: i) a fused ring to the thiadiazine framework is necessary to maintain the anti-HCMV action, and ii) the sulfamido moiety in the main heterocycle is crucial to avoid cytotoxicity.
Are the 2-Isomers of the Drug Rimantadine Active Anti-Influenza a Agents?
Authors: Zoidis Grigoris , Kolocouris Nicolas , Foscolos George B , Kolocouris Antonios , Fytas George , Karayannis P , Padalko Elizaveta , Neyts Johan , De Clercq Erik
Published: March 2015

There is a lack of information in the medical chemistry literature concerning the anti-influenza A activity of the drug rimantadine's 2-isomer (2-rimantadine). We now present results showing that, although 2-adamantanamine (2-amantadine) 3 is only moderately active, some 2-rimantadine analogues are effective anti-influenza A virus agents in vitro. The 2-rimantadine analogues and their spirocyclobutane and spirocyclopentane congeners were synthesized through interesting routes. The 2-rimantadine analogues were 2–4 times more potent than rimantadine 2 against influenza virus A H2N2 strain; their spirocyclobutane congeners proved equally active to rimanta-dine 2. Two compounds exhibited a similar activity and one of the compounds was was fourfold more potent than rimantadine 2 against H3N2 strain.
Mechanistic Effect of NMSO3 on Replication of Human Immunodeficiency Virus
Authors: Nakamura Mariko , Terada Masaki , Kamada Minori , Yokono Akira , Nakamori Shogo , Ohno Tsuneya
Published: March 2015

NMSO3, a sulphated sialyl lipid, was evaluated for its efficacy against human immunodeficiency virus type 1 (HIV-1). The compound exhibited concentration-dependent inhibition of HIV-1 replication in primary infection cell culture systems. Substantial inhibition was observed at concentrations of NMSO3 that showed little cytotoxicity. NMSO3 also exhibited anti HIV-1 activity in chronically HIV-1 infected cultures. The production of progeny viruses was completely abolished without cytotoxicity by continuous addition of NMSO3 to chronically infected U937 cells. Furthermore, in attempting to define the inhibitory mechanism of NMSO3, we investigated its effect on several steps of the HIV-1 replication cycle. NMSO3 competes with gp120 for binding to CD4 receptors on cells and inhibits the entry of HIV-1. By epitope analysis of the human CD4 molecule, NMSO3 inhibits the binding of antibodies, which recognize the D1 domain of CD4. Moreover, semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) showed that the integrated provirus is transcriptionally inactive in NMSO3-treated cells, supporting the lack of progeny in the culture supernatant of chronically HIV-1-infected cells treated with NMSO3. These findings indicate that NMSO3 has a unique mechanism of action against HIV-1 in both primary and chronic infection, and may be a valuable compound for the treatment of HIV-1 infection.
Design and Synthesis of Phosphonoacetic Acid (PPA) Ester and Amide Bioisosters of Ribofuranosylnucleoside Diphosphates as Potential Ribonucleotide Reductase Inhibitors and Evaluation of Their Enzyme Inhibitory, Cytostatic and Antiviral Activity
Authors: Manfredini Stefano , Solaroli Nicola , Angusti Angela , Nalin Federico , Durini Elisa , Vertuani Silvia , Pricl Sabrina , Ferrone Marco , Spadari Silvio , Focher Federico , Verri Annalisa , De Clercq Erik , Balzarini Jan
Published: March 2015

Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonu-cleotides to deoxyribonucleotides, we have now prepared and evaluated 5′-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 μM for the UDP analogue 5′-deoxy-5′- N-(phosphon-acetyl)uridine sodium salt (amide) to 600 μM for the CDP analogue 5′- O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100–500 μM concentrations, whereas ADP analogue 5′- N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5′-deoxy-5′- N-(phos-phon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 μM) against HSV-2 and a modest inhibitory activity (EC50: 110 μM) against HIV-1, respectively.
Antiherpes Virus Activities of New 6–19 Carbon-Bridged Steroids and Some Synthetic Precursors
Authors: Petrera Erina , Joselevich Maria , Ghini Alberto , Burton Gerardo , Coto Celia E
Published: March 2015

Three synthetic 6,19-carbon bridged steroids: 3β,20β-diacetyloxy-5α-chloro-19a(R)-hydroxy-6,19-methanopregnane, 3β,20β-diacetyloxy-5α-chloro-6,19-methanopregnane, 6,19-methanopregn-4-ene-3,20-dione and four synthetic precursors: 3β,20β-diacetyloxy-19-hydroxypregn-5-ene, 3β,20β-diacetyloxy-pregn-5-en-19-al, 3β,20β-diacetyloxy-19(E)-(methoxymethylidene)-pregn-5-ene and 20β-acetyloxy-3β-hydroxy-19(E)-(methoxymethylidene)-pregn-5-ene were tested against herpes virus replication in cell cultures. Several compounds were cytotoxic for stationary cells. Antiviral studies performed with all compounds against HSV-1 indicated a dose-dependent virus susceptibility with selectivity indexes (SI) values in the range 1.7–183.2. Selected compounds were also tested against HSV-2 and the SI values obtained were in the range of 31–273. Attempts to reveal the step of virus multiplication affected by pregnanes were performed with one compound. HSV-1 virus incubation with the compound did not alter the ability of virus particles to infect cells; moreover, neither virus adsorption nor penetration appeared to be affected. The drug must be present during at least the first 7 h of the virus cycle to inhibit more than 90% of virus production. All these results suggest that these novel molecules interfere with an intracellular step of virus multiplication, thus behaving like true antivirals.
Synthesis and Antiviral Activity of 1,3-Disubstituted Uracils against HIV-1 and HCMV
Authors: Maruyama Tokumi , Kozai Shigetada , Yamasaki Tetsuo , Witvrouw Myriam , Pannecouque Christophe , Balzarini Jan , Snoeck Robert , Andrei Graciella , De Clercq Erik
Published: March 2015

The development of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) is an efficient strategy for finding new therapeutic agents against human immunodeficiency virus (HIV). A large number of 6-substituted uracil derivatives have been prepared in order to explore new NNRTIs. However, there are few approaches to anti-HIV agents from 1,3-disubstituted uracil derivatives. Therefore, we tried to prepare several 1,3-disubstituted uracils, which were easily obtainable from uracil by preparation under alkali and Mitsunobu conditions, and examined their antiviral activity against HIV-1 and human cytomegalovirus (HCMV). We found that 1-benzyl-3-(3,5-dimethylbenzyl)uracil and 1-cyanomethyl-3-(3,5-dimethylbenzyl)-4-thiouracil showed powerful inhibition against HCMV and HIV-1, respectively.
Nucleoside Reverse Transcriptase Inhibitor Usage and the Incidence of Peripheral Neuropathy in HIV/AIDS Patients
Authors: Dragovic Gordana , Jevtovic Djordje
Published: March 2015

Peripheral neuropathy (PN) is a well-known adverse effect of treatment with nucleoside reverse transcriptase inhibitors (NRTIs). We performed a prospective follow-up study in HIV-infected patients on antiretroviral therapy containing NRTIs. The objective of this study was to examine the incidence of PN among patients using NRTI drugs. Data were obtained using medical records of patients continuously monitored for the efficacy and toxicity of antiretroviral therapy. The incident cases of PN were examined. Incidence rates of PN were calculated for each antiretroviral regimen that included zidovudine, zalcitabine, lamivudine, didanosine (ddI), stavudine (d4T) or didanosine+ stavudine. Poisson regression was used to compare the relative risk of PN for each regimen. A total of 112 HIV-infected patients were treated with at least one NRTI-containing regimen. Thirty-two cases of PN were recorded. The lowest incidence rate (IR) of 0.13 per 100 person-years occurred in patients treated with didanosine. The highest IR for PN occurred with the didanosine+stavudine combination (IR=0.18 per 100 person-years). Compared to didanosine alone, the relative risk of PN was 1.77 (95%CI, 0.52–2.12) for stavudine, and 1.76 (95%CI, 0.95–2.49) for didanosine+stavudine. Other risk factors for PN included a low CD4 cell count and female sex. Our data show that the risk of PN is almost twice as high when stavudine is used alone or in combination with didanosine. The use of stavudine alone or in combination with didanosine should probably not be routinely recommended if other treatment options are available.
Mechanisms of Hepatitis C Virus Infection
Authors: Moriishi Kohji , Matsuura Yoshiharu
Published: March 2015

Hepatitis C virus (HCV) is the major causative agent of chronic non-A, non-B hepatitis. The life cycle of HCV is largely unknown because a reliable culture system has not yet been established. HCV presumably binds to specific receptor(s) and enters cells through endocytosis, as do other members of Flaviviridae. The viral genome is translated into a precursor polyprotein after uncoating, and viral RNA is synthesized by a virus-encoded polymerase complex. Progeny viral particles are released into the luminal side of the endoplasmic reticulum and secreted from the cell after passage through the Golgi apparatus. Understanding the mechanisms of HCV infection is essential to the development of effective new therapies for chronic HCV infection. Several host membrane proteins have been identified as receptor candidates for HCV. Recent advances using pseudotype virus systems have provided information surrounding the initial steps of HCV infection. An HCV RNA replicon system has been useful for elucidating the replication mechanism of HCV. In this review, we summarize our current understanding of the mechanisms of HCV infection and discuss potential antiviral strategies against HCV infection.
Potent and Selective Inhibition of HIV and SIV by Prostratin Interacting with Viral Entry
Authors: Witvrouw Myriam , Pannecouque Christophe , Fikkert Valery , Hantson Anke , Van Remoortel Barbara , Hezareh Marjan , De Clercq Erik , Brown Stephen J
Published: March 2015

Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against human immunodeficiency virus type 1 (HIV-1). However, the antiviral mechanism of prostratin is not well defined. In the present study, we report that prostratin exhibits potent antiviral activity against different strains of HIV-1 (subtypes B and D), a clinical HIV isolate (L1), HIV-2 (ROD and EHO) and SIV (MAC251) with EC50-values ranging from 0.02–0.09 μg/ml. Prostratin was equally active against HIV strains resistant to the polyanionic binding inhibitor dextran sulphate, the fusion inhibitor T-20 (enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs). In contrast, prostratin lost 4.4- and 6.8-fold of its effect against the HIV strains resistant to AMD3100 and the quaternary ammonium salt QAS10+, respectively. As shown by time-of-addition experiments, prostratin needs to be present at the time of viral adsorption to exert its antiviral activity. We selected an HIV strain (NL4.3/PROS) resistant to prostratin in MT-4 cells. The sensitivity of NL4.3/PROS towards prostratin, dextran sulphate and QAS10+ was reduced by 3.2, 4.1 and >50-fold, respectively. However, NL4.3/ PROS was still sensitive to AMD3100, T-20, NRTIs (zidovudine and nevirapine) and a PI (ritonavir). Recombination of the gp160-gene of the NL43/ PROS strain in a NL4.3 wild-type molecular clone fully rescued its phenotypic resistance. DNA sequencing of the NL4.3/PROS strain revealed mutations throughout the gp120 gene previously associated with resistance towards other HIV entry inhibitors. We concluded that prostratin inhibits the entry step of the replication cycle of HIV by interacting with a cellular target necessary for viral entry.
Challenges for the Development of Ribonucleoside Analogues as Inducers of Error Catastrophe
Authors: Graci Jason D , Cameron Craig E
Published: March 2015

RNA viruses are responsible for numerous human diseases; some of these viruses are also potential agents of bioterrorism. In general, the replication of RNA viruses results in the incorporation of at least one mutation per round of replication, leading to a heterogeneous population, termed a qua-sispecies. The antiviral nucleoside ribavirin has been shown to cause an increase in the mutation frequency of RNA viruses. This increase in mutation frequency leads to a loss of viability due to error catastrophe. In this article, we review lethal mutagenesis as an antiviral strategy, emphasizing the challenges remaining for the development of lethal mutagenesis into a practical clinical approach.
Synthesis and Evaluation of ATP-Binding Site Directed Potential Inhibitors of Nucleoside Triphosphatases/ Helicases and Polymerases of Hepatitis C and other Selected Flaviviridae Viruses
Authors: Bretner Maria , Schalinski Sarah , Haag Annemarie , Lang Melanie , Schmitz Herbert , Baier Andrea , Behrens Sven-E , Kulikowski Tadeusz , Borowski Peter
Published: March 2015

5′-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine (FSBI) were obtained by acylation of the 5′-OH of adenosine, guanosine, inosine, and ribavirin with 4-fluorosulphonylbenzoyl chloride (FSBCl) in HMPA. The above derivatives were tested as inhibitors of nucleoside triphosphatase (NTPase)/helicase activities of Flaviviridae: hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and dengue virus (DENV) and polymerase activity of HCV and WNV. When the unwinding activity of viral NTPase/helicases was tested under standard conditions, only weak inhibition was obtained with FSBI (IC50≥120 μM) and in the case of FSBG even an activation was seen. The preincubation of the NTPase/helicases with the 5′-O-FSB derivatives increased the inhibitory effect. Screening of the 5′-O-FSB derivatives on inhibition of the WNV and HCV RNA polymerases employing GTP or UTP substrates revealed rather modest inhibitory effect. FSBI exhibited the highest inhibitory activity against WNV (IC50=70 μM with UTP substrate) and HCV polymerase (IC50=80 μM with GTP substrate). Other 5′-O-FSB derivatives were very weak inhibitors or completely failed to show any activity against HCV and WNV enzymes. In contrast to the NTPase/helicases the preincubation of the polymerases did not influence the inhibition.
Synthesis of β-Enantiomers of N4-Hydroxy-3′-Deoxy-Pyrimidine Nucleosides and Their Evaluation against Bovine Viral Diarrhoea Virus and Hepatitis C Virus in Cell Culture
Authors: Hollecker Laurent , Choo Hyunah , Chong Youhoon , Chu Chung K , Lostia Stefania , McBrayer Tamara R , Stuyver Lieven J , Mason J Christian , Du Jinfa , Rachakonda Suguna , Shi Junxing , Schinazi Raymond F , Watanabe Kyochi A
Published: March 2015

N4-Hydroxycytidine (NHC) was recently reported to have anti-pestivirus and anti-hepacivirus activity. It is thought that this nucleoside acts as a weak alternative substrate for the hepatitis C virus (HCV) polymerase. In addition to NHC, 3′-deoxyuridine (3′-dU) was found to inhibit bovine diarrhoea virus (BVDV) production by 1 log10 at 37.2 μM. These initial findings prompted the synthesis of β-D and β-L analogues of (i) base-modified 3′-deoxy-NHC; (ii) 3′-deoxyuridine; and 3′-deoxycytidine. The antiviral activity of these 42 nucleosides was evaluated against BVDV and HCV bicistronic replicon in cell culture. Among the NHC analogues, the antiviral activity observed for the β-L-3′-deoxy-5-fluoro-derivative 1-(3-deoxy-β-L- erythro-pentofuranosyl)-5-fluoro-4-hydrox-yaminopyrimidin-2( 1H)-one and the β-D-3′-deoxy-5-iodo-derivative 1-(3-deoxy-β-D-erythro-pentofuranosyl)-5-iodocytosine in the replicon system (1 log10 reduction at 100 μM) was due to the concomitant toxicity towards intracellular ribosomal RNA levels (CC90 equal or lower than the EC90). In conclusion, none of the newly synthesized derivatives exhibited enhanced antiviral activity compared to the parent nucleoside NHC.
Gene Silencing of HIV-1 by RNA Interference
Authors: Takaku Hiroshi
Published: March 2015

Short interfering RNAs (siRNAs) are as effective at targeting and silencing genes by RNA interference (RNAi) as long double-stranded RNAs (dsRNAs). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. siRNAs are also promising reagents for developing gene-specific therapeutics. Specifically, the inhibition of HIV-1 replication is particularly well-suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on recent advances in siRNA-based silencing technologies.
Design and Synthesis of Novel Dihydroxyindole-2-Carboxylic Acids as HIV-1 Integrase Inhibitors
Authors: Sechi Mario , Angotzi Gianfranco , Dallocchio Roberto , Dessì Alessandro , Carta Fabrizio , Sannia Luciano , Mariani Alberto , Fiori Stefano , Sanchez Tino , Movsessian Leah , Plasencia Carmen , Neamati Nouri
Published: March 2015

In a search for new HIV-1 integrase (IN) inhibitors, we synthesized and evaluated the biological activity of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and a series of its derivatives. These compounds were designed as conformationally constrained analogues of the acrylate moiety of caffeic acid phenethyl ester (CAPE). DHICA, an intermediate in the biosynthesis of melanins, was prepared as a monomeric unit by a novel synthetic route. In order to perform coherent SAR studies, two series of DHICA amides were synthesized. First, to validate the utility of a previously identified three-point pharmacophore based on CAPE in inhibitor design, we prepared a series of benzyl- or phenylethylamine substituted derivatives lacking and containing hydroxyl groups. Second, dimers of DHICA containing various aminoalkylamine linkers were also prepared with a goal to increase potency. All compounds were tested against purified IN and the C65S mutant in enzyme-based assays. They were also tested for cytotoxicity in an ovarian carcinoma cell line and antiviral activity in HIV-1-infected CEM cells. Seven compounds inhibited catalytic activities of purified IN with IC50 values below 10 μM. Further computational docking studies were performed to determine the title compounds' mode of interaction with the IN active site. The residues K156, K159 and D64 were the most important for potency against purified IN.
New Synthetic Glutathione Derivatives with Increased Antiviral Activities
Authors: Palamara Anna Teresa , Brandi Giorgio , Rossi Luigia , Millo Enrico , Benatti Umberto , Nencioni Lucia , Iuvara Alessandra , Garaci Enrico , Magnani Mauro
Published: March 2015

A series of glutathione (GSH) derivatives with aliphatic chains of different lengths, coupled by peptides bound to the α-NH2 group of Glu, were synthesized. When added to several cell lines, the C6 (n-hexanoyl), C8 (n-octanoyl) and C12 (n-dodecanoyl) derivatives were toxic while the C2 (n-ethanoyl) and C4 (n-butanoyl) derivatives were not. Preliminary experiments were performed to investigate the potential antiviral activity of the C2 and C4 derivatives compared to GSH. The C4 derivative was the most potent and fully characterized. GSH-C4 is a poor substrate of GSH metabolizing enzymes; once oxidized by disulphide-bound formation, C4 is slowly reduced by GSH-reductase. GSH-C4 completely abrogated Sendai virus replication at 7.5 mM with an EC50 of 3.6 mM, compared to 7.5 mM for GSH. GSH-C4 completely inhibited herpes simplex virus (HSV-1) virus production in Vero cells at 10 mM, while the same dose of GSH caused only a 2.5 log10 reduction. Furthermore, the GSH-C4 treatment (7.5 mM) was able to markedly reduce the cytopathic effect of HSV-1 in Vero cells. Thus, GSH derivatives with increased hydrophobic properties are more effective antiviral agents against Sendai and HSV-1 viruses than GSH, suggesting their usefulness in antiviral therapy.