bluuebunny/crossref_metadata_2025 huggingface dataset:
The Intracellular Formation of a Mononucleotide of the Anti-Influenza Agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896)
Authors: Birch G. M. , Colacino J. M. , Ehlhardt W. J. , Balzarini J.
Published: May 2015

LY217896 is a substituted thiadiazole compound with anti-influenza activity in vitro and in the mouse model of infection. LY297336 is a ribosylated (N-4) derivative of LY217896. A highly polar intracellular metabolite of LY217896 was isolated by HPLC, and mass spectral analysis and treatment of the metabolite with alkaline phosphatase showed that it was a monophosphate (LY307987) derived from LY217896. The formation of LY307987 was inhibited by 43 and 63% when 10 μm of LY217896 was incubated with 100 μM of 8-aminoguanosine (8AGuo) and guanine (Gua), respectively, whereas inosine (Ino) and hypoxanthine (Hx) had no effect on the formation of LY307987. LY217896 inhibited the incorporation of [14C]-Hx into nucleic acids in cells which metabolize LY217896; however, LY217896 did not inhibit the formation of inosine 5′-monophosphate (IMP) from Hx in a cell-free HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-catalysed reaction. Incubation of MDCK cells with 10 μm of LY217896 resulted in an 8-fold increase in the level of intracellular IMP. At 100 μm, neither LY217896 nor LY297336 inhibited inosine 5′-monophosphate dehydrogenase (IMPDH) and only cellular extracts which contained intracellular metabolites of LY217896 inhibited IMPDH. Quantification of the 5-phosphorylribose pyrophosphate (PRPP) levels in BS-C-1, MDCK, and MCN cells showed a positive correlation between PRPP concentration and cellular metabolism of LY217896. Combination studies of LY217896 with 2′,3′-dideoxyinosine (ddlno) or 2′,3′-dideoxyguanosine (ddGuo) showed that LY217896 enhanced the antiretroviral activities of these dideoxynucleosides, which is consistent with an inhibitory effect on IMPDH.
Stereochemical Aspects of the anti-HCMV Activity of Cytidine Nucleoside Analogues
Authors: Mansour T. S. , Cimpoia A. R. , Jin H. , Hunter P. J. , Evans C. A. , Tse H. L. A. , Gillard J. W. , Borthwick A. D. , Knight D. J. , Coates J. A. V.
Published: May 2015

The remarkable selectivity of the β-L enantiomers of 2′,3′-dideoxycytidine analogues against the viral polymerases of HIV and HBV has stimulated our interest in targeting β-L enantiomers of anti-HCMV cytidine analogues. Indeed, Ara-C, FIAC and DMDC are cytidine analogues with β-D configuration that show significant potency as anti-HCMV agents but lack selectivity. β-L enantiomers have therefore been synthesized and evaluated together with four other nucleoside analogues, and the β-L. enantiomers were found not to be inhibitory to HCMV replication. However, the three α-L isomers, α-L-Ara-C, α-L-Xylo-C and α-L-FMAU, emerged with activity against HCMV and have provided new approaches for the treatment of viral diseases with nucleoside analogues possessing the unusual L-configuration.
Synthesis and Antiviral Evaluation of Fluorinated Dipyridodiazepinones and Dipyridodiazepines (Nevirapine Derivatives)
Authors: Boyode B. P. , Sinet M. , Farese A. , Forestier-Roux M.-A. , Condom R. , Ayi I. A. , Kirn A. , Moog C. , Guedj R.
Published: May 2015

The synthesis of trifluoromethyldipyridodiazepinones, which are analogues of nevirapine, one of the non-nucleoside inhibitors of the HIV-1 RT enzyme, is described. The condensation of 3-amino-2-chloro-4-tri-fluoromethyl pyridine with 2-chloronicotinoyl chloride followed by a cyclization with cyclopropylamine provided the corresponding trifluoromethylated dipyridodiazepinone. Similarly, the dipyridodiazepine analogues were synthesized by condensation of 3-amino-2-chloro-4-trifluoromethylpyridine with the O-mesyl derivative of 2-chloro-3-pyridincarbinol followed by reaction with cyclopropylamine. The inhibitory effect of these compounds on the HIV-1-induced cytopathic effect in MT4 cells has been evaluated.
Oxathiin Carboxanilide Derivatives: A Class of Non-Nucleoside HIV-1-Specific Reverse Transcriptase Inhibitors (NNRTIs) that are Active against Mutant HIV-1 Strains Resistant to other NNRTIs
Authors: Balzarini J. , Jonckheere H. , Harrison W.A. , Dao D.C. , Anné J. , De Clercq E. , Karlsson A.
Published: May 2015

The HIV-1-specific oxathiin carboxanilide derivative 1-methylethyl 2-chloro-5-[[(5,6-dihydro-2-methyl-1,4-oxathiin-3-yl)carbonyl]amino]benzoate (NSC 615985) (designated UC84) has potent activity against HIV-1(IIIB) (50% effective concentration: 0.015 μg ml−1). UC84 was found to select for a 138-Lys mutant virus strain in HIV-1-infected CEM cell cultures. When the 138-Lys mutation was introduced solely in the p51 subunit of the p51/p66 reverse transcriptase (RT) heterodimer by site-directed mutagenesis, the enzyme proved 10-fold more resistant to UC84 than when the amino acid mutation was introduced solely in the p66 subunit of the p51/p66 RT heterodimer. These data provided clear evidence for a structural and functional role of the p51 subunit in the sensitivity/resistance of the enzyme to UC84. UC84 also proved to be virtually inactive against mutant HIV-1 strains containing the 100-lle, 106-Ala, 138-Lys or 181-Cys mutation in their RT. However, minor structural changes in the molecule, such as replacement of the oxygen of the amide moiety by sulfur, or the isopropyl ester moiety by cyclopentyl or a secondary butyl, or the methyl group of the oxathiin part by ethyl, made the compound markedly more inhibitory to one or several HIV-1 mutant strains. For example, compound 131 (1-methylethyl 2-chloro-5-[[(5,6-dihydro-2-methyl-1,4-oxathiin-3-yl)thioxomethyl]amino]benzoate was only 2-fold more active than the parent compound UC84 against wild-type HIV-1, but 30- to 100-fold more inhibitory to HIV-1 mutant strains that contained the 100-11e, 106-A1a, 138-Lys or 181-Cys in their RT. These findings should be taken into account when selecting suitable drug candidates for the treatment of HIV-1 infections, particularly those that have developed resistance to other non-nucleoside RT inhibitors (NNRTIs).
The Effects of Delayed-Onset Chemotherapy using Famciclovir or Valaciclovir in a Murine Immunosuppression Model for HSV-1
Authors: Field H. J. , Thackray A. M.
Published: May 2015

BALB/c mice were immunosuppressed using a dosing regimen of cyclosporin-A previously shown to interfere with T-cell function. HSV-1 was then inoculated into the skin of the ear pinna and the infection allowed to progress for 5 days. In untreated mice, virus replication was observed in both ear tissue and the central nervous system (brainstem). Virus replication continued at high titre in both sites for 15 days, the duration of the experiment. This model was used to investigate the efficacy of famciclovir (FCV) and vaiaciclovir (VACV), which are the oral prodrugs of penciclovir and acyclovir, respectively, on an established HSV-1 infection. Mice were treated orally with either FCV or VACV at 50 mg kg−1, from day 5 to day 10 post-infection. Within 2 days of the onset of therapy, virus titres in both skin and neural tissues were reduced significantly, and by day 10 post-infection less than 101 p.f.u. per tissue was detectable compared with approximately 104 p.f.u. in the untreated control mice. However, when VACV therapy was stopped, there was a recurrence of infectious virus replication within the ear pinna and the brainstem 2 days later. No recurrence of virus replication was observed in mice that had been treated with FCV. In addition, FCV treatment was superior to VACV at reducing the incidence of clinical signs and disease. Furthermore, mice receiving FCV gained weight more quickly than infected controls and reached uninoculated control weights by the end of the study period. In contrast, VACV had no such beneficial effects. These results confirm that VACV and FCV have different effects on the pathogenesis of HSV. The reasons for this appear to result from critical differences in the mechanism of action of the two compounds at a cellular level. We suggest that these data have important implications for the treatment of severe herpesvirus infections in man.
Sulfated Alkyl Oligosaccharides Inhibit Human Immunodeficiency virus in vitro and Provide Sustained Drug Levels in Mammals
Authors: Nakashima H. , Inazawa K. , Ichiyama K. , Ito M. , Ikushima N. , Shoji T. , Katsuraya K. , Uryu T. , Yamamoto N. , Juodawlkis A. S. , Schinazi R. F.
Published: May 2015

This study provides an estimate of the relative anti-human immunodeficiency virus (HIV) activities of synthetic sulfated alkyl oligosaccharides in vitro and of their mechanism of action, and an assessment of the levels of alkyl oligosaccharides in small mammals. The antiviral activities of the compounds against several human immunodeficiency virus type-1 and type-2 strains were determined in human CD4+ cells, including primary lymphocytes and macrophages. Laser flow cytometry and a cell-based syncytium assay were used to elucidate the anti-binding/fusion properties of the oligosaccharides. The sulfated alkyl laminarioligosaccharide DL-110 was shown to be the most potent and selective anti-HIV agent in culture with a median inhibitory concentration of 0.2 μM in primary human lymphocytes. This compound did not markedly interact with the CD4+ receptor on lymphocytes at 50 μM, but demonstrated potent anti-syncytium properties in vitro at submicromolar concentrations. DL-110 had no anti-coagulation activity at 38 μM. Mice, rabbits and beagle dogs were given an intravenous injection of test compounds and the drug levels in serum were quantified. When 32 mg kg−1 of DL-110 was administered to mice, significant antiviral concentrations in serum were achieved even 12 h after treatment. Similarly, prolonged antiviral effects were noted in rabbits and dogs 24 h after injection of DL-110. The half-life of DL-110 in mice, rabbits and dogs was estimated to be 5 h. DL-110 and some of its derivatives are promising candidates for further evaluation of the prophylaxis and therapy of HIV infections.
Synthesis and Anti-Herpes Activity of a Series of Indolizines
Authors: Foster C. , Ritchie M. , Selwood D. L. , Snowden W.
Published: May 2015

A systematic screening study identified 2-phenyl-indolizine [1] as having significant human cytomegalovirus (HCMV) activity (IC50 = 7-19 μM). Preliminary mode of action studies on infected cells treated with [1] indicated supression of HCMV-related polypeptides with little effect on the host cells. A series of analogues were synthesized, some of which showed improved HCMV activity although with lower therapeutic indices than [1]. Several analogues demonstrated significant varicella zoster virus activity, with 2-(4-cyanophenyl)indolizine [5] having an IC50 of 5 μM and a therapeutic index of 20 over cell toxicity in the MRC 5 cell line.
Basis of Relative Insensitivity of HIV-Infected JM Cells to AZT
Authors: Snyder R. D. , Brennan T. , Taylor D. L. , Tyms A. S.
Published: May 2015

The basis of azidothymidine (AZT) insensitivity in the human JM T-cell line has been investigated. It was shown that uptake of radiolabelled thymidine or AZT into cellular acid-soluble pools of JM cells was about 10-fold lower than that seen in AZT-sensitive HeLa cells. Thymidylate kinase, however, was apparently not inhibited by AZTMP in JM cells to the extent observed in most cells and, as a result, AZTTP formation proceeded at a greater rate than in HeLa cells, which exhibited accumulation primarily of AZTMP. Thus the deficit in phosphorylation in JM cells cannot solely account for the decreased AZT sensitivity. Instead, it was shown that JM cells excreted AZTMP into the culture medium and that, whereas HeLa cells continued to accumulate AZT nucleotides over time, JM cells did not. This excretion of AZTMP in JM cells also led to a failure to sustain lowered competing TTP pools. It is concluded that it may not be appropriate to use the JM cell line for testing of novel anti-HIV nucleotides designed to circumvent requirements for phosphorylation.
Synthesis and in vitro Antiviral Properties of Amphiphilic Dinucleoside Phosphate Derivatives of 2′,3-dideoxycytidine (ddC)
Authors: Schott H. , Häussler M. P. , Gowland P. , Bender A. , von Briesen H. , Schwendener R. A.
Published: May 2015

N4-hexadecyl-5′-0-(4-monomethoxytrityl)-2′-deoxycytidine-3′-hydrogenphosphonate and 5′-0-(4-monomethoxytrityl)-2′-deoxythymidine-3′-0-hydrogenphosphonate were condensed with 2′,3′-dideoxycytidine (ddC) according to the hydrogenphosphonate method to yield N4-hexadecyl-2′-deoxycytidylyl-(3′-5′)-2′,3′-dideoxycytidine (N4-hexadecyldC-ddC) and 2′-deoxythymidylyl-(3′-5′)-N4-palmitoyl-2′,3′-dideoxycytidine (dT-N4-palmddC). N4-palmitoyl-2′,3′-dideoxycytidine (N4-palmddC) was synthesized by reacting palmitic anhydride with ddC. Both dinucleoside phosphates have amphiphilic properties and represent a new class of ddC derivatives in which in the case of the dinucleosides, the ddC-5′-monophosphate is masked with lipophilic residues of variable stability. The ddC derivatives can be solubilized in water by micelle formation and, because they have lipophilic residues, they can be incorporated into the lipid membranes of liposomes. The ddC derivatives were shown to have antiviral activities comparable to those of AZT and ddC when tested in vitro against HIV-1-infected HeLa and H9 cells as well as infected human monocytes/macrophages.
Potent and Orally Bioavailable HIV-1 Proteinase Inhibitors Containing the 2-aminobenzylstatine Moiety
Authors: Billich A. , Scholz D. , Charpiot B. , Gstach H. , Lehr P. , Peichl P. , Rosenwirth B.
Published: May 2015

In order to design HIV proteinase inhibitors which combine antiviral potency in HIV-infected cells with good oral bioavailability, new derivatives of 2-aminobenzylstatine containing HIV-1 proteinase inhibitors were synthesized. Compounds showing the desired profile emerged from a series of modifications at the P3′ moiety of the parent inhibitor [1], and are characterized by the presence of hydroxy or methoxy substituents at the C-terminal benzylamide. The most potent congeners, compounds [15] and [19], were evaluated in more detail and proved inhibitory to HIV-1 replication in primary T4 lymphocytes with EC90 = 2.2 and 2.7 nM, respectively. They also exhibited adequate oral bioavailability in the range of [13] to 42% in mice and rats. Thus, further investigation of this type of HIV proteinase inhibitor seems warranted.
Antiviral Activity of Bicyclic Pyrimidine Nucleosides
Authors: Loakes D. , Brown D. M. , Mahmood N. , Balzarini J. , De Clercq E.
Published: May 2015

A number of pyrimidine nucleosides, which may show two hydrogen bonding modes, have been prepared and tested for antiviral activity against a series of viruses. Whilst none of the compounds described showed significant activity against human immunodeficiency virus (HIV), the bicyclic 2′-deoxynucleoside, [2], derived from the base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, was shown to inhibit herpes simplex virus type 1 (HSV-1) at similar concentrations as BVDU1 and ACV. Compounds 13, 6-(2-deoxyribofuranosyl)-6H,8H-2-methyl-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, and 14, N4-hydroxy-5-(2-chloroethyl)-2′-deoxyuridine, were as active as ACV against varicella-zoster virus (VZV).
Treatment Parameters for the Successful Management of Varicella Zoster Virus
Authors: Nathwani D.
Published: May 2015

Antiviral therapy has an important role to play in the management of shingles. However, one needs to define clearly when the therapy is effective, who is likely to benefit from it, and whether or not its use can be justified from an economic point of view. Early medical consultation is of paramount importance if the clinical effectiveness and economic benefits from antiviral therapy are to be optimized.
A Proposal that Herpesviruses are Co-Factors of HIV Disease
Authors: Griffiths P. D.
Published: May 2015

There is now extensive evidence that herpesviruses can interact with human immunodeficiency virus (HIV) in vitro. To determine if such interactions could be operative in vivo, evidence from AIDS autopsy series are reviewed. Using basic histopathological techniques, cytomegalovirus (CMV) and HIV coinfection of various individual ceils has been demonstrated. Using cell culture, CMV has been detected in 66% and herpes simplex virus in 11% of patients. Using immunocytochemistry, human herpesvirus type 6 (HHV-6) has been found in virtually all tissue sections. The hypothesis that these viruses contribute to the death of individuals with AIDS is supported by the results of two double-blind, placebo-controlled randomized trials of high-dose aciclovir and an observational study of the same drug. All three studies have shown improved survival. Thus, the case is made that herpesviruses act as co-factors of HIV disease, and that inhibition of such infections represents a potential way of reducing mortality in those with AIDS.

This paper was presented as the case for in a debate on ‘Herpesviruses as co-factors of HIV disease’. The case against was made by Don Jeffries (see page 22)
Antiviral Activity of Inhibitors of Pyrimidine De-Novo Biosynthesis
Authors: Wachsman M. , Hamzeh F. M. , Assadi N. B. , Lietman P. S.
Published: May 2015

Evaluation of the elevation of host cell biosynthesis of deoxynucleoside triphosphates (dNTP's) induced by human cytomegalovirus (HCMV) infection as a target for antiviral therapeutics was carried out. The concentrations of all four intracellular dNTP's rose rapidly following HCMV infection, and were markedly above baseline by 8 h post infection (p.i.). All four deoxynucleoside triphosphates remained elevated above baseline for at least 72 h p.i. The effects of inhibitors of the de-novo pathway of pyrimidine biosynthesis on HCMV viral replication-were quantified by DNA dot blot. All pyrimidine biosynthesis inhibitors examined inhibited the HCMV DNA replication at concentrations that were non-toxic to the cell. These drugs were also more effective against HCMV, which is highly dependent on host denovo synthesis, than against HSV-1 which encodes enzymes capable of increasing the supply of dNTP's. The antiviral effect of brequinar, an inhibitor of one of the enzymes of the de-novo pathway (dihydroorotate dehydrogenase), was examined to determine if it coincided with a decrease in dNTP's. HCMV-infected fibroblasts and uninfected control cells were treated with a concentration of brequinar able to inhibit HCMV DNA levels 90%. It was found that brequinar markedly lowered the levels of dTTP found in treated cells compared to untreated cells in both HCMV-infected and uninfected cells.
Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine in the Presence of Ganciclovir or Foscarnet
Authors: Palmer S. , Rasmussen H. , Harmenberg J. , Cox S.
Published: May 2015

Comparisons were made between the intracellular phosphorylation of 3′-Azido-3′-deoxythymidine (AZT) alone and in combination with ganciclovir (GCV) or foscarnet (PFA) in lymphocytes, uninfected fibroblasts and CMV-infected fibroblasts. The effects of AZT and the combinations of AZT with GCV or PFA on cellular dNTP pools were also examined. The phosphorylation of AZT was not altered by the presence of GCV or PFA in lymphocytes, and neither AZT nor the combinations of AZT with GCV or PFA changed the levels of cellular dNTP pools in these cells. AZT was phosphorylated to a greater extent in lymphocytes when compared to fibroblasts, but the proportion of AZT di- and triphosphates was greater in fibroblasts. The infection of fibroblasts with CMV enhanced AZT phosphorylation and increased the levels of cellular dNTP pools. GCV caused a specific reduction in AZT phosphorylation in CMV-infected fibroblasts, with a seven-fold drop in AZT triphosphate compared to AZT alone. GCV did not affect AZT phosphorylation in uninfected fibroblasts, nor did GCV reduce the dTTP pool compared to AZT alone. The effects of GCV upon AZT phosphorylation in CMV-infected cells may shed light on the antagonistic effects of GCV upon the anti-HIV activity of AZT, and are of importance for the development of effective combination therapies for the treatment of AIDS patients infected with CMV.
Inhibition of SIV and HIV-2 Replication in Cynomolgus Monkeys by (-)9-[4-Hydroxy-2-(Hydroxymethyl)-Butyl]Guanine (H2G)
Authors: Böttiger D. , Johansson N.G. , Lind P. , Lindborg B. , Noréen R. , Putkonen P. , Vrang L. , Wahren B. , Öberg B.
Published: May 2015

The antiherpes compound (-)9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) has been found to suppress the multiplication of SIVsm and HIV-2 in cynomolgus monkeys. This was seen as a delay in the appearance of viral antigen in serum during the primary infection at drug concentrations of 3×10 mg kg−1 day−1 and higher, when H2G was given subcutaneously. These effects of H2G on SIVsm and HIV-2 replication in monkeys were similar to those observed using the same dose of 3′-azidothymidine (AZT). A complete prevention of HIV-2 infection was observed in one of four animals treated with 3×10 mg kg−1 day−1 of H2G. The enantiomeric mixture (+)H2G at 3×25 mg kg−1 day−1 also delayed the appearance of SIVsm antigen but the (+)enantiomer of H2G at 3×10 mg kg−1 day−1 had no effect on primary SIVsm infection in monkeys, indicating that only the (−)enantiomer (H2G) was inhibitory and that this effect was not influenced by the presence of the (+)enantiomer. No adverse effects on blood chemistry or haematology were observed in monkeys given 25 mg kg−1 day−1 of H2G for 9 weeks or 3×25 mg kg−1 day−1 for 10 days.
Phosphoramidates as Potent Prodrugs of anti-HIV Nucleotides: Studies in the Amino Region
Authors: McGuigan C. , Cahard D. , Salgado A. , De Clercq E. , Balzarini J.
Published: May 2015

Novel phosphoramidate derivatives of the anti-HIV nucleoside analogues AZT and d4T have been prepared by phosphorochloridate chemistry. These materials are designed to act as labile membrane-soluble prodrugs of the bio-active free nucleotides. All compounds were fully characterised by a range of methods and were subjected to evaluation in vitro of their anti-HIV efficacy. A notable feature of the current study was that any attempt to replace the amino acid moiety of the phosphoramidate with a simple amine lead to a marked, virtually total loss of activity. Such simple phenyl alkylamino phosphate derivatives of either d4T or AZT inhibit HIV replication at cytotoxic concentrations and have no detectable antiviral selectivity. This clearly highlights the vital role played by the amino acid in the antiviral efficacy of the blocked phosphoramidates.
Acetylfurans as Bio-Isosteres for 3-Methylisoxazole. The Effect of Acetylfuran Positional Isomers on Antirhinoviral Activity
Authors: Bailey T. R. , Diana G. D. , Draper T. L. , Pevear D. C.
Published: May 2015

A panel of four isomeric acetylfuran analogues of disoxaril were prepared. Using a 96-well tissue culture inhibitory dose assay (TCID), these acetylfuran isomers were screened against 15 rhinovirus serotypes. Comparison of the results obtained suggested that antiviral activity was related to hydrogen bond participation by the carbonyl oxygen within the viral pocket. Modelling acetylfuran conformations using an X-ray crystal structure template of 1 in HRV-14 demonstrates that three of the four isomers can adopt the desired conformation for this hydrogen bonding, and the fourth isomer 15 is prohibited by steric constraints from achieving the necessary conformation. These observations are consistent with the hypothesis that broad-spectrum antirhinoviral activity may require a hydrogen bond acceptor at that region of the inhibitor.
Hepatitis B: New Approaches for Antiviral Chemotherapy
Authors: Locarnini S. A. , Civitico G. M. , Newbold J. E.
Published: May 2015

Progress in the development of effective therapeutic regimes for chronic hepatitis B has been slow, mainly due to the lack of promising lead compounds and useful assays for high throughput in-vitro screening. Nucleoside analogue chemotherapy has targeted the inhibition of the hepatitis B virus (HBV) polymerase and achieved inhibition of this unique viral enzyme. The persistence and resistance of HBV covalently closed circular (or supercoiled) DNA, the key replicative intermediate and sole transcriptional template, to existing treatments also poses challenges for the effective development of antiviral chemotherapy. In spite of these difficulties, the process of viral DNA replication, as well as supercoiled DNA generation and processing, is now being elucidated at the molecular level, presenting unique opportunities for new drug targeting and design. This review attempts to highlight these new approaches to the development of treatment regimes for this important disease.
Boronic Acid Derivatives Targeting HIV-1
Authors: Chen X. , Bastow K. , Goz B. , Kucera L. , Morris-Natschke S.L. , Ishaq K.S.
Published: May 2015

A series of novel boronic acid derivatives containing either a pyrimidine or purine base was synthesized. The preparation involved the condensation of 4-bromobutyl boronic acid with the appropriate base. These acyclic nucleosides were designed as potential antiviral agents especially targeting the human immunodeficiency virus. Two analogues, 6-chloro-9-(4-dihydroxyborylbutyl)purine and 2,6-dichloro-9-(4-dihydroxyborylbutyl)purine, exhibited EC50 values of 7.7 μM and 0.99 μM, respectively, in an HIV-1 syncytial plaque reduction assay.
Antiviral Activity of 2-Hydroxy Fatty Acids
Authors: Harper D.R. , Gilbert R.L. , O'Connor T.J. , Kinchington D. , Mahmood N. , Mcllhinney R.A.J. , Jeffries D.J.
Published: May 2015

Following an earlier demonstration of an antiviral effect against varicella-zoster virus (VZV, human herpesvirus 3) using 2-hydroxymyristic acid (2-hydroxytetradecanoic acid; 2-HM), an inhibitor of protein myristoylation, both 2-HM and 2-hydroxypalmitic acid (2-hydroxyhexadecanoic acid; 2-HP) have been tested against a range of viruses. Although both compounds inhibit the replication of varicella-zoster virus (VZV; human herpesvirus 3) they do not inhibit the replication of closely related herpesviruses. They do, however, inhibit the replication of both poliovirus (a member of the Picornaviridae) and the human immunodeficiency virus type 1 (HIV-1; a member of the Retroviridae). Neither compound is toxic to adherent cells by dye uptake assay, although limited toxicity is apparent to non-adherent cell lines at high concentrations. The mechanisms underlying these effects are discussed. A diminished effect of 2-hydroxymyristic acid when the compound is dissolved in dimethyl sulphoxide (DMSO) rather than ethanol is reported, and the implications for the use of DMSO as a ‘universal solvent’ for compound screening noted. Finally, it is suggested that targeting of ‘virus-essential’ cellular functions may provide an alternative route for inhibiting viral replication.
Mechanism of the Antiviral Activity of New Aurintricarboxylic Acid Analogues
Authors: Reymen D. , Witvrouw M. , Esté J. A. , Neyts J. , Schols D. , Andrei G. , Snoeck R. , Cushman M. , Hejchman E. , De Clercq E.
Published: May 2015

Various new aurintricarboxylic acid (ATA) polymer analogues have been evaluated for their antiviral activity against a wide array of DNA and RNA viruses, and their mechanism of action against human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1). Most of the polymers exhibited marked antiviral activity against a variety of enveloped viruses, but not against non-enveloped viruses. The ATA polymers displayed the most pronounced activity against HIV-1, HCMV and human herpesvirus type 6 (HHV-6). Their action against HCMV and HIV could be ascribed to inhibition of the initial attachment of virus particles to the cells. Using radiolabelled virus, we proved that the polymers inhibit the binding of HCMV to HEL fibroblasts. By flow cytometric analysis, we demonstrated that these new polymers interfere with (i) the binding of OKT4A monoclonal antibody (mAb) to the cellular CD4 receptor, (ii) the binding of anti-gp120 mAb to HIV-1 glycoprotein (gp) 120, and (iii) the adsorption of HIV-1 virions and recombinant HIV-1gp120 (rgp120) to MT-4 cells. The presence of a salicylic acid substituent on the central bridging carbon in the parent compound ATA seems to play an important role in the anti-HIV activity of these ATA related polymer analogues.
Effect of Herpesvirus Inhibition on Primary SIV Infection in Cynomolgus Monkeys
Authors: Böttiger D. , Öberg B.
Published: May 2015

Foscarnet and (-)9-[4-hydroxy-2-(hydroxymethyl)butyl] guanine (H2G) have already been shown to inhibit herpesviruses in vitro and also to inhibit viral antigen production in primary SIV infection in monkeys. Attempts have been made to determine if these invivo effects on SIV were due to a direct effect on SIV or were mediated through inhibition of endogenous transactivating herpesviruses. The possible involvement of herpesviruses in primary SIVsm infection in monkeys was studied by the use of various inhibitors of herpesvirus replication. Subcutaneous injections of 3 × 5 mg kg−1 day−1 of aciclovir, 3 × 5 mg kg−1 day−1 of ganciclovir and 3 × 28 mg kg−1 day−1 of phosphonoacetic acid had no effect on primary SIVsm infection in cynomolgus monkeys. These doses of aciclovir, ganciclovir and phosphonoacetic acid are inhibitory to several herpesviruses. The results suggest that the effects of foscarnet and H2G on primary SIVsm infection in monkeys are direct and not mediated through inhibition of a replicating herpesvirus.
In-Vitro Virucidal and Virustatic anti HIV-1 Effects of Extracts from Persea Americana Mill, (Avocado) Leaves
Authors: Wigg M.D. , Al-Jabri A.A. , Costa S.S. , Race E. , Bodo B. , Oxford J.S.
Published: May 2015

Aqueous (PA1) and methanolic extracts (PA2a–d; PA3) from the tropical tree Persea americana Mill. (Lauraceae), were evaluated for their cellular toxicity and anti-HIV-1 activity both in virustatic and virucidal assays. With the exception of PA3 and PA2d, all extracts showed anti-HIV-1 activity at concentrations which were not toxic for the H9 indicator cells. From the methanol insoluble extract (PA2) four different fractions (PA2a–d) were obtained using reverse-phase column chromatography, and two of the fractions (b and c) showed detectable virucidal effect. One fraction (PA2a) showed virustatic effects inhibiting HIV syncytium formation and viral p24 antigen formation at concentrations which were not toxic for the indicator cells. The results demonstrate for the first time that extracts from P. americana leaves have moderate anti-HIV-1 activity in vitro.
The Hydrophobic Mannose Derivative 1B6TM Efficiently Inhibits Borna Disease Virus in Vitro
Authors: Stoyloff R. , Bode L. , Wendt H. , Mulzer J. , Ludwig H.
Published: May 2015

α-D-Mannnose occupies the terminal position on the N-linked carbohydrate side chain of BDV-specific gp17 (Stoyloff et al., 1994). A hydrophobic derivative of this sugar residue, the 1-0-benzyl-6-0-trityl-α-D-mannnopyranoside (1B6TM), showed a potent and selective inhibition of BDV-replication in vitro, using a range of host-cell/virus systems. When tested in comparison with the unmodified sugar, 1B6TM inhibited the infection in a dose-dependent manner up to 100% without effecting cell viability. After removal of the compound, the antiviral effect remained for several hours. These results suggest that simple modified carbohydrate molecules of BDV-specific sugar residues are able to interfere with virus replication.
Ingenol Derivatives are Highly Potent and Selective Inhibitors of HIV Replication in Vitro
Authors: Fujiwara M. , Ijichi K. , Tokuhisa K. , Katsuura K. , Wang G.-Y.-S. , Uemura D. , Shigeta S. , Konno K. , Yokota T. , Baba M.
Published: May 2015

Ingenol 3,5,20-triacetate has recently been identified as a highly potent and selective inhibitor of HIV replication in vitro. To evaluate the potential of ingenol derivatives as anti-HIV agents, several ingenol derivatives have been synthesized and investigated for their anti-HIV activities, structure-activity relationships, and possible mechanisms of action. Among the ingenol derivatives, 13-hydroxyingenol-3-(2,3-dimethylbutanoate)-13-dodecanoate (RD4-2138) proved to be a highly potent and selective inhibitor of HIV replication. Its 50% effective concentration for viral replication in MT-4 cells was 0.07-0.5 nM depending on viral strains, including HIV-2. This concentration was approximately 105-fold lower than its cytotoxic threshold. RD4-2138 was also inhibitory to the syncytium formation induced by cocultivation of Molt-4 cells with Molt-4/IIIB cells (Molt-4 cells chronically infected with HIV-1). Some correlation was observed with the ingenol derivatives between their inhibitory effects on HTLV-IIIB replication and surface CD4 expression in MT-4 cells, suggesting that the mechanism of inhibition is in part attributed to the inhibition of virus adsorption through down-regulation of CD4 molecules in the host cells. However, such correlation was not identified between the inhibition of HTLV-IIIB and the activation of protein kinase C. Thus, they might have a potential as effective anti-HIV agents when toxicity in vivo could be elucidated.
A Diarylsulphone Non-Nucleoside Reverse Transcriptase Inhibitor with a Unique Sensitivity Profile to Drug-Resistant Virus Isolates
Authors: Buckheit R.W. , Fliakas-Boltz V. , Russell J.D. , Snow M. , Pallansch L.A. , Yang S.S. , Bader J.P. , Khan T.N. , Zanger M.
Published: May 2015

Structure-activity relationship evaluations with a series of diarylsulphone non-nucleoside reverse transcriptase (RT) inhibitors indicated that the steric properties of the molecule and compound lipophilicity primarily contributed to the overall level of activity of the compounds against human immunodeficiency virus type 1 (HIV-1). The most active compounds in the diarylsulphone series had an orthonitro group and yielded anti-HIV activity at sub-micromolar concentrations. Compounds of the diarylsulphone class exhibited antiviral properties similar to other members of the pharmacologic class of HIV-1 specific non-nucleoside reverse transcriptase inhibitors, including activity in a wide variety of established and primary human cells, activity against a wide variety of laboratory and clinical virus isolates, and activity when challenged at high multiplicity of infection. Synergistic inhibition of HIV-1 was observed when the diarylsulphone NSC 667952 was used with the nucleoside analogues AZT, ddl, 3TC and d4T, the protease inhibitor KNI 272 and the sulphonated dye resobene; additive effects were observed when NSC 667952 was used with the nucleoside analogue ddC and other non-nucleoside RT inhibitors. The diarylsulphones exhibited a unique sensitivity profile when evaluated against both virus isolates and purified reverse transcriptase containing non-nucleoside reverse transcriptase inhibitor resistance-engendering mutations. Unlike other members of the class of non-nucleoside compounds, NSC 667952 remained active against virus isolates with the L100I amino acid change in the RT. The compound was, however, highly sensitive to Y181C., K103N and K101E amino acid changes in the RT. The diarylsulphone selected for resistant virus populations which possessed the Y181C amino acid change in the reverse transcriptase and which exhibited enhanced sensitivity to the non-nucleoside inhibitors calanolide A and costatolide.
Phenotypic Resistance of Herpes Simplex Virus Type 1 Strains Selected in Vitro with Antiviral Compounds and Combinations Thereof
Authors: Morfin F. , Snoeck R. , Andrei G. , De Clercq E.
Published: May 2015

Several drug-resistant herpes simplex virus type 1 (HSV-1) strains were obtained under the selective pressure of various antiherpetic drugs used alone or in combination. Their susceptibility to a wide range of antiviral compounds was determined. Strains selected under the pressure of brivudin (BVDU) or 1-β-D-arabinofuranosyl-( E)-5-(2-bromovinyl)uracil (BVaraU) alone were composed of two virus populations: (1) virus resistant to BVDU and BVaraU but not to acyclovir (ACV) or ganciclovir (GCV), which is suggestive of an alteration in the thymidylate kinase activity associated with the viral thymidine kinase (TK) (responsible for the phosphorylation of BVDU-monophosphate to BVDU-diphosphate); and (2) virus resistant to BVDU, BVaraU, ACV and GCV, which is indicative of an alteration in the viral TK activity that converts BVDU, BVaraU and other nucleoside analogues such as ACV and GCV to their monophosphate derivatives. Strains resistant to TK-dependent drugs (i.e. ACV, GCV, BVDU and BVaraU) as well as double-mutant strains with decreased sensitivity to both TK-dependent compounds and the pyrophosphate analogues foscarnet (PFA) and phosphonoacetic acid (PAA) (suggestive of mutations at the level of the DNA polymerase) were recovered under the selective pressure of ACV alone or in combination with BVDU or BVaraU. Combinations of BVDU or BVaraU with PFA or PAA led to strains resistant only to BVDU and BVaraU or double-mutant strains resistant to BVDU, BVaraU and the pyrophosphate analogues, but not to strains resistant to other TK-dependent drugs. Interestingly, strains resistant to ACV, BVDU, GCV and/or the pyrophosphate analogues PFA and PAA remained sensitive to the (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC) and adenine (HPMPA).
Pyridazino[3,4-b][1,5]Benzoxazepin-5(6H)-ones: Synthesis and Biological Evaluation
Authors: Barth B. , Dierich M. , Heinisch G. , Jenny V. , Matuszczak B. , Mereiter K. , Planer R. , Schöpf I. , Stoiber H. , Traugott T. , Aufschnaiter P. v.
Published: May 2015

Starting from 3,6-dichloro-4-pyridazinecarboxylic acid chloride a series of novel pyridazino[3,4- b][1,5]benzoxazepin-5(6 H)-ones as 1,2-diazine isosters of nonnucleoside reverse transcriptase inhibitors was prepared via N-(2-hydroxyphenyl)-3,6-dichloro-4-pyridazinecarboxamides. The inhibition of HIV-1 reverse transcriptase was evaluated.
Inhibitory Effect of Penciclovir-Triphosphate on Duck Hepatitis B Virus Reverse Transcription
Authors: Dannaoui E , Trépo C , Zoulim F
Published: May 2015

The aim of this study was to investigate the mechanism of inhibition of hepatitis B virus replication by penciclovir-triphosphate, the active metabolite of famciclovir. A recently developed in vitro translation assay for the expression of an enzymatically active duck hepatitis B virus (DHBV) reverse transcriptase was used to assess the inhibitory activity of penciclovir-triphosphate (PCV-TP) in comparison with other guanosine analogue triphosphates. Acyclovir-triphosphate (ACV-TP), the chiral triphosphates of penciclovir (PCV), ( R)-PCV-TP and ( S)-PCV-TP, and carbocyclic 2′-deoxyguanosine-TP (CDG-TP) did inhibit reproducibly minus strand DNA synthesis to different extents. CDG-TP was the most potent inhibitor of dGTP incorporation. The inhibitory effect of these compounds against the incorporation of the first nucleotide of minus strand DNA, dGMP, was similar to that observed with DNA chain elongation. 2′,3′-dideoxyguanosine-TP (ddG-TP), ACV-TP and both ( R) and ( S)-PCV-TP inhibited the incorporation of the next nucleotides in the short DNA primer, whereas CDG-TP did not. These results demonstrate that PCV-TP inhibits hepadnavirus reverse transcription by inhibiting the synthesis of the short DNA primer. The data obtained with the inhibition of the enzymatic activity of the DHBV polymerase provides a new insight into the mechanism of action of penciclovir-triphosphate on HBV replication.
Influenza Virus-Inhibitory Effects of a Series of Germanium- and Silicon-Centred Polyoxometalates
Authors: Huffman JH , Sidwell RW , Barnard DL , Morrison A , Otto MJ , Hill CL , Schinazi RF
Published: May 2015

A series of germanium- or silicon-centred heteropolytungstates (polyoxometalates) with the Barrel, Keggin or double Keggin structure were evaluated in vitro for their effects against influenza A (IV-A) and B (IV-B) viruses. Their 50% effective concentrations (EC50) against recent isolates of IV-A (H1N1) and IV-B ranged from 0.1 to 7.8 μM against IV-A (H3N2), the EC50 concentrations were often 10-fold higher. Recent clinical isolates of IV-A were generally more susceptible to these antiviral effects than older, laboratory-adapted strains. These experiments used inhibition of viral CPE in MDCK cells as determined microscopically and by Neutral Red (NR) uptake. Virus yield reduction studies indicated the 90% effective concentrations (EC90) ranged from 0.2 to 32 μM against these viruses. Cytotoxic or cell inhibitory concentrations (CC50), determined by NR uptake and total cell count, ranged from 38 to 189 μM, indicating high selective indices for some of these compounds. Altering time of addition of an active compound relative to infecting cells with IV-A (HINl) showed greatest efficacy when given early in viral replication. Five of the most active polyoxometalates were evaluated against IV-B infections in mice using intraperitoneal treatment beginning 4 h prior to virus exposure. Two of the compounds, one with the Barrel structure and the other with a double Keggin structure, were particularly inhibitory, preventing deaths, reducing arterial oxygen decline and lowering lung consolidation. Lung virus titres were reduced by a maximum of 0.7 log10. Therapy initiated 8 h post-virus exposure was not effective against this in vivo infection.
Antiviral Activity of DG-35-VIII, a Potent Inhibitor of the Protease of Human Immunodeficiency Virus
Authors: Grobelny D , Chen Q , Tyssen D , Tachedjian G , Sebire K , Buchanan L , Birch C
Published: May 2015

The protease of human immunodeficiency virus (HIV) is an important target for antiretroviral drug therapy. The synthesis and in vitro antiviral activity of a novel protease inhibitor, DG-35-VIII, which contains an hydroxyethylhydrazide core unit, is described. DG-35-VIII had potent activity against HIV-1 and related viruses (HIV-2 and simian immunodeficiency virus) in an acutely infected T lymphocyte line (MT-2) and was also active in cells chronically infected with HIV-1, where it inhibited processing of the Pr55gag and Pr160 gag-pal precursor proteins.
The Inhibitory Effect of ddC on Human Immunodeficiency Virus Replication Diminishes in Cells that are Chronically Exposed to the Drug
Authors: Brandi G , Puddu P , Casabianca A , Cianfriglia M , Magnani M
Published: May 2015

One possible explanation for the failure of human immunodeficiency virus type 1 (HIV-1) antiretroviral inhibitors to block the clinical progression of the infection may be a failure to maintain adequate drug levels at the site of viral replication. We have previously found that exposure of human monoblastoid cells (U937) for several months to a therapeutically relevant concentration (0.1 μM) of 2′,3′-dideoxycytidine (zalcitabine, ddC) allowed the isolation of a drug-resistant cell line characterized by a normal drug transport but a reduced ability to accumulate 2′,3′-dideoxycytidine 5′-triphosphate (the active antiretroviral form of the drug). In this paper we show that the drug-resistant cells were indistinguishable from normal cells in terms of surface CD4 receptors. The susceptibility of parental and ddC-resistant U937 cells to infection by HIV-1 was similar, as measured by proviral DNA formation. However, HIV-1 p24 production and the number of infectious virus particles produced were significantly lower in the drug-resistant compared to control cells. Addition of 0.1 μM ddC inhibited viral production by up to 92% in the control cells but had no effect on ddC-resistant cells. Thus, human cells exposed to therapeutically relevant ddC concentrations for several months show a reduced ddC anabolism and allow ddC-sensitive HIV-1 to replicate in the presence of inhibitory ddC concentrations.
T Cell Costimulation by Derivatives of Benzoic Acid
Authors: Kinchington D , Ng T , Mathews N , Tisdale M , Devine D , Ayuko WO
Published: May 2015

A number of analogues of benzoic acid were evaluated in a T cell costimulation assay. One compound, the sodium salt of 2-chloro-5-nitrobenzoic acid (CNBA-Na) was chosen for further study and was found to be a potent costimulator of anti-CD3-induced proliferation of both H9 lymphoblastoid cells ( P<0.001) and human peripheral blood mononuclear cells ( P=0.001) in a dose-dependent manner. The costimulatory effect of CNBA-Na on CD3-triggered DNA synthesis did not enhance human immunodeficiency virus replication in infected cells. Studies with blocking monoclonal antibodies against B7-1 or B7-2 indicated that the immunopotentiatory effect of CNBA-Na required a macromolecular interaction between CD28 (a costimulatory receptor on T cells) and its counter receptor B7 expressed on antigen-presenting cells. The discovery that this low molecular weight compound causes T cell proliferation highlights a potentially novel therapeutic approach to immunodeficiency diseases.
A Non-Radioactive Microtitre Plate Reverse Transcriptase (RT) Assay, Based on Immobilized Template, for Screening of RT Activity Inhibitors and Evaluation of their Mode of Action
Authors: Shao X , Ekstrand DHL , Bhikhabhai R , Kallander CFR , Gronowitz JS
Published: May 2015

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjugated polyadenylic acid (prA), was investigated for simple analyses of the RT inhibiting capacity and mode of action of various substances. Three different technical procedures using the assay were evaluated: (i) direct lC50 determinations with various substances, using four different combinations of primer and dNTP amounts; (ii) analyses of the capacity of the substances to interfere with the binding of RT to template or template-primer (BIC50); (iii) analyses of the capacity of the substances to destroy the template-primer in presence or absence of RT (TDC50). The assay was found to be useful for all three purposes using small amounts of recombinant RT. In the IC50 analyses, the test substances gave values similar to those reported for soluble RT assays, and the values varied in accordance with their known mode of action in relation to the combination of primer and dNTP amount used. Only one of the substances, prG, in addition to DNA and RNA gave true RT binding inhibition. The template destruction assay showed that chain terminating substances gave destruction at low inhibitor concentrations. Furthermore, this destruction was RT-dependent, in contrast to the destruction obtained with substances that can base-pair with the template or primer. For optimum information on mode of action of a given substance all three assay procedures should be used. The use of the assay in relation to the screening and analyses of new RT inhibitory substances and characterization of RT in primary isolates or plasma is discussed.
Reverse Transcriptase-Associated Ribonuclease H Activity as a Target for Antiviral Chemotherapy
Authors: Rausch JW , Grice SFJ Le
Published: May 2015

The availability of highly purified recombinant enzymes and model heteropolymeric nucleic acid substrates now allows more precise evaluation of the ribonuclease H (RNase H) activity associated with human immunodeficiency virus (HIV) reverse transcriptase. In addition to degrading the RNA–DNA replicative intermediate, this C-terminal domain of around 130 residues supports highly specialized events that cannot be complemented by host-coded enzymes during retrovirus replication. RNase H activity should therefore be considered a plausible candidate for therapeutic intervention. Events during HIV replication requiring precise RNase H-mediated hydrolysis, the methodologies available to study these events, and their potential for therapeutic intervention are reviewed here.