bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening of Compounds for Activity against HIV: A Collaborative Study
Authors: Holmes H. C. , Mahmood N. , Karpas A. , Petrik J. , Kinchington D. , O'Connor T. , Jeffries D. J. , Desmyter J. , De Clercq E. , Pauwels R. , Hay A.
Published: May 2015

The collaborative study was undertaken to examine the sensitivity of a range of tests used in assessing the antiviral activities of compounds against human immunodeficiency virus (HIV). A panel of 20 compounds with diverse antiviral activities against HIV were tested under code at three antiviral testing centres supported by the Medical Research Council's AIDS Directed Programme and at the European Community Centralised Facility (ECCF) for New Antiviral Compounds against AIDS in Belgium. Compounds known to have major anti-HIV activity ranked high in all assays, with the exception of the glucosidase inhibitors and certain nucleoside analogues. Results of two assays based on MT4 cells (centre IV) showed a high degree of similarity, despite the use of distinct HIV-1 (HTLV-IIIB) and HIV-2 (ROD) viruses. Considerable similarity was also observed between the assays based on HTLV-IIIRF in C8166 cells (centres I and II). Other assays performed at centre II and at centre III had enhanced sensitivity for glycosidase inhibitors. The differences in anti-HIV activity that were observed may be attributable to specific properties of the cell lines used and particular testing methodologies. The use of more than one type of assay is advisable in order not to miss compounds with low to moderate activity against HIV.
Synthesis, anti-HIV and anti-Proliferative Activity of New Urea and Nitrosourea Derivatives of Amino Acids
Authors: Gallant G. , Salvador R. , Dulude H.
Published: May 2015

A series of N-methyl, N-(2-chloroethyl) and N-propargyl urea and nitrosourea derivatives of amino acids were synthesized and tested for anti-HIV and anti-proliferative activity. All the agents tested showed only a weak activity to counteract the cytopathic effects of the HIV-1 virus on a T4 lymphocyte cell line (CEM-IW). At high concentration, the N-methyl and N-propargyl ureas were cytotoxic. The nitrosoureas failed to suppress cell proliferation of uninfected CEM-IW cells. The lack of activity and cytotoxicity of the nitrosoureas in this model could be explained by their short chemical half-life.
Anionic Compounds as Inhibitors of African Swine Fever Virus Replication in Vero Cells
Authors: García-Villalón D. , Gil-Fernández C.
Published: May 2015

The anionic compounds, aurintricarboxylic acid, Evans blue, glycyrrhizic acid, taurolithocholic acid, and fuchsin acid, have been tested for their capacity to inhibit the replication of African swine fever virus (ASFV) in Vero cells. Aurintricarboxylic acid suppressed ASFV cytopathic effects at 5μg ml−1 and emerged as the most efficacious inhibitor with a selectivity index (S.I.) of 400, followed by Evans blue with 40, Fuchsin acid with 20, taurolithocholic acid with 10, and glycyrrhizic acid with 5.

All the compounds inhibited the virus adsorption or penetration to the cells in some degree, but inhibition was also found when they were added after virus adsorption, indicating that other mechanism (or mechanisms) are involved in viral inhibition.
HIV-1 Integrase: High-Level Production and Screening Assay for the Endonucleolytic Activity
Authors: Billich A. , Schauer M. , Frank S. , Rosenwirth B. , Billich S.
Published: May 2015

The integration protein of the human immunodeficiency virus type 1 was purified from recombinant bacteria overproducing this enzyme. The final step of purification, namely chromatography on polyUsepharose, yielded a homogeneous protein preparation showing specific DNA cutting and joining activities. For a convenient assay of the endonuclease reaction, a 21-mer duplex oligonucleotide corresponding to the U5-LTR end of the viral DNA was radiolabeled at the dinucleotide that is removed by the enzyme. After the reaction, assay mixtures were passed through DEAE-cellulose filters which bind the substrate, but not the short radiolabeled product. Thus, an enzyme-dependent decrease of bound radioactivity was observed with time. Reaction rate was linearly dependent on enzyme concentration and the amount of substrate used was far below saturating concentrations. The reaction showed a pH-optimum at 7.5 and was strictly dependent on the presence of Mn2+. The presence of reducing agents like 2-mercaptoethanol was not essential for enzymatic activity. The assay was used to test selected compounds for their inhibitory potential against integrase. Typical inhibitors of DNA-topoisomerases did not inhibit the endonuclease reaction, with the exception of the intercalative agent actinomycin D which blocked the reaction with an IC50-value of 3 μM. Dextran sulphate inhibited the enzyme with an IC50 = 1.6 μg ml−1.
5′Prime;-Ester Prodrugs of the Varicella-Zoster Antiviral Agent, 6-Methoxypurine Arabinoside
Authors: Moorman A. R. , Chamberlain S. D. , Jones L. A. , de Miranda P. , Reynolds D. J. , Peoples M. E. , Krenitsky T. A.
Published: May 2015

The potent and selective activity of 6-methoxypurine arabinoside (9-[β-D-arabinofuranosyl]-6-methoxy-9H-purine; 1) and its pharmacokinetic limitations have been described previously. In an attempt to circumvent first-pass catabolism following oral administration, a series of 5′-esters (2a-t) was prepared by dicyclohexyl-carbodiimide-promoted condensation with formic acid in the case of 2a, or by direct acylation of the parent nucleoside with the corresponding acyl chloride. These compounds were evaluated in rats for efficacy as oral prodrugs of 1 by measuring the amount of 1 excreted in the urine. Both aliphatic and aromatic esters exhibited a range of systemic availabilities. The substituted benzoate esters showed a correlation between the amount of 1 excreted and the electron-donating properties of the substituent. A similar relationship was observed for the relative stabilities of this series of esters to non-enzymatic hydrolysis at neutral pH. No clear relationship between systemic availability and solubility, partition coefficient, or stability was observed for the remaining esters of 1. Although significant enhancements in systemic availability were observed with some of the 5′-esters of this series, their limited water solubility precluded their use in intravenous formulations.
Inhibition of the Binding of HIV rgp120 to CD4 by Dyes
Authors: Weaver J. L. , Pine P. Scott , Anand R. , Bell S. , Aszalos A.
Published: May 2015

We have found that several dye compounds have the ability to inhibit the binding of HIV rgpl 20 to the Leu3a epitope of CD4 on PBL. One of these compounds, selected for further testing as the best candidate, can inhibit the growth of HIV In vitro. The tested dyes have varying degrees of specificity and efficacy in inhibiting the binding of rgpl 20. These results may point towards compounds that can be useful therapeutics against HIV.
Amino Acid Ester Prodrugs of Acyclovir
Authors: Beauchamp L. M. , Orr G. F. , de Miranda P. , Bumette T. , Krenitsky T. A.
Published: May 2015

Eighteen amino acid esters of the antiherpetic drug, acyclovir, were synthesized as potential prodrugs for oral administration. The esters were examined for in vitro antiviral activity against herpes simplex virus Type 1 (HSV-1). They were found to have less potency than the parent compound. Their efficiencies as prodrugs were evaluated in rats by measuring the urinary recovery of acyclovir. Ten prodrugs produced greater amounts of the parent drug in the urine. The L-amino acid esters were better prodrugs than the corresponding D- or D, L-isomers, suggesting the involvement of a stereoselective transporter. The L-valyl ester, 256U87, was the best prodrug. Sixty three per cent of its administered dose was excreted as acyclovir in the urine, a considerable improvement over acyclovir itself, for which this value was 19%. Since 256U87 was stable in aqueous solutions, its conversion to acyclovir in vivo was probably enzyme catalyzed. This L-valyl ester prodrug of acyclovir is now undergoing clinical evaluation.
Metabolism of 3′-Azido-3′-Deoxythymidine and 3′-Fluoro-3′-Deoxythymidine in Combination in Human Immunodeficiency Virus Infected Lymphoblastoid Cells
Authors: Cox S.
Published: May 2015

A combination of 3′-azido-3′-deoxythymidine (AZT) with 3′-fluoro-3′-deoxythymidine (FLT) has been shown previously to give synergistic inhibition of human immunodeficiency virus replication and greatly reduced cytotoxicity in vitro. The phosphorylation of the compounds, and their effect upon the natural deoxynucleoside triphosphate pools, were compared in CEM, H9, and HIV-infected H9 lymphoblastoid cells, both for the compounds when used alone and when combined together.

Higher levels of FLT triphosphate than AZT triphosphate, and higher levels of AZT monophosphate than FLT monosphosphate, were formed in all cell types. Both compounds were phosphorylated most efficiently in CEM cells, whereas they were least efficiently phosphorylated in infected H9 cells.

Owing to competition, the phosphorylation of both analogues was reduced when used in combination, compared to the phosphorylation of the separate compounds. The phosphorylation of the separate compounds was therefore at a maximum and was not increased by combining the compounds. The two compounds competed equally with each other for phosphorylation when used at a ratio of AZT: FLT of 5: 1.

Both analogues severely reduced the deoxynucleoside triphosphate pools in uninfected and human immunodeficiency virus-infected H9 cells, but not in CEM cells. The effects of the two compounds were similar to those found when the compounds were combined, and thus H9 cells were shown to be much more sensitive to the effects of the analogues upon deoxynucleoside triphosphate pools than CEM cells were.

Thus the synergistic combination of 3′-azido-3′-deoxythymidine and 3′-fluoro-3′-deoxythymidine was shown to have a similar metabolism and a similar effect upon cellular deoxynucleoside triphosphate pools to the individual compounds.
Purine Arabinosides as Inhibitors of Human Haemopoietic Progenitor Cells
Authors: Averett D. R. , Steinberg H. N. , Koszalka G. W. , Spector T. , Krenitsky T. A.
Published: May 2015

Four purine arabinosides that inhibit varicella-zoster virus (VZV) replication in vitro were tested as inhibitors of colony formation by progenitor cells from normal human bone marrow. In general, erythroid burst forming cells (BFU-E) were more sensitive to inhibition by these compounds than were either erythroid colony forming cells (CFU-E) or granulocyte/macrophage colony forming cells (CFU-GM). A 50% reduction in colony formation (IC50) was observed for BFU-E in the presence of 8 μM 6-methoxypurine arabinoside. Adenine arabinoside and hypoxanthine arabinoside had IC50 values of 1 μM and 4 μM respectively, whereas 6-ethoxypurine arabinoside was not inhibitory (IC50 > 50 μM). Enzyme studies showed that both 6-methoxypurine arabinoside and adenine arabinoside were converted to hypoxanthine arabinoside by adenosine deaminase. 6-Ethoxypurine arabinoside was a much less efficient substrate. When the BFU-E assays were performed in the presence of an inhibitor of adenosine deaminase, 6-methoxypurine arabinoside became non-inhibitory. In contrast, adenine arabinoside became much more inhibitory (IC50 = 0.03 μM). The potency of hypoxanthine arabinoside was unaffected. Thus, incubation of 6-methoxypurine arabinoside and adenine arabinoside under conditions appropriate for the BFU-E assay resulted in the in situ conversion of these compounds to hypoxanthine arabinoside. Biotransformation of compounds must be considered in the assessment of toxicity in vitro.
Inhibition of Moloney Murine Leukaemia Virus Transcription by a Phospholipase-C Inhibitor Affecting Trans-Acting Factors
Authors: Music L. , Sauer G.
Published: May 2015

The propagation and the transcription of Moloney murine leukaemia virus (Mo-MuLV) can be inhibited by the antiviral compound tricyclodecan-9-yl-xanthate (code name: D609), which inhibits phospholipase-C (PLC) and, as a consequence, the activation of protein kinase-C (PKC) isoenzyme(s). The frans-acting factors LVa, LVb, and LVc were shown to be affected; it was not possible to retrieve them after treatment with D609 from Mo-MuLV producer cells, by virtue of the binding affinity to their consensus sequences. In contrast, the binding efficiency of the other three known transacting factors (core, NF1 and GRE), which in addition to the viral transcription, play a role in the regulation of cellular mRNA synthesis remained unimpaired. Neither LVa, LVb, nor LVc was found to be phosphorylated, which suggests that these are not targets of PKC. Only one phosphorylated DNA-binding protein was identified with an apparent molecular weight of 34kDa. This protein co-purified irrespective of the recognition sequences that we used (LVa, LVb, LVc, core, and NF1). Direct evidence is provided for the inhibition of the TPA-induced phosphorylation of the 34 kDa protein by D609. We suggest that the binding of LVa, LVb, and LVc to the DNA is mediated by the 34 kDa protein in its phosphorylated state.
2-Ester Prodrugs of the Varicella-Zoster Antiviral Agent, 6-Methoxypurine Arabinoside
Authors: Chamberlain S. D. , Moorman A. R. , Jones L. A. , de Miranda P. , Reynolds D. J. , Koszalka G. W. , Krenitsky T. A.
Published: May 2015

6-Methoxypurine arabinoside [9-(β-D-arabinofuranosyl)-6-methoxy-9H-purine; 1] is a potent and selective agent against varicella-zoster virus in vitro. Studies in the rat indicated that extensive first pass metabolism of this nucleoside occurred after oral dosing, presumably from high levels of adenosine deaminase in the intestine. Only 3.8% of an oral dose of 6-methoxypurine arabinoside was recovered in the urine. In an attempt to improve these unfavourable pharmacokinetics, a series of twenty-five 2′-esters of 6-methoxypurine arabinoside were prepared either by direct acylation or by a protection, acylation, and deprotection sequence. These esters were evaluated in the rat for their suitability as prodrugs on the basis of urinary recovery of 1 after oral dosing. The straight-chain aliphatic esters, but not the branched chain or aromatic esters, improved this urinary recovery. The 2′-valerate, hexanoate, heptanoate, and octanoate esters (2d–g, respectively) were the most effective, with urinary recoveries of 1 above 15%. The water solubility of the valerate ester was high (107 mM at 37°C and pH 7.2), but solubilities progressively decreased with the longer chain esters. The combination of high water solubility and improved metabolic stability upon oral dosing made 2d the prodrug of choice for both oral and intravenous dosing.
In vitro Anti-Herpesvirus Activities of 5-Substituted 2′-Deoxy-2′-Methylidene Pyrimidine Nucleosides
Authors: Machida H. , Sakata S. , Ashida N. , Takenuki K. , Matsuda A.
Published: May 2015

New pyrimidine deoxyribonucleoside analogues, 2′-deoxy-2′-methylideneuridine (DMDU), 2′-deoxy-2′-methylidenecytidine (DMDC), and their 5-substituted derivatives were tested for the anti-herpesvirus activities and anti-proliferative activity. E-5-(2-Bromovinyl)uracil derivative (BV-DMDU) and its cytosine congener were synthesized from 1-β-D-arabinofuranosyl- E-5-(2-bromovinyl)uracil (BV-araU). 5-Bromo, 5-iodo, 5-methyl, and 5-ethyl derivatives of DMDU and BV-DMDU showed activities against herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The corresponding DMDC derivatives had no or only weak antiviral activity. Among the 2′-deoxy-2′-methylidene pyrimidine nucleosides, BV-DMDU showed the most potent and selective anti-VZV activity. BV-DMDU was more potent than acyclovir, but less active than BV-araU. BV-DMDU was inactive against human diploid and tumour cells. DMDC and F-DMDC (5-fluoro derivative) were potent inhibitors of HSV-1, herpes simplex virus type 2, VZV, and human cytomegalovirus (HCMV) and also had significant anti-proliferative activity. Their potency against HCMV was better than that of ganciclovir and araC. Some DMDU derivatives also showed anti-HCMV activity, but they had anti-proliferative activity. The anti-HCMV activity of these DMDC and DMDU compounds was generally more potent than those against HSV-1 and VZV thereof, suggesting the participation of cellular kinase in their antiviral action.
Prevention of Activation of HIV-1 by Antiviral Agents in OM-10.1 Cells
Authors: Feorino P. M. , Butera S. T. , Folks T. M. , Schinazi R. F.
Published: May 2015

The development of a reliable and simple system for evaluating compounds that could prevent activation of latent HIV would allow us to devise new therapeutic approaches. These compounds could eventually be used in combination with drugs that are effective against acute and chronic infections. The OM-10.1 cell line is a chronically infected clone which remains CD4+ until HIV-1 activation with tumour necrosis factor-α. A variety of compounds are known to have antiviral properties against either acutely or chronically infected cells were evaluated for their ability to inhibit HIV induced expression in these cells. We also examined the effect of several compounds that interact with biochemical pathways that may interfere with or enhance the reactivation process. These included nucleoside analogues, cytokines, steroidal and nonsteroidal anti-inflammatory agents, polyoxometalates, a TAT inhibitor, various natural products (including nerve growth factor, N-acetyl-L-cysteine, taxol, and interferons), TIBO, porphyrins, and various oligomers. CD4 cellular expression and supernatant reverse transcriptase activity were quantitated as markers of induced viral expression. Among the 58 compounds evaluated, 3′-fluoro-3′-deoxythymidine (FLT), interferon γ, Ro 5–3335 (a TAT inhibitor) and desferrioxamine were modest and selective inhibitors of HIV-1 activation.
Inhibitory Effects of Novel Mannose-Binding Lectins on HIV-Infectivity and Syncytium Formation
Authors: Animashaun T. , Mahmood N. , Hay A. J. , Hughes R. C.
Published: May 2015

Several new lectins were isolated and characterized with respect to their composition and sugar binding specificities and their ability to prevent HIV-1 infection. Twelve of the 13 mannose-specific lectins were inhibitory to varying degrees. The most effective were Machaerium biovulatum agglutinin (MBA) and M. lunatus agglutinin (MLA) which at 0.4 μg ml−1 prevented the cytopathic effect of the virus. Lower protection was obtained with Bowringia mildbraedii agglutinin (BMA), Galanthus nivalis agglutinin (GNA), Lablab niger agglutinin (LNA) and Dolichos lablab agglutinin (DLA). All these lectins are more protective than Con A while MBA is nearly 10 times more potent than any previously reported lectin. In each case the selective antiviral activity appears to be due to interaction with virus and not with some component on the target cell. MBA and GNA immobilized on Sepharose specifically bound gp120.

Studies of binding to glycoproteins confirmed the recognition of particular isomers of high mannose oligosaccharides Man9 to Man7GlcNAc2 by BMA and of Man5GlcNAc2 glycopeptides by GNA. By contrast, MBA did not bind oligomannosidic structures but did interact with ovalbumin, a glycoprotein rich in hybrid-type glycans.
Pharmacokinetic Evaluation of anti-HIV Drug Interactions: Effect of Zidovudine on 2′-3′-Dideoxyinosine Kinetics in Monkeys
Authors: Qian M. , Swagler A. R. , Mehta M. , Vishwanathan C.T. , Gallo J. M.
Published: May 2015

The current investigation was conducted to determine if zidovudine (AZT) altered the pharmacokinetics of dideoxyinosine (ddl) in non-hurnan primates, an appropriate animal model for AZT and ddl pharmacokinetics in human. Each of nine animals received 20 mg kg−1 of ddl intravenously in the absence and presence of two different dosage regimens of AZT. For each combination regimen, AZT was administered as a combined i.v. bolus-constant rate infusion regimen for 30 min that produced AZT plasma concentrations of about 4 μg ml−1 in six animals (low dose group) and 11 μg ml−1 in three others (high dose group). Serial blood samples were collected, and pharmacokinetic parameters for ddl were calculated based on plasma ddl concentrations measured by HPLC techniques.

The pharmacokinetics of ddl given alone in the first phase of the low ( n = 6) and high ( n = 6) dose AZT groups, resulted in a mean elimination half-life 1.54 and 1.9h, a mean total clearance of 0.62 and 0.731 h−1 kg−1, and a mean steady state volume of distribution of 1.02 and 0.891 kg−1, respectively. Following combined ddl and AZT administrations, in both the low and high dose AZT groups, plasma concentration-time profiles of ddl were similar for each monkey, and no statistical differences were observed in the pharmacokinetic parameters compared to those obtained when ddl was given alone. The fact that AZT does not alter the pharmacokinetics of ddl at the range of AZT dose studied provides a basis for rational dosage design for combined ddl and AZT treatments in HIV infection.
Frequent Occurrence of HIV-Inhibitory Sulphated Polysaccharides in Marine Invertebrates
Authors: Beutler J. A. , McKee T. C. , Fuller R. W. , Tischler M. , Cardellina J. H. , Snader K. M. , McCloud T. G. , Boyd M. R.
Published: May 2015

Aqueous extracts of many marine invertebrates have exhibited some activity in the National Cancer Institute's primary screen for anti-HIV cytopathicity. Using a variety of techniques, including gel permeation, size exclusion and ion exchange chromatography, toluidine blue metachromicity, 13C-NMR spectroscopy and combustion analyses, we have determined that this activity is largely due to sulphated polysaccharides. Because of the wide occurrence of this class of compounds in these organisms we sought a method for the rapid dereplication of sulphated polysaccharides. It was critical that the method selected for dereplication allow differentiation of anionic polysaccharides from other AIDS-antiviral chemotypes. After evaluating a variety of methods, we found that the most efficient strategy appeared to be precipitation of the polysaccharide fraction from aqueous ethanolic solutions of the crude aqueous extracts.
HIV Replication in CD4-Negative Cell Lines: Effect of Cloning, CD4 Expression and Inhibition by Dextrin Sulphate
Authors: Beddows S. , Bieniasz P. , Shaunak S. , Weber J.
Published: May 2015

HIV-1 infects CD4 negative (CD4) cell lines with low efficiency. Infected CD4 cells have a low copy number of HIV proviruses per cell and require a high multiplicity of infection. Following CD4 transfection, most human cell lines permit high efficiency HIV entry and replication. We have compared entry and inhibition of HIV-1 into CD4 cells and their equivalent CD4 positive (CD4+) transfectants. Entry of HIV-1 into both CD4+ and CD4− was completely inhibited by a novel sulphated polysaccharide, dextrin sulphate (DS) at 100 μg ml−1, whereas anti-CD4 antibodies only inhibited HIV infection of CD4+ cells. One glial cell line, U251SP-CD4, expressed surface CD4, but this did not increase HIV-1 susceptibility compared to the CD4− U251SP cell line. Subclones of the CD4− cell lines TE671 and U251SP were no more permissive for infection than their corresponding parental line. HIV-1 infected CD4− cells have a significantly lower provirus copy number than CD4+ cells, confirming that the block to HIV-1 replication is predominantly at entry. The action of DS was examined in conjunction with soluble recombinant CD4 (srCD4); DS was found to potentiate the inhibiting effect of srCD4.
Anionic Liposomes Inhibit Human Immunodeficiency Virus Type 1 (HIV-1) Infectivity in CD4+ A3.01 and H9 Cells
Authors: Konopka K. , Davis B. R. , Larsen C. E. , Düzgüneş N.
Published: May 2015

Immunodeficiency viruses undergo fusion with liposomes containing anionic phospholipids (Larsen etal., 1990). We have investigated the effect of liposomes composed of cardiolipin, phosphatidylserine or phosphatidylinositol, on the infectivity of three strains of HIV-1 in A3.01 and H9 cells, measured by p24 (gag) production in the medium. The infectivity of HIV-1 in A3.01 or H9 cells was inhibited by the presence of cardiolipin liposomes during a 2 h infection period, with IC50's of 23.0, 4.8, and 5.0 μM phospholipid, respectively, for the different strains. Liposomes composed of phosphatidylserine or phosphatidylinositol were ineffective under similar conditions. However, prolonged pre-incubation of the virus with these liposomes also inhibited infectivity. Inhibition of virus binding to cells could not account for the inhibition of infectivity. We propose that the fusion products of HIV-1 and anionic liposomes are impaired in their ability to fuse with the plasma membrane.
(1′R, 2′S, 3′R)-9-(2′, 3′-Dihydroxycyclopentan-1′-yl)-Adenine and −3-Deaza-Adenine: Analogues of Aristeromycin Which Exhibit Potent Antiviral Activity with Reduced Cytotoxicity
Authors: Hasobe M. , Liang H. , Ault-Riche D. B. , Borcherding D. R. , Wolfe M. S. , Borchardt R. T.
Published: May 2015

Two synthetic analogues of aristeromycin, which were shown in a separate study to be inhibitors of S-adenosylhomocysteine hydrolase and devoid of substrate activity with adenosine kinase and adenosine deaminase, were found in this study to inhibit vaccinia virus replication in murine L929 cells and to have reduced cytotoxicity compared with that of the parent compound. Aristeromycin was shown to produce cytocidal effects on murine L929 cells, whereas the synthetic analogues produced cytostatic effects on cell growth. The antiviral effects of these synthetic analogues are correlated with their ability to elevate the intracellular ratio of S-adenosylhomocysteine/ S-adenosylmethionine. These results confirm that S-adenosylhomocysteine hydrolase is the molecular target which mediates the antiviral effects of aristeromycin and that transformation of aristeromycin by cellular adenosine kinase mediates its cytocidal properties.
In vitro Activity of Acyclic Nucleoside Phosphonate Derivatives against Feline Immunodeficiency Virus in Crandell Feline Kidney Cells and Feline Peripheral Blood Lymphocytes
Authors: Hartmann K. , Balzarini J. , Higgins J. , De Clercq E. , Pedersen N. C.
Published: May 2015

Several novel fluorinated acyclic nucleoside phosphonates [i.e. 9-(3-fluoro-2-phosphonylmethoxy propyl)adenine (FPMPA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)-2,6-diaminopurine (FPMPDAP)] were evaluated for their inhibitory effect against feline immunodeficiency virus (FIV) replication in Crandell feline kidney (CrFK) cells and feline peripheral blood lymphocytes (PBL) in vitro. Whereas 3-azido-3-deoxythymidine (AZT) was not able to achieve complete suppression of viral antigen expression and reverse transcriptase activity in the FIV-infected cell culture supernatants at 25 μM, FPMPA, FPMPDAP, and the 9-(2-phosphonylmethoxyethyl)purine derivatives PMEA and PMEDAP fully protected FIV-infected cells at μM. Both FPMPA and FPMPDAP were endowed with a higher antiviral potency and/or therapeutic selectivity than PMEA and PMEDAP in inhibiting FIV infection, mainly due to a markedly lower toxicity for the cell cultures.
Cell-Based and Biochemical Analysis of the anti-HIV Activity of Combinations of 3′-azido-3′-deoxythymidine and Analogues of TIBO
Authors: Buckheit R. W. , White E. L. , Germany-Decker J. , Allen L. B. , Ross L. J. , Shannon W. M. , Janssen P. A. J. , Chirigos M. A.
Published: May 2015

The toxicity of 3′-azido-3′-deoxythymidine (AZT) and the appearance of drug-resistant mutants in patients treated with AZT emphasizes the critical importance of the development of alternative strategies for the therapy of AIDS patients. Combination antiviral chemotherapy provides an attractive therapeutic strategy since the dose of the individual agents may be lowered to reduce toxicity and the use of two potent antiviral agents may limit the development of drug resistance. Two analogues of tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO) potently and selectively inhibit the replication of HIV-1 in cell culture. In combination with AZT, either of the two TIBO compounds, R82913 and R86183, was highly synergistic in cell culture against HIV-1. However, in biochemical enzyme inhibition assays, utilizing recombinant HIV-1 reverse transcriptase, synergy was not detected at the enzymatic level. These results suggest that one of these two known inhibitors of HIV-1 reverse transcriptase may have a secondary mechanism of action distinct from inhibition of the reverse transcriptase.
Antiviral Properties of the HIV-1 Proteinase Inhibitor RO 31-8959
Authors: Galpin S. , Roberts N. A. , O'Connor T. , Jeffries D. J. , Kinchington D.
Published: May 2015

The peptide derivative Ro 31-8959 has been shown to be a potent inhibitor of HIV proteinase with an IC50 of 2 × 10−9M, against HIV-1RF in acutely infected lymphoblastoid cells. This inhibition was not overcome by increasing the infectious dose or by extending the culture time. Similar antiviral activity was also obtained against HIV-2, SIV and several AZT-resistant strains of HIV-1. The time of addition of the inhibitor could be delayed for 22 h without significant loss of activity, supporting its mode of action as taking place late in the replication cycle of HIV-1. Ro 31-8959 also showed activity against chronically infected cells.
Drug Combination of AZT and ddl: Synergism of Action and Prevention of Appearance of AZT-Resistance
Authors: Antonelli G. , Dianzani F. , Bellarosa D. , Turriziani O. , Riva E. , Gentile A.
Published: May 2015

Both 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxynosine (ddl) strongly inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Here, it is shown that combination of AZT and ddl at concentrations that are readily achievable in vivo synergistically inhibit HIV-1 replication in C8166 cells and peripheral blood mononuclear cells. The synergism is significant even when the effect of AZT and ddl alone was negligible.

Our findings show that AZT-resistance is less likely to occur when a combination of AZT and ddl is used. Particularly, generation of AZT-resistant strains by in vitro selection is prevented, or delayed, by the combination of AZT plus ddl.

Taken together these observations provide a rationale for combination of AZT and ddl in the therapy of AIDS patients.
The Antiviral Agent Hypericin has in vitro Activity against HSV-1 Through Non-Specific Association with Viral and Cellular Membranes
Authors: Weber N. D. , Murray B. K. , North J. A. , Wood S. G.
Published: May 2015

The antiviral and virucidal compound, hypericin, was studied regarding its activity and possible mechanism against herpes simplex virus (HSV-1). It was determined that hypericin caused slight inhibition of viral adsorption to and penetration of Vero cells. Additionally, yield reduction assays suggested that hypericin was most effective against HSV-1 as a virucidal agent rather than as an intracellular antiviral agent. Fluorescence microscopy revealed that hypericin initially associated with cytoplasmic membranes and that over the course of time it became concentrated in intracellular membranous regions, probably the Golgi apparatus or endoplasmic reticulum (ER). These concentration events failed to inhibit glycosylation of either viral or cellular proteins and were effectively blocked by compounds which inhibit endocytosis or membrane cycling between the ER and Golgi. Based on fluorescence studies, it was determined that hypericin had non-specific affinity for protein and higher affinity for detergent and lipid. The evidence suggested that strong, non-specific association with membranes, both viral and cellular, are probably the basis of hypericin's virucidal and antiviral activity.
An Assay for HIV RNA in Infected Cell Lysates, and its use for the Rapid Evaluation of Antiviral Efficacy
Authors: Bacheler L. T. , Paul M. , Otto M. J. , Jadhav P. K. , Stone B. A. , Miller J. A.
Published: May 2015

A rapid, high-capacity assay for evaluating the potency of anti-HIV compounds was devised. This assay measures cell associated viral RNA levels 3 days after infection of susceptible T-cell lines grown in individual microtitre plate wells. Viral RNA was quantified by a sandwich hybridization assay, the first step of which was performed directly in crude infected cell lysates prepared in guanidinium isothio-cyanate. Levels of cell-associated viral RNA were shown to correlate with the yield of infectious virus and this correlation formed the basis of the test. Antiviral potencies of a large series of compounds tested in this RNA hybridization assay correlated closely with potency values determined by a sensitive but slower and more labour-intensive yield-reduction assay. Both laboratory strains and selected clinical isolates of HIV can be detected in this RNA hybridization assay.
Selective Inhibition of Myxovirus Replication by a Novel Series of Cholesterol-Naphthalenesulfonic Acid Hybrid Molecules
Authors: Ikeda S. , Wong M.F. , Mohan P. , De Clercq E.
Published: May 2015

A novel series of hybrid molecules with cholesterol and naphthalenesulfonic acid have been synthesized and evaluated for their inhibitory activity against myxoviruses (respiratory syncytial virus [RSV], influenza A and B virus), Naphthalenesulfonic acids (compounds 1–3) and cholesterols (compounds 4 and 5) did not inhibit myxovirus-induced cytopathic-ity. However, hybrid molecules (compounds 6 and 7) proved active against RSV and influenza A virus but not influenza B virus. The antiviral effects of the hybrid compounds 6 and 7 were comparable to those of dextran sulfate. Mixtures of naphthalenesulfonic acid and cholesteryl chloroformate (compounds 1 and 5 molecules, corresponding to the hybrid molecule 6; and compounds 2 and 5, corresponding to the hybrid molecule 7) did not show antiviral activity. The mode of action of the cholesterol-linked naphthalenesulfonic acids can be attributed to inhibition of virus-cell fusion (influenza A virus) or inhibition of both virus-cell binding and fusion (RSV).
Modified Cyclodextrin Sulphates(mCDS11) have Potent Inhibitory Activity against HIV and High Oral Bioavailability
Authors: Otake T. , Schols D. , Witvrouw M. , Naesens L. , Nakashima H. , Moriya T. , Kurita H. , Matsumoto K. , Ueba N. , De Clercq E.
Published: May 2015

Modified cyclodextrin sulphate (mCDS) in which lipophilic groups were introduced to cyclodextrin sulphate (CDS) was synthesized and proved more inhibitory to the replication of HIV-1 and HIV-2 than CDS or dextran sulphate (DS). The anti-coagulant activity of mCDS was lower than that of DS. Cyclodextrin phosphate (CDP) showed anti-HIV activity similar to that of CDS, and its anti-coagulant activity was even lower than that of mCDS. Flow cytometric analysis suggested that the mechanism of the anti-HIV-1 action of CDS, mCDS, and CDP is based on inhibition of HIV-1 binding to the cells. The peak blood concentration after oral administration of mCDS11(potassium tris[6-benzylthio-6-deoxy]-β-cyclodextrin hexadecasulphate) to rabbits was about 1000 times higher than the concentration showing anti-HIV activity. The retention time in the blood was also long (blood half-life: 4 h). These results point to the potential usefulness of oral mCDS administration in the prophylaxis and/or therapy of HIV infections.
Haloalkyl Phosphate Derivatives of AZT as Inhibitors of HIV: Studies in the Phosphate Region
Authors: McGuigan C. , Turner S. , Nicholls S. R. , O'Connor T. J. , Kinchington D.
Published: May 2015

Novel haloalkyl phosphate derivatives of the anti-HIV nucleoside analogue AZT were prepared by phosphorochloridate chemistry. These materials were designed to act as labile membrane-soluble prodrugs of the bio-active free nucleotides. In vitro evaluation revealed the compounds to have a pronounced and selective antiviral action, which varied greatly with the structure of the phosphate moiety. By comparison to simple dialkyl phosphates, which are inactive against HIV-1, the introduction of halogen atoms into the alkyl (phosphate) chains led to anti-HIV activity. Although halogen substitution in just one alkyl chain was sufficient for biological activity, substitution in the second alkyl chain further enhanced activity. Conversely, stabilization of the second chain, by conversion to a phosphonate, led to a reduction in activity. In one case, the diastereo-isomers resulting from mixed stereochemistry at the phosphate centre were separated, and found to differ in activity by one order of magnitude. Lastly, the bis(mono- and di-chloroethyl) phosphates were prepared and found to display moderate anti-HIV activity.
In vitro and in vivo antiviral (RNA) evaluation of orotidine 5′-monophosphate decarboxylase inhibitors and analogues including 6-azauridine-5′-(ethyl methoxyalaninyl)phosphate (a 5′-monophosphate prodrug)
Authors: Gabrielsen B. , Kirsi J. J. , Kwong C. D. , Carter D. A. , Krauth C. A. , Hanna L. K. , Huggins J. W. , Monath T. P. , Kefauver D. F. , Blough H. A. , Rankin J. T. , Bartz C. M. , Huffman J. H. , Smee D. F. , Sidwell R. W. , Shannon W. M. , Secrist J. A.
Published: May 2015

A series of 29 pyrimidines comprising analogues of 6-azauridine (e.g. 2- and 4-thio-6-azauridine), 6-substituted uridines (including several known inhibitors of orotidine 5′-monophosphate decarboxylase, ODCase, e.g. pyrazofurin), and 6-azauridine-5′-(ethyl methoxyalaninyl) phosphate (a potential prodrug of 6-AU-5′-MP) were synthesized and evaluated in vitro and in vivo against five RNA viruses: Japanese encephalitis (JE), yellow fever (YF), sandfly fever (SF), Punta Tora (PT) and Venezuelan equine encephalomyelitis (VEE) viruses. 2-Thio-6-azauridine demonstrated the best In vitro activity against all five viruses. However, in vivo activity was not observed in JE-, PT- and VEE-infected mice. The phosphate prodrug of 6-azauridine was significantly more effective than the parent compound in the PT virus mouse model. Optimum in vivo dose/route/schedule was determined for pyrazofurin in PT-virus-infected mice.
Synthesis of 2′,3′,5′-trideoxyuridine-5′-methylphosphonic Acid and its Diphosphate Derivative. Study of the Interaction with HIV-1 reverse Transcriptase
Authors: Benzaria S. , Maury G. , Gosselin G. , Rittinger K. , Divita G. , Goody R. S. , Imbach J.-L.
Published: May 2015

2′,3′,5′-Trideoxyuridine-5′-methylphosphonate, [8], was synthesized from ddU. The 5′,6′ carbon-carbon bond was formed by condensing the 5′-aldehyde of ddU and a Wittig reagent. The binding interaction of the diphosphate derivative [10] of the phosphonate [8] with HIV-1 reverse transcriptase (RT) was studied using methods based primarily on fluorescence spectroscopy. From the quenching of intrinsic tryptophan fluorescence of RT during its titration against [10], a dissociation constant of 24 μm was calculated at 25°C. In the presence of a DNA/DNA template/primer of defined sequence and RT, [10] and a fluorescent derivative of ddTTP competed for binding to RT without incorporation of ddU-5′-methylphosphonate. In the presence of a 0.5 mm concentration of [10], the RT activity measured with Poly(rA)/(dT)15 and [3H] dTTP was lowered to 65%. All our observations are consistent with suppression of the catalysis of bond formation between the OH at the primer 3′-end and α-P of [10] after formation of the complex between RT, template/primer and [10].
Characterization of a Mutant HIV-1 Reverse Transcriptase Resistant to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione (TIBO R82150)
Authors: Samanta H. , Rose R. , Patick A. K. , Bechtold C. M. , Trimble J. , Li H. , Lee A. , Colonno R. J. , Lin P. F.
Published: May 2015

A virus strain resistant to R82150, a non-nucleoside reverse transcriptase (NNRT) inhibitor (tetrahydro-imidazo [4,5, 1- jk] [1,4] benzodiazepine-2(1 H)-thione), was isolated following serial passage of HIV-1 RF in CEM-SS cells. The virus is cross-resistant to another non-nucleoside reverse transcriptase inhibitor, TGG-II-23A [1,4-dimethyl-1-[5,5-dimethyl-2-oxazoionyl]-naphthalen-2-one), but remains susceptible to AZT, DDI, D4T and phosphonoformate (PFA). DNA sequencing of reverse transcriptase genes from resistant virus indicated that R82150 selects for amino acid alterations Y181C and V108I. In vitro mutagenized reverse transcriptase and recombinant HIV-1 (pNL4-3) carrying either of the mutations have been generated. Genotypic and phenotypic analyses identified V108I as an unreported R82150-associated mutation. Both reverse transcriptase and viral resistance assays indicated that the resistance conferred by the V108I mutation is 7-fold less than that conferred by Y181C.
The Effect of Acyclovir on the Acute and Latent Murine Gammaherpesvirus-68 Infection of Mice
Authors: Sunil-Chandra N. P. , Efstathiou S. , Nash A. A.
Published: May 2015

Mice inoculated intranasally with murine gammaherpesvirus-68 were used to evaluate the efficacy of acyclovir (ACV) in the treatment of acute and latent infections. Effectiveness was measured by infectious virus assay of the lung (site of active replication) and infectious centre assay of spleen cells (site of latency). Intraperitoneal administration of ACV at 6-h intervals starting soon after inoculation was more effective in reducing infectious virus in the lung than was treatment with 12-hourly injections commencing 3 days post-infection. Further, ACV treatment during acute infection resulted in an approximately 10-fold reduction in the number of infectious centres in the spleen as compared to placebo-treated animals. However, once latency was established, ACV treatment was not effective in reducing the number of infectious centres in the spleen. This is the first report demonstrating that ACV can be used to minimize the replication of murine gammaherpesvirus in mice at the site of primary infection, resulting in a reduction in the number of latently infected spleen lymphocytes.
Inhibition of Respiratory Syncytial Virus Replication in vitro by a Pyrazole Dicarboxamide Analogue of Ribavirin
Authors: Woods J. M. , Marr C. L. P. , Penn C. R.
Published: May 2015

1-β-D-Ribofuranosylpyrazole-3,4-dicarboxamide (GR-92938X) was found to be an inhibitor of respiratory syncytial virus in vitro, with a potency equivalent to that of ribavirin. The compound was more specific as, unlike ribavirin, it did not inhibit influenza A, influenza B or parainfluenza 2 virus. It was phosphorylated by adenosine kinase and, unlike ribavirin, it did not inhibit cellular inosine monophosphate dehydrogenase.
Comparative Anti-Retrovirus and Anti-Hepadnavirus Activity of Three Different Classes of Nucleoside Phosphonate Derivatives
Authors: Balzarini J. , Kruining J. , Heijtink R. , De Clercq E.
Published: May 2015

The prototype compounds of three different classes of nucleoside phosphonates [i.e. 9-(2-phosphonomethoxyethyl)adenine (PMEA), 9-(2-phosphonomethoxyethoxy)adenine (PMEoA) and 9-[(2R,5R)-2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4API)] were investigated and compared for their antiviral activities. The three test compounds showed a marked inhibition of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in CEW and MT-4 cell cultures [50% effective concentration (EC50): 0.8-14μm]. D4API was 2- and 15-fold more inhibitory than PMEA and PMEoA, respectively. In contrast, the activity of PMEA against human hepatitis B virus (HHBV) in human hepatoma Hep G2 2.2.15 cells was 5- and 10-fold more pronounced than the activities of PMEoA and D4API, respectively (EC50 1.2μm versus 10 and 6 μm, respectively). The inhibitory activity of D4API against Moloney murine sarcoma virus (MSV)-induced C3H/3T3 cell transformation was superior to the activities of PMEA and PMEoA by at least one order of magnitude (EC50 for D4API 1.3μM, versus 2.8 and 14 μM for PMEA and PMEoA, respectively). The markedly greater inhibitory effect of D4API on MSV in vitro was in agreement with our In vivo findings that D4API inhibited MSV-induced tumour formation in newborn mice and delayed the MSV-associated animal death at a lower dose than PMEA or PMEoA. Both PMEA and D4API emerged as promising compounds that warrant further investigation for their anti-retrovirus and anti-hepadnavirus activities in vivo.
In vitro anti-HIV and Cytotoxicological Evaluation of the Triple Combination: AZT and ddC with HIV Proteinase Inhibitor Saquinavir (Ro 31-8959)
Authors: Craig J. C. , Whittaker L. , Duncan I. B. , Roberts N. A.
Published: May 2015

A combination of three anti-HIV compounds (AZT, ddC and saquinavir) has been studied in vitro. In confirmation and extension of previous studies, a greater than additive effect was found against both HIV-1GB8 and HIV-1N1T, when any two compounds were combined. Synergy of action was also found against both virus strains when all three compounds were employed together. Cytotoxicological studies indicated that a high therapeutic index could be maintained for this treatment. The potential benefits of improved anti-HIV surveillance, reduced risk of the development of resistance to the treatment and reduced requirement for drugs in quantities that produce toxic side-effects support the extension of clinical trials of saquinavir to investigate this combination.
Relationship of the Molecular Size and Charge Density of Polyoxometalates to Their anti-gp120-CD4-binding Activity
Authors: Judd D. A. , Schinazi R. F. , Hill C. L.
Published: May 2015

The ability of several classes of polyoxometalates to inhibit the interaction between HIV-1 gp120 and CD4 was assessed. No clear relationship was found between binding inhibition and the negative charge density on the anion portion of the polyoxometalate. However, a weak correlation was found with molecular size. There was a molecular weight threshold of 3800 g mol-1 above which no significant increase in potency was gained; the binding inhibition was nearly quantitative above this molecular weight.
The X-ray Crystal Structure of the Sulfonated Azo Dye Congo Red, a Non-Peptidic Inhibitor of HIV-1 Protease which also Binds to Reverse Transcriptase and Amyloid Proteins
Authors: Ojala W. H. , Ojala C. R. , Gleason W. B.
Published: May 2015

Congo Red is a sulfonated azo dye and widely used biological stain that has recently been the focus of intense interest because it has been shown to bind to proteins involved in viral recognition and replication. Congo Red also finds wide use as a histological stain for amyloid proteins of the type found in neurodegenerative conditions such as Alzheimer's disease, transmissible spongiform encephalopathies in cattle and mink, and scrapie in sheep. Congo Red has been demonstrated to protect normal prion protein from being converted to the protease-resistant form, an important step in the pathology of the so-called ‘slow viral’ diseases. The range of biological molecules to which Congo Red binds makes it an important lead compound in drug development, for example in the development of new anti-HIV and anti-Alzheimer's therapeutic agents. In this report we present the first high-resolution structure of Congo Red: the low-temperature (173 K) X-ray crystal structure determination of its calcium salt. Two conformations of the molecule are found in the same crystal structure, one in which the central biphenyl group assumes a twisted (25°) conformation, and one in which the biphenyl group is planar and is located on a crystallo-graphic inversion centre. In both conformations the sulfonate groups are oriented anti with respect to the long molecular axis and assume eclipsed conformations with respect to the naphthalene rings. A comparison is made with a published structure [Turned, W.G., and Finch, J.T. (1992) J Mol Biol 227: 1205-1223] in which Congo Red is bound to porcine insulin, this complex serving as a model for amyloid binding. The results illustrate the conformational flexibility possessed by the biphenyl spacer, which allows the hydrophobic portion of the molecule to assume an optimum fit in the hydrophobic binding pockets of target proteins. A model is presented for the binding of Congo Red to the HIV protease in which the sulfonate groups interact with the side-chains of arginine residues. This proposed binding mode is consistent with the observed binding for other sulfonated aromatic inhibitors such as Evans Blue.