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Antiviral Research (v.67, #3)
Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors by Mike Westby; Grace R. Nakayama; Scott L. Butler; Wade S. Blair (pp. 121-140).
The identification of novel HIV-1 inhibitors is facilitated by screening campaigns that combine the right screening strategy with a large diverse collection of drug-like compounds. Cell-based screening approaches offer some advantages in the quest for novel inhibitors because they can include multiple targets in a single screen and in some cases reveal targets and/or structures not captured in biochemical assays. However, follow-up activities for cell-based screens are often more complicated and resource intensive when compared to biochemical screens. Alternatively, biochemical screens usually offer the advantage of focusing on a single target with a well-defined set of follow-up assays. In this review we cover multiple cell-based and biochemical assay formats, many of which were designed to identify inhibitors that act through new mechanisms. Some of the assays discussed have been utilized in antiviral screens while others might be formatted for HTS or utilized as secondary assays in a screening campaign. As drug discovery efforts in the pharmaceutical industry shift away from traditional strategies, new approaches such as those presented here are likely to play a significant role in the identification of next generation HIV-1 inhibitors.
Keywords: Abbreviations; beta-Gal; beta-galactosidase; CAT; chloramphenicol acetyltransferase; CHO; Chinese Hamster Ovary; CV-N; cyanovirin-N; dsDNA; double-stranded DNA; Ec; ecdysone; EIA; enzyme immunoassay; ELISA; enzyme-linked immunosorbent assay; Env; envelope; FLIPR; Fluorometric Imaging Plate Reader; FP; fluorescence polarization; FRET; fluorescence resonance energy transfer; GFP; green fluorescent protein; HOS; human osteosarcoma; HR; heptad repeat; HTRF; homogeneous time-resolved fluorescence; HTS; high throughput screening; IC; indicator cell line; IN; integrase; ITC; infected T-cells; LTR; long terminal repeat; μARCS; Microarray Compound Screening Technology; NC; nucleocapsid; NHEJ; non-homologous end joining; NNRTI; non-nucleoside reverse transcriptase inhibitors; NRTI; nucleoside analog reverse transcriptase inhibitors; PEc; ecdysone promoter; PIC; preintegration complex; PI; protease inhibitor; PR; protease; RNase H; ribonuclease H; RRE; rev response element; RT; reverse transcriptase; SEAP; secreted alkaline phosphatase; SPA; scintillation proximity assay; TC; T-cells; 7-TM GPCR; 7-transmembrane g-protein coupled receptor; TRF; time-resolved fluorescenceHIV; Antiviral screen; Novel inhibitors; Drug discovery
Viral resistance in shrimp that express an antisense Taura syndrome virus coat protein gene by Yuanan Lu; Piera S. Sun (pp. 141-146).
Taura syndrome virus (TSV) is a major cause of mortality and morbidity in shrimp, and has a profound economic impact on commercial U.S. shrimp farming. This paper describes the stable expression of an antisense Taura syndrome virus-coat protein (TSV-CP) gene construct in shrimp zygotes, via transfection using jetPEI reagent, over a period of at least 236 days. The transgenic shrimp showed no statistically significant difference from normal control shrimp in terms of weight gain or their appearance, morphology, swimming and eating activities. When challenged with live TSV, the transgenic shrimp exhibited increased resistance to the TSV infection (83% survival rate) as compared to control animals (44% survival rate). This work demonstrates that transgenic shrimp, which stably express an antisense transcript from the TSV-CP gene, are partially resistant to TSV infection. These data may have an important implication for commercial shrimp farming.
Keywords: Virus resistance; Transgenic shrimp; Taura syndrome virus; Antisense RNA; TSV coat-protein; Transfection
In vitro activity of cycloSal-nucleoside monophosphates and polyhydroxycarboxylates against orthopoxviruses by A. Sauerbrei; C. Meier; A. Meerbach; M. Schiel; B. Helbig; P. Wutzler (pp. 147-154).
Because variola virus might be used as a pathogen in biological attacks, there is an urgent need to provide effective antiviral drugs for the treatment of orthopoxvirus infections. Thus, the aim of the present study was to test the antiviral activity of 3 pro-nucleotides of the acyclic nucleoside analogues aciclovir (ACV), 3 of penciclovir (PCV) and 38 of the cyclic nucleoside analogue brivudin (BVDU), on the basis of cycloSaligenyl-nucleoside monophosphate approach against vaccinia virus and cowpox virus in vitro. In further experiments, 13 synthetic humic acid-like polymers, so-called polyhydroxycarboxylates, were examined. Antiviral screening was performed by means of the plaque reduction assay and for quantification of the cytotoxicity of the test compounds the XTT-based tetrazolium reduction assay EZ4U was used. As result, three cycloSal-monophosphate derivatives of ACV proved to be potent inhibitors of both vaccinia virus and cowpox virus replication in vitro. Among the tested monophosphate derivatives of cycloSal-PCV and cycloSal-BVDU, selected substances showed a promising antiviral activity against vaccinia virus and cowpox virus. For the polyanionic compounds, no relevant antiviral activity was detected. In conclusion, by the delivery of nucleoside monophosphates from neutral, membrane-permeable prodrugs on the basis of the cycloSaligenyl-nucleotide concept, different ACV, PCV and BVDU derivatives can act as potent and selective inhibitors of orthopoxvirus replication. However, most of the cycloSal-monophosphate derivatives of BVDU had a higher cytotoxicity than their parent nucleosides.
Keywords: Vaccinia virus; Cowpox virus; Antiviral activity; Pro-nucleotides; Polyanionic compounds
Effect of resveratrol on herpes simplex virus vaginal infection in the mouse by John J. Docherty; Ming Ming Fu; Jennifer M. Hah; Thomas J. Sweet; Seth A. Faith; Tristan Booth (pp. 155-162).
Resveratrol (3,5,4′-trihydroxystilbene) is a natural component of certain foods, such as grapes, that, when topically applied, has been shown to limit HSV-1 lesion formation in the skin of mice [Antiviral Res. 61:19–26, 2004]. To determine if it is active on genital HSV infection, the vagina of mice were infected with HSV-2 or HSV-1 and treated with a cream formulation of resveratrol. Mice were evaluated daily for extravaginal disease and vaginal swabs were taken regularly and assayed for infectious virus. Initial studies demonstrated that 19% resveratrol cream administered intravaginally five times a day for 5 days beginning 1h after infection significantly reduced HSV-2 replication beginning on day 1 of infection and prevented extravaginal disease when compared to animals treated with placebo. When resveratrol was tested at a concentration of 6.25% and 12.5% administered five times a day, 6.25% limited virus replication only on day 1 and delayed development of extravaginal disease by 1 day. However, 12.5% resveratrol inhibited HSV-2 replication beginning on day 1 and abolished extravaginal disease. If the number of applications per day was reduced to three for 5 days, 12.5% resveratrol inhibited HSV-2 replication only on day 1, while 19% resveratrol inhibited it throughout the 9-day assay period. When the animals with three treatments per day were examined for extravaginal disease, it was found that 12.5% resveratrol was ineffective when compared to placebo, while animals treated with 19% resveratrol did not exhibit extravaginal disease. When treatment was delayed 6h, 12.5% resveratrol did not inhibit HSV-2 replication or extravaginal lesion formation, but 19% resveratrol did. When resveratrol was used to treat vaginal HSV-1 infection, it was found that 12.5% resveratrol did not limit replication or prevent extravaginal lesion formation. In contrast, 19% resveratrol did significantly limit vaginal HSV-1 replication and reduced extravaginal lesion formation, but the latter was not significant. Mortality rates in placebo-treated animals was 37%, 6.25% resveratrol-treated animals was 40%, 12.5% resveratrol-treated animals was 24%, and 19% resveratrol-treated animals was 3%. Collectively, these results demonstrate that resveratrol cream inhibits or reduces HSV replication in the vagina of mice and limits extravaginal disease.
Keywords: Resveratrol; Herpes simplex virus; Mouse
A flavonoid from medicinal plants blocks hepatitis B virus-e antigen secretion in HBV-infected hepatocytes by Min Soo Shin; Eun Hwa Kang; Young Ik Lee (pp. 163-168).
A flavonoid molecule that showed a unique anti-HBV function was isolated from Phyllanthus urinaria. The molecular formula was determined as C14H6O8 based on FAM-MS analysis and the structure was determined by NMR. The identified flavonoid molecule, ellagic acid, showed unique anti-HBV functions. Ellagic acid did not inhibit either HBV polymerase activity, HBV replication or block HBsAg secretion. Rather, ellagic acid blocks effectively HBeAg secretion in HepG2 2.2.15 cells (IC50=0.07μg/ml). Since HBeAg is involved in immune tolerance during HBV infection, ellagic acid, a newly identified functional anti-HBV compound, may be a new candidate therapeutic against immune tolerance in HBV-infected individuals.
Keywords: Hepatitis B virus-e antigen; Phyllanthus urinaria; NMR spectrometer; HBeAg secretion
Erratum to “Extracts and molecules from medicinal plants against herpes simplex viruses� [Antiv. Res. 67 (2005) 107–119]
by Mahmud Tareq Hassan Khan; Arjumand Ather; Kenneth D. Thompson; Roberto Gambari (pp. 169-169).