High-capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay.
AUTOR(ES)
Jansen, R W
RESUMO
An integrated assessment system specific for hepatitis B virus (HBV) Dane particle DNA was developed to examine the activity of potential anti-HBV compounds in chronic HBV-producing HepG2-derived 2.2.15 cells. Cell culture, immunoaffinity purification, polymerase chain reaction, and hybrid-capture detection were performed in the microtiter format to facilitate increased throughput by automation. The high sensitivity afforded by the assay provided quantitative detection of less than 0.5 fg of extracellular HBV DNA from 25 microliters of cell culture supernatants, and drug-induced reductions in HBV titers greater than 100-fold were easily measured. Fluorometric determination of total cellular DNA from the same 96-well proliferating cell cultures allowed simultaneous evaluation of inhibition of cell growth, thus providing the ability to assess the overall selectivities of candidate compounds in a single experiment. The potent activities of three anti-HBV compounds, the (+) and (-) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxythiolane-5-yl]cytosine (FTC) and D-carbocyclic-2'-deoxyguanosine (CDG), were confirmed by this method. (-)-FTC was more active than its (+) enantiomer (50% inhibitory concentrations, 0.033 +/- 0.006 and 0.723 +/- 0.160 microM [standard error of the mean; SEM], respectively), while both enantiomers demonstrated a lack of cytotoxicity at 200 microM. CDG was more potent (50% inhibitory concentration, 0.0063 +/- 0.0007 microM [SEM]) but was also significantly more toxic, inhibiting cell growth by 50% at 32 +/- 6 microM (SEM). These results demonstrate the usefulness of this immunoaffinity-based, quantitative polymerase chain reaction system as a high-capacity in vitro tool for assessment of anti-HBV compound selectivity.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=187690Documentos Relacionados
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