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| The goal of our preclinical anti-hepatitis drug development program is the cost-effective and expeditious evaluation of the efficacy, toxicity, range and mechanism of action of your compound. Development of anti-hepatitis compounds has clearly followed a different path compared with the anti-retroviral or anti-herpetic compounds due to the lack of appropriate in vitro cell-based antiviral models. Our laboratory is working vigorously to develop new models which will expedite research in the anti-hepatitis field. |
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Anti- Hepatitis B Virus (HBV) Evaluation
Evaluation in HepG2-2.2.15 cells
HepG2-2.2.15 cells contain a stably transfected HBV genome. Thus, compounds blocking late steps of viral replication, such as transcription, translation, pregenome encapsidation, reverse transcription, particle assembly and maturation can be evaluated in this particular cell line.
a. Measurement of viral DNA released from treated cultures.
Each viral particle possesses a genome of one molecule of relaxed circular (RC) viral DNA. The amount of RC DNA released from cultured cells is affected by not only the quantity of the particles but also by the quality of the particles. To measure this particular form of DNA, we have developed two quantitative assays which can be used for anti-HBV evaluation. These assays include:
- Dot blot assay. This assay involves a semi-quantitative evaluation of negative strand of RC DNA within viral particles and is suitable for primary, high capacity screening.
- Real time quantitative PCR assay. This assay quantifies with high sensitivity the actual copy number of virion-associated RC molecules.
b. Measurement of HBsAg released from treated cultures.
To quantify the level of particles released from cultures, we have used RIA to detect the amount of HBsAg released from cultures. Distinct from the measurement of virion-associated RC, the quantification will only be affected by the amount of small envelope glycoprotein, a component of HBV virions, which are released from the infected cells.
c. Measurement of relaxed circular DNA in the cytoplasm and closed circular DNA (cccDNA) in the nucleus.
As a part of screening assay, the intracellular replicative intermediates in the cytoplasm and ccc DNAs in the nuclei are measured either semi-quantitatively by dot blot hybridization., or quantitatively using the real time PCR assay.
d. Examination of toxicity of compounds
In parallel with either of the assays described above, the toxicity of compounds to the target cells is evaluated by use of the tetrazolium dye XTT. |
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Mechanism of Action Evaluation
In addition to screening assays, we have developed a series of assays to determine the mechanism of anti-HBV action of test compounds.
Primary human hepatocytes
This culture system is used to confirm anti-HBV activity in the primary screening assay, to evaluate the ability of compounds to block attachment and internalization, and to evaluate the ability of the compounds to block conversion of viral genomic RC DNA into cccDNA. Due to limited resources of human liver tissues, similar assays have been developed for DHBV to dissect each early event.
HepG2-2.2.15 cell line
This cell line is used to define the mechanism of compounds active in the late phase of viral replication. Late phase hepatitis virus replication events include:
- Viral transcription: Northern blot analysis of total viral RNA
- Particle assembly: Capsid gel electrophoresis
- Pre-genome encapsidation: Northern blot analysis of particle-associated pre-genomic RNA
- Viral DNA synthesis: Southern blot analysis of cytoplasmic replicative intermediates
- Viral particle release: Southern blot analysis of virion-associated viral DNA and/or RIA analysis of virion-associated HBsAg
Again, the assays for mechanistic studies on the late phase of DHBV are also available.
In addition, a transient transfection system has been used for studying the action of test compounds on the late phase of replication of another animal hepatitis B virus, woodchuck hepatitis B virus.
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Anti-Hepatitis C Virus Evaluation
Anti-HCV Replicon-Based Evaluations
The current standard for evaluation of anti-HCV activity is efficacy in the replicon model.
Molecular target-based assays
Molecular target based assays which are currently being used in our laboratory include:
1. Protease assay: A biochemical assay is used to examine the activity of the compounds on HCV NS3/4A protease activity. The activity of compounds against NS2/3 protease is evaluated using a cell-based assay.
2. RNA polymerase assay: A biochemical assay is used to determine the activity of your compounds on NS5B RNA polymerase activity.
3. IRES assay: The accumulation of viral proteins are monitored to determine the compounds effect on initiation of viral protein translation mediated through the virus’s internal ribosome entry site (IRES).
4. Virus entry assay: This is a cell- based assay and can be used to study whether compounds or antibody block viral entry into cells.
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Surrogate Animal Virus-Bovine Viral Diarrhea Virus (BVDV) Assay
At present, there is no in vitro replication system available for HCV. Instead, a surrogate animal virus, BVDV, is used for drug screening. We have developed a high throughput cytoprotection assay, which evaluates activity of compounds against BVDV replication in vitro. Ribavirin, a compound approved for combination therapy for treatment of hepatitis C, exhibits activity in this assay and is used as a positive antiviral control compound. |
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| Copyright © 2006, ImQuest BioSciences. | | | |
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