Joseph M. Colacino

The identification and development of antiviral agents for the treatment of chronic hepatitis B virus infection

Hepatitis B virus (HBV) is the leading cause of chronic hepatitis throughout the world. Notwithstanding the availability of a safe and effective vaccine, the world prevalence of HBV has not declined significantly, thus resulting in the need for a selective antiviral agent. HBV is a small, partially double-stranded DNA virus which replicates through an RNA intermediate. Most efforts to develop anti-HBV agents have been targeted to the viral DNA polymerase which possesses reverse transcriptase activity. Currently, the most promising anti-HBV agents are nucleoside analogs which interfere with viral DNA replication. Although earlier nucleoside analogs such as vidarabine (ara-A) and fialuridine (FIAU) have displayed unacceptable toxicities, newer analogs such as lamivudine (3TC), bis-POM PMEA (GS-840), lobucavir, and BMS-200,475 have demonstrated clinical utility. In particular, the use of lamivudine has generated considerable interest in the development of other L-enantiomeric nucleoside analogs for use against HBV. Here, we provide an overview of HBV structure and replication strategy and discuss the use of cell culture systems, in vitro viral polymerase systems, and animal models to identify and evaluate anti-HBV agents. We also discuss the various classes of nucleoside analogs in terms of structure, mechanism of action, status in clinical development, ability to select for resistant HBV variants, and use in combination therapies. Finally, we present a discussion of novel antiviral approaches, including antisense and gene therapy, and address the various challenges to successful anti-HBV chemotherapeutic intervention.

Approaches and Strategies for the Treatment of Influenza Virus Infections

Influenza A and B viruses belong to the Orthomyxoviridae family of viruses. These viruses are responsible for severe morbidity and significant excess mortality each year. Infection with influenza viruses usually leads to respiratory involvement and can result in pneumonia and secondary bacterial infections. Vaccine approaches to the prophy-laxis of influenza virus infections have been problematic owing to the ability of these viruses to undergo antigenic shift by exchanging genomic segments or by undergoing antigenic drift, consisting of point mutations in the haemagglutinin (HA) and neuraminidase (NA) genes as a result of an error-prone viral polymerase. Historically, antiviral approaches for the therapy of both influenza A and B viruses have been largely unsuccessful until the elucidation of the X-ray crystallographic structure of the viral NA, which has permitted structure-based drug design of inhibitors of this enzyme. In addition, recent advances in the elucidation of the structure and complex function of influenza HA have resulted in the discovery of a number of diverse compounds that target this viral protein. This review article will focus largely on newer antiviral agents including those that inhibit the influenza virus NA and HA. Other novel approaches that have entered clinical trials or been considered for their clinical utility will be mentioned.

Physical structure and molecular cloning of equine cytomegalovirus DNA.

Restriction endonuclease (RE) mapping studies and molecular hybridization analyses were conducted to determine the molecular structure of the genome of equine cytomegalovirus (ECMV). The ECMV genome is a linear, double-stranded DNA with a molecular size of 126 +/- 0.6 MDa (189 kbp). A library of cloned BamHI, EcoRI, and HindIII fragments of the viral genome was used to construct RE maps. Individual 32P-labeled cloned DNA fragments were hybridized to Southern blots of viral genomic DNA digested to completion with BamHI, EcoRI, HindIII, or SalI. These analyses revealed that the ECMV genome consists of a 97-MDa unique long region which is bracketed by repeated sequences. At one terminus of the genome, a 21.3-MDa segment of repeated sequences with no apparent unique sequences was identified. At the other terminus, a 6-MDa unique region bracketed by 2.4-MDa repeat segments was identified. No submolar RE fragments were identified upon digestion of the ECMV genome with BamHI, EcoRI, HindIII, SalI, or other REs, including BclI, BglII, NruI, and XbaI. The genome possesses only two termini as judged by lambda exonuclease digestion and by T4 DNA polymerase end-labeling of the intact DNA followed by digestion with BamHI, EcoRI, HindIII, SalI, BclI, BglII, NruI, or XbaI. In addition, Southern blot analysis of DNA extracted from ECMV-infected rabbit kidney cells revealed that only one viral DNA fragment within the intracellular viral DNA pool contains fused genomic termini. Taken together, these observations indicate that the ECMV genome does not isomerize and suggest that the genome of ECMV may be unique among those of the herpesviruses and especially those of the betaherpesviruses (cytomegaloviruses) since it contains regions of extensive internal homology yet does not undergo isomerization. Lastly, the relatively small size of the viral genome indicates an evolutionary diversification among the cytomegaloviruses.

Selection of Influenza A and B Viruses for Resistance to 4-Guanidino-Neu5Ac2en in Cell Culture

The reassortant influenza viruses, A/NWS-G70c with N9 neuraminidase (NA) and B/HK/8/73 (HG) with B/Lee/40 NA, were selected for resistance to 4-guanidino-Neu5Ac2en (4-GuDANA) by passaging the virus in stepwise increases in the concentration of 4-GuDANA. In the NA of resistant viruses, the absolutely conserved Glu 119, which lies in a pocket beneath the active site of the enzyme and interacts with the guanidinium moiety of 4-GuDANA, was changed to Gly. The mutant NA was >200-fold more resistant to 4-GuDANA than was the wild-type enzyme. During 72 h in cell culture, resistant A and B viruses displayed much less NA activity than did wild-type viruses but did undergo multicycle replication. While emergence of resistance to 4-GuDANA has not been observed in vivo, these results demonstrate that the development of resistance is possible and can be mediated by a single amino acid change in the active site of the viral NA.

Hepatitis prevention and treatment.

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Drug discovery and development of antiviral agents for the treatment of chronic hepatitis B virus infection.

A safe and effective vaccine for hepatitis B virus (HBV) has been available for nearly twenty years and currently campaigns to provide universal vaccination in developing countries are underway. Nevertheless, chronic HBV infection remains a leading cause of chronic hepatitis worldwide and there is a strong need for safe and effective antiviral therapies. Attempts to identify and develop antiviral agents to treat chronic HBV infection remains focused on nucleoside analogs such as 3TC (lamivudine), adefovir dipivoxil, (bis-POMPMEA), and others. However, advances in our understanding of the molecular biology of HBV and the development of new assays for HBV polymerase activity, such as the reconstitution of active HBV polymerase in vitro, should facilitate large screening efforts for non-nucleoside reverse transcriptase inhibitors. Recent advances have furthered our understanding of clinical resistance to lamivudine, have provided new approaches to treatment, and have offered new perspectives on the major challenges to the identification and development of antiviral agents for chronic HBV infection. Here, in an update to our previous review article that appeared in this series [59a], we focus on recent advances that have occurred in the areas of virus structure and replication, in vitro viral polymerase assays, cell culture systems, and animal models.