Melinda G. Hollingshead

Thiazolobenzimidazole: Biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

SYNTHESIS AND ANTIVIRAL EVALUATION OF ANALOGS OF ADENOSINE-N1-OXIDE AND 1-(BENZYLOXY)ADENOSINE

The activity of a series of compounds related to adenosine-N1-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.

An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus

Abstract A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Activity of Selected Amaryllidaceae Constituents and Related Synthetic Substances Against Medically Important RNA Viruses

RNA viruses comprising the families, Togaviridae, Bunyaviridae, Arenaviridae and Filoviridae cause hemorrhagic, encephalitic or febrile disease and represent significant health hazards to persons living in or traveling through endemic regions of the world. These viruses include: a) Japanese encephalitis (JE), yellow fever (YF) and dengue type 1–4 viruses (flaviviruses); b) Rift Valley fever (RVF), sandfly fever (Sicilian, SF), Hantaan (hemorrhagic fever with renal syndrome, HFRS) and Crimean-Congo hemorrhagic fever (CCHF) viruses (bunyaviruses); c) Venezuelan equine encephalitis virus (VEE, alphavirus); d) Lassa (LAS) and Junin (JUN) viruses (arenaviruses); and e) Ebola (EBO) and Marburg (MAR) viruses (filoviruses). In general, these viruses are associated with insect (mosquito, tick) or rodent vectors, occur primarily in Asia, Africa or Central/South America, may require an increased level of biohazard protection, and are often referred to as “exotic” viruses in the context of “tropical” medicine (Figure 1). These diseases occur world-wide but the development of appropriate antiviral chemotherapies is not considered economically feasible by the private sector. The drug discovery program of the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) seeks to develop antiviral chemotherapeutic agents against several of these viruses. This task is hampered by a limited knowledge of many of the diseases and viruses, the remote location of disease outbreaks with the inherent problems in conducting clinical studies and the requirement for BL3 and BL4 (“spacesuit”) laboratories for biocontainment.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Failure of Castanospermine to Reduce Spleen or Serum Virus Titres in Rauscher Murine Leukaemia Virus-Infected Mice

The effects of orally administered castanospermine (CS) and azidothymidine (AZT) on spleen and serum virus titres and on spleen weights of mice inoculated with Rauscher murine leukaemia virus (R MuLV) were evaluated. Virus titres of samples from CS-treated mice did not differ appreciably from saline-treated mice, despite a significant inhibition of splenomegaly. In contrast, AZT treatment resulted in significant inhibition of splenomegaly and a reduction of virus titres.

Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with Rauscher murine leukemia virus

Rauscher murine leukemia virus induces an erythroleukemia in susceptible strains of mice that is associated with splenomegaly and viremia. This animal model has been used for evaluating the in vivo efficacy of potential anti-HIV agents. The in vivo antiviral activity of therapeutic agents has usually been determined by measuring a reduction in the spleen weights of compound-treated mice or by quantitating viremia with the UV-XC plaque assay. The UV-XC assay, however, is time-consuming and labor-intensive. Virions of Rauscher murine leukemia virus, like other retroviruses, contain the enzyme reverse transcriptase. Quantitating the level of this enzyme in infected mouse sera provides a more rapid measure of viremia in the animal. We have examined the effects of several reagents, including detergent, KCl, EGTA, dGMP, spermine, as well as protease and RNase inhibitors, on the reverse transcriptase assay. The optimized assay method was effective in evaluating the antiviral activity of AZT in the Rauscher murine leukemia virus in vivo model. The assay is also amenable to automation if large numbers of assays are required.