The overall objective of this Project is two-fold: (i) to identify by in vitro evaluation new compounds which have the potential to become useful antiviral drugs and (ii) to investigate how promising compounds act at the cellular and biochemical level. We shall evaluate for antiviral activity and cytotoxicity new series of nucleoside analogs and related heterocyclics (benzimidazole carbamates and imidazoles) prepared in the laboratory of Dr. L.B. Townsend as described in Research Project I. Evaluations will be performed in vitro for activity against human cytomegalovirus (HCMV) plus other herpesviruses and for cytotoxicity in uninfected cells. New compounds which are active against either of the viruses and low in cytotoxicity plus promising compounds already discovered shall be evaluated more extensively including studies to elucidate their biochemical mode of action. All new compounds prepared in Research Project I will be evaluated in a series of primary in vitro tests for their capacity to inhibit the replication of HCMV in plaque reduction or ELISA assays. Many compounds also will be tested against other human herpesviruses--including ganciclovir (DHPG)-resistant HCMV. All compounds also will be tested in vitro for cytotoxicity utilizing visual scoring, effects on cell growth, and effects on labeled precursor uptake. Those compounds which show potent antiviral activity and low toxicity will be tested further including virus yield reduction assays, uninfected cell growth rates, and cell plating efficiency. Those compounds with potent and selective activity against HCMV will be sent to our collaborators at Burroughs Wellcome for evaluation in vitro on the growth of human narrow progenitor cells and to our Core Laboratory for in vivo antiviral evaluation. The mode of antiviral action of newly discovered and existing promising antiviral compounds also will be investigated. Because we already know that two series of active nucleoside analogs act by different mechanisms and because we shall be studying new non-nucleoside heterocycles, different approaches will be used depending on the nature of the compound. Approaches will include but not be limited to the following: (i) determination of the in vitro metabolism of active compounds. (ii) Determination if active compounds are selective inhibitors of viral DNA synthesis. (iii) Characterization of the mechanism by which viral DNA synthesis is inhibited. Studies would include the effects of triphosphates of the active nucleoside analogs on HCMV DNA polymerase and the mammalian DNA polymerases. (iv) Investigation of the effects of compounds such as the benzimidazoles which act by mechanisms other than DNA synthesis inhibition. Such studies will include isolation of drug-resistant mutants, electron microscopic studies of viral particles, drug metabolism in resistant viruses, selected biochemical characterization, and marker rescue studies. We also shall investigate mechanisms of cytotoxicity in selected antiviral compounds including DNA flow cytometry, labeled precursor uptake, and kinase substrate specificity studies.
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