The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 M) and Epstein-Barr virus (50% inhibitory concentration of 0.9 M), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di-and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10 6 cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10 6 cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (K i of 2.8, 2.2, and 0.3 M, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [ (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) is an acyclic guanosine analog with structural similarities to acyclovir (ACV){9-[(2-hydroxyethoxy)methyl]guanine}, the established treatment for herpesvirus infections (6), and penciclovir (PCV) [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine], a compound recently licensed in some countries for the treatment of herpes zoster (29). The racemic mixture of (RS)H2G [previously abbreviated (Ϯ)2HM-HBG (1, 3, 17)] was originally shown to have good activity against herpes simplex virus (HSV) (18) and varicella-zoster virus (VZV) in vitro (3) and in vivo (17), and subsequent studies with the separated isomers showed that this activity resided predominantly with the R isomer (1, 25), now called H2G (25,27). H2G has activity against HSV type 1 (HSV-1) and type 2 (HSV-2) (1) and has been reported to have activity against human herpesvirus type 6 (4).Such a broad-spectrum anti-herpesvirus activity makes H2G an interesting candidate for clinical development, particularly for the treatment of VZV infection, where its impressive activity could lead to improved efficacy over current therapies.Strains of VZV that are deficient in thymidine kinase (TK) are resistant to H2G (1, 3), suggesting a mechanism of activation involving selective phosphorylation by herpesvirus-induced TKs, as is the case for ACV and PCV. This is support...

Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.

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The overexpression of a gene in a heterologous system is often the prelude to or the prerequisite of the elucidation and characterization of a given protein, in particular where the protein is difficult to obtain in sufficient quantity from natural sources. Prokaryotic expression systems, in particular Escherichia coli have been exploited successfully for a number of viral proteins. A large number of E. coli expression systems are currently commercially available that offer the investigator a large number of choices with regard to promoter choice, site of expression, fusions, and so forth. Since the variety and number of expression systems available are extensive, it is not within the scope of this chapter to discuss them fully, and the final choice of expression system used, is often arrived at empirically and often reflects the investigator's "favored system."