Cellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE  Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

ST-246 was evaluated for activity against cowpox virus (CV), vaccinia virus (VV), and ectromelia virus (ECTV) and had an in vitro 50% effective concentration (EC 50 ) of 0.48 M against CV, 0.05 M against VV, and 0.07 M against ECTV. The selectivity indices were >208 and >2,000 for CV and VV, respectively. The in vitro antiviral activity of ST-246 was significantly greater than that of cidofovir, which had an EC 50 of 41.1 M against CV and 29.2 M against VV, with selectivity indices of >7 and >10, respectively. ST-246 administered once daily by oral gavage to mice infected intranasally with CV beginning 4 h or delayed until 72 h postinoculation was highly effective when given for a 14-day duration using 100, 30, or 10 mg/kg of body weight. When 100 mg/kg of ST-246 was administered to VV-infected mice, a duration of 5 days was sufficient to significantly reduce mortality even when treatment was delayed 24 h postinoculation. Viral replication in liver, spleen, and kidney, but not lung, of CV-or VV-infected mice was reduced by ST-246 compared to levels for vehicle-treated mice. When 100 mg/kg of ST-246 was given once daily to mice infected by the intranasal route with ECTV, treatment for 10 days prevented mortality even when treatment was delayed up to 72 h after viral inoculation. Viral replication in target organs of ECTV-infected mice was also reduced.National preparedness for bioterrorist events includes the development of rapid detection techniques, improved vaccination strategies, and antimicrobial chemotherapeutics with differing modes of action or targets. Preparing for a weaponized variola virus release is one component necessary for national security, and the development of highly effective, nontoxic antiviral agents that have proven efficacy when given postexposure is essential. Although cidofovir (CDV) has been approved under investigational new drug application for treatment of smallpox or complications of vaccination, its use would be limited since it is not orally bioavailable and produces nephrotoxicity.Previous evaluation of ST-246 for activity against orthopoxviruses has shown both in vitro and in vivo efficacies (13). When evaluated in vitro against vaccinia virus (VV), cowpox virus (CV), ectromelia virus (ECTV), monkeypox virus, camelpox virus, and variola virus, ST-246 inhibited virus replication by 50% at a concentration (50% effective concentration [EC 50 ]) of Յ0.07 M. In animal models using lethal infections with ECTV or VV, ST-246 was reported to be nontoxic and effective against mortality when given orally twice daily at 50 mg/kg of body weight for 14 days beginning before or shortly after infection. ST-246 was also evaluated in the nonlethal mouse tail lesion model by use of intravenous VV to mimic the primary viremic phase of viral infection and systemic lesional disease. When ST-246 was administered by oral gavage at 15 or 50 mg/kg twice daily for 5 days, the tail lesions were significantly reduced (13).The current studies expand upon the previous in vitro and in vivo findings and furt...

Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log 10 . In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.Orthopoxvirus diseases continue to pose challenges to researchers preparing for a bioterrorist release of biological weapons of mass destruction. Rapid diagnostics for smallpox and the development of effective antiviral chemotherapies are two essential components for national preparedness (16,17). The in vitro and in vivo activities of cidofovir (CDV) against orthopoxviruses are well documented (3,4,8,9,15,18,(20)(21)(22)(23)(24); however, its usefulness is limited by the requirement for intravenous administration and dose-limiting nephrotoxicity (14). Orally active compounds with prolonged therapeutic levels in blood and reduced toxicity would be desirable for mass distribution in response to an actual release of the smallpox virus.Results from earlier studies with acyclovir and ganciclovir showed that alkoxyalkyl esters of the monophosphates of ganciclovir or acyclovir have improved oral bioavailabilities compared to those of the unmodified parent compounds and are effective against cytomegalovirus and herpes simplex virus infections (13). To improve the oral bioavailability of CDV, a novel series of analogs were synthesized by esterification with long-chain alkoxyalkanols. Hexadecyloxypropyl-CDV (HDP-CDV) and octadecyloxyethyl-CDV (ODE-CDV) were synthesized and evaluated in vitro for their efficacies against cowpox virus (CV) and vaccinia virus (VV) infections. As reported previously (15), HDP-CDV had 50% effective concentrations (EC 50 s) of 0.52 and 0.62 M against CV and VV, respectively, whereas the EC 50 s of CDV were 42 and 31 M, respectively. ODE-CD...

The combination of ST-246 and hexadecyloxypropyl-cidofovir or CMX001 was evaluated for synergistic activity in vitro against vaccinia virus and cowpox virus (CV) and in vivo against CV. In cell culture the combination was highly synergistic against both viruses, and the results suggested that combined treatment with these agents might offer superior efficacy in vivo. For animal models, ST-246 was administered orally with or without CMX001 to mice lethally infected with CV. Treatments began 1, 3, or 6 days postinfection using lower dosages than previously used for single-drug treatment. ST-246 was given at 10, 3, or 1 mg/kg of body weight with or without CMX001 at 3, 1, or 0.3 mg/kg to evaluate potential synergistic interactions. Treatment beginning 6 days post-viral inoculation with ST-246 alone only increased the mean day to death at 10 or 3 mg/kg but had no effect on survival. CMX001 alone also had no effect on survival. When the combination of the two drugs was begun 6 days after viral infection using various dosages of the two, a synergistic reduction in mortality was observed. No evidence of increased toxicity was noted with the combination either in vitro or in vivo. These results indicate that combinations of ST-246 and CMX001 are synergistic both in vitro and in vivo and suggest that combination therapy using ST-246 and CMX001 for treatment of orthopoxvirus disease in humans or animals may provide an additional benefit over the use of the two drugs by themselves.

The high prevalence of Herpesviruses in the population and the maintenance of lifelong latent reservoirs are challenges to the control of herpetic diseases, despite the availability of antiviral pharmaceuticals that target viral DNA replication. In addition to oral and genital lesions, herpes simplex virus infections and recurrent reactivations from the latent pool can result in severe pathology including neonatal infection and mortality, blindness due to ocular keratitis, and viral-induced complications in immunosuppressed individuals. Herpesviruses, like their cellular hosts, are subject to the regulatory impacts of chromatin and chromatin modulation machinery that promotes or suppresses gene expression. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 and JMJD2s. Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and a block to infection and reactivation in vitro. Here, the concept of epigenetic suppression of viral infection is demonstrated in three animal models of herpes simplex virus infection and disease. Inhibition of LSD1 via treatment of animals with the monoamine oxidase inhibitor tranylcypromine results in suppression of viral lytic infection, subclinical shedding, and reactivation from latency in vivo. Phenotypic suppression is correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Given the expanding development of epipharmaceuticals, this approach has substantial potential for anti-herpetic treatments with distinct advantages over the present pharmaceutical options.

N-Methanocarbathymidine [(N)-MCT] is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. The antiviral activity of this molecule is dependent on the type I thymidine kinase (TK) in herpes simplex virus and also appears to be dependent on the type II TK expressed by cowpox and vaccinia viruses, suggesting that it is a substrate for both of these divergent forms of the enzyme. The drug is also a good inhibitor of viral DNA synthesis in both viruses and is consistent with inhibition of the viral DNA polymerase once it is activated by the viral TK homologs. This mechanism of action explains the rather unusual spectrum of activity, which is limited to orthopoxviruses, alphaherpesviruses, and Epstein-Barr virus, since these viruses express molecules with TK activity that can phosphorylate and thus activate the drug. The compound is also effective in vivo and reduces the mortality of mice infected with orthopoxviruses, as well as those infected with herpes simplex virus type 1 when treatment is initiated 24 h after infection. These results indicate that (N)-MCT is active in vitro and in vivo, and its mechanism of action suggests that the molecule may be an effective therapeutic for orthopoxvirus and herpesvirus infections, thus warranting further development.The most commonly used therapies for herpesvirus infections derive their specificity primarily through selective phosphorylation by viral kinases. Acyclovir (ACV) and penciclovir are phosphorylated by the thymidine kinases (TK) encoded by herpes simplex virus (HSV) and varicella-zoster virus, and the active triphosphate metabolites are potent inhibitors of the viral DNA polymerases (3,5,12,13). These compounds are ineffective against betaherpesviruses since they do not encode TK homologs, yet a closely related nucleoside, ganciclovir (GCV), is specifically phosphorylated by the human cytomegalovirus UL97 kinase (21, 39). Indeed, this strategy could be utilized against any virus provided that (i) a nucleoside could be identified that is a substrate for a viral kinase and (ii) the phosphorylated metabolite is an inhibitor of the viral polymerase. Orthopoxviruses are ideal candidates for such an approach since they encode highly conserved DNA polymerases and they fortuitously express enzymes with TK activity (17). The type II TK homologs in orthopoxviruses differ significantly from the type I homologs encoded by herpesviruses in several respects, including molecular weight, quaternary structure, and a more limited substrate specificity (4). ACV, GCV, and penciclovir are not substrates for this enzyme, and as a consequence, the drugs are inactive against orthopoxviruses. In order to identify new antiviral agents for the treatment of orthopoxvirus infections, we sought to identify nucleoside analogs that were dependent on the cowpox virus (CV) TK for their antiviral activity and, by inference, should be selective inhibitors of this virus and other viruses that express enzymes with TK activity. Herein, we de...

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Orthopoxviruses, including variola and monkeypox, pose risks to human health through natural transmission or potential bioterrorist activities. Since vaccination has not recently been utilized for control of these infections, there is renewed effort in the development of antiviral agents not only for postexposure smallpox therapy but also for treatment of adverse reactions following vaccination. The objectives of this study were to expand on the results of others that cidofovir (CDV) is effective in mice inoculated with cowpox virus (CV) or vaccinia virus (VV) and to document the efficacy of single and interval dosing beginning prior to or after infection, particularly including evaluations using suboptimal doses of CDV. We utilized BALB/c or SCID mice inoculated with CV or VV as models for systemic poxvirus infections. BALB/c mice were inoculated intranasally with CV or VV and treated with CDV prior to or after virus inoculation. CDV, at concentrations as low as 0.7 to 6.7 mg/kg of body weight/day for 5 days, conferred significant protection when treatment was initiated as late as 72 to 96 h postinfection. A single-dose pretreatment or posttreatment with CDV at 3 to 100 mg/kg was effective when given as early as 5 days prior to infection or as late as 3 days after infection with either VV or CV. Interval treatments given every third day beginning 72 h postinfection using 6.7 or 2 mg of CDV/kg also proved effective against CV infections. When SCID mice were inoculated intraperitoneally with CV or VV and treated for 7 to 30 days with CDV, all the mice eventually died during or after cessation of treatment; however, significant delays in time to death and reduction of virus replication in organs occurred in most treated groups, and no resistance to CDV was detected.