Oral Activity of a Methylenecyclopropane Analog, Cyclopropavir, in Animal Models for Cytomegalovirus Infections

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We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC 50 ) levels of <50 M. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC 50 s of 0.5 to 44 M, and all were active against MCMV by PR (0.3 to 54 M). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 M). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 M), and three compounds were active against HHV-8 (5.5 to 16 M). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC 50 s of 0.7 to 8 M. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.Human cytomegalovirus (HCMV) infects approximately 40 to 80% of the population, and serious life-threatening manifestations are associated with congenital infection and immunosuppression (5). Currently, therapeutic agents such as ganciclovir (GCV), foscarnet (PFA), and cidofovir (CDV) are used to treat HCMV infections, and while GCV is highly effective in treating CMV retinitis, pneumonia, and gastrointestinal disease, long-term therapy is generally required because infection often recurs upon cessation of therapy. The development of drug resistance and severe side effects due to drug toxicity may also occur during long courses of treatment and may limit the clinical use of these drugs. The increased use of immunosuppression for cancer chemotherapy and organ transplantation presents an ever-increasing need for more effective and less toxic antiviral drugs.A relatively new series of nucleoside analogs, the methylenecyclopropanes were modeled after allene analogs, which have been shown to exhibit antiviral activity (23). The first generation can be regarded as bioisosteric analogs of acyclovir where the C-O-C moiety was replaced by the methylenecyclopropane moiety. In a similar fashion, the second generation of methylenecyclopropane analogs are related to ganciclovir (25). In both cases, the Z-and E-isomeric series of analogs were generated. We have reported previously that ...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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In Vitro Activity and Mechanism of Action of Methylenecyclopropane Analogs of Nucleosides against Herpesvirus Replication

Kern

Kushner

Hartline

et al. 2005

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“…In addition, CPV is also active against several HCMV strains that are resistant to GCV or PFA (14). Further experimentation in vivo with CPV demonstrated a 2 to 5 log 10 reduction in titers of murine cytomegalovirus, resulting in reduced mortality in severe combined immunodeficient (SCID) mice and reduced viral replication in human fetal tissue implanted in SCID mice infected with HCMV (15). Toxicology studies performed in vivo demonstrated few to no adverse effects at therapeutic concentrations, making CPV a good clinical candidate for the treatment of systemic HCMV infections (16).…”

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Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells

Gentry

Drach

2014

H uman cytomegalovirus (HCMV) is a widespread pathogen infecting between 40% and 80% of the population worldwide (1). Although individuals with competent immune systems rarely manifest any symptoms, HCMV infections can result in severe interstitial pneumonia, encephalitis, and gastroenteritis in immunocompromised individuals (2). HCMV is also the most common congenital infection in the United States and results in over 4,000 cases of severe mental disabilities, hearing, and/or vision loss in infants each year (3, 4). Drugs currently approved by the FDA for the treatment of systemic HCMV infections are ganciclovir (GCV; Fig. 1) and its oral prodrug valganciclovir, foscarnet (PFA), and cidofovir (5-8). The mechanism of action for each of these drugs involves inhibition of the viral DNA polymerase, resulting in inhibition of HCMV DNA synthesis and viral replication (5). However, long-term chemotherapy for HCMV is generally required due to recurrence of infection upon cessation of treatment. As such, the selection of strains with decreased drug susceptibility is common (6, 9-11). Because adverse effects occur with a high rate of incidence (up to 30% of patients) (12), there is a need for new compounds with a greater therapeutic index for the treatment of systemic HCMV infection.We have previously demonstrated that cyclopropavir (CPV; Fig. 1), a methylenecyclopropane guanosine nucleoside analog, is approximately 10-fold more active in vitro (50% effective concentration [EC 50 ] ϭ 0.46 M) than GCV (EC 50 ϭ 4.1 M) without any observed increase in cytotoxicity (13). In addition, CPV is also active against several HCMV strains that are resistant to GCV or PFA (14). Further experimentation in vivo with CPV demonstrated a 2 to 5 log 10 reduction in titers of murine cytomegalovirus, resulting in reduced mortality in severe combined immunodeficient (SCID) mice and reduced viral replication in human fetal tissue implanted in SCID mice infected with HCMV (15). Toxicology studies performed in vivo demonstrated few to no adverse effects at therapeutic concentrations, making CPV a good clinical candidate for the treatment of systemic HCMV infections (16).We and others have previously established that the mechanism of action of CPV is similar to that of GCV, namely, phosphorylation to a monophosphate (MP) by the viral pUL97 protein kinase (17)(18)(19), additional phosphorylation to a triphosphate (TP) by an endogenous cellular kinase (20), and viral DNA synthesis inhibition resulting in inhibition of viral replication (14,21). Although enzymatic conversion of CPV to a triphosphate by the pUL97 viral protein kinase and an endogenous cellular kinase has been established (17,20), this conversion has not been observed in virusinfected cells. Therefore, the goal of this study was a comparison of the metabolism of CPV and the current standard for HCMV chemotherapy, GCV, in HCMV-infected cells. MATERIALS AND METHODSViral strain and chemicals. HCMV strain Towne was kindly provided by M. F. Stinski, University of Iowa. GCV was kindly pro...

“…Maribavir (MBV) remains experimental after phase III clinical trials showed low toxicity but inadequate antiviral efficacy at the dose tested (2,3). Additionally, methylenecyclopropane nucleoside analogs, such as cyclopropavir (CPV), are being investigated for clinical use (4,5). The CMV UL97 kinase (pUL97) has important roles in the initial phosphorylation of GCV and CPV that is essential for the antiviral activity of these drugs and as a direct antiviral target for the kinase inhibitor MBV, since UL97 shutoff results in severe viral growth impairment in vitro (6).…”

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Cytomegalovirus UL97 Kinase Catalytic Domain Mutations That Confer Multidrug Resistance

Chou

Ercolani

Marousek

et al. 2013

cHuman cytomegalovirus UL97 kinase mutations that commonly confer ganciclovir resistance cluster in different parts of the gene than those conferring resistance to maribavir, an experimental UL97 kinase inhibitor. The drug resistance, growth, and autophosphorylation phenotypes of several unusual UL97 mutations in the kinase catalytic domain were characterized. Mutations V466G and P521L, described in clinical specimens from ganciclovir-treated subjects, conferred a UL97 kinase knockout phenotype with no autophosphorylation, a severe growth defect, and high-level ganciclovir, cyclopropavir, and maribavir resistance, similar to mutations at the catalytic lysine residue K355. Mutations F342S and V356G, observed after propagation under cyclopropavir in vitro, showed much less growth attenuation and moderate-to high-level resistance to all three drugs while maintaining UL97 autophosphorylation competence and normal cytopathic effect in cell culture, a novel phenotype. F342S is located in the ATP-binding P-loop and is homologous to a c-Abl kinase mutation conferring resistance to imatinib. UL97 mutants with relatively preserved growth fitness and multidrug resistance are of greater concern in antiviral therapy than the severely growth-impaired UL97 knockout mutants. Current diagnostic genotyping assays are unlikely to detect F342S and V356G, and the frequency of their appearance in clinical specimens remains undefined.