Synthesis and Enantioselectivity of the Antiviral Effects of (<i>R,Z</i>)-,(<i>S,Z</i>)-methylenecyclopropane Analogues of Purine Nucleosides and Phosphoralaninate Prodrugs: Influence of Heterocyclic Base, Type of Virus and Host Cells

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Human cytomegalovirus (HCMV), a betaherpesvirus, is a widespread pathogen infecting between 40 and 80% of the population. Although immunocompetent individuals rarely manifest any symptoms, HCMV can result in severe disease, such as interstitial pneumonia, mental retardation, and hearing loss in immunocompromised and immunologically immature individuals (26, 50). Currently, therapeutic agents such as ganciclovir (GCV), foscarnet (PFA), cidofovir, and fomivirsen are used for the treatment or prophylaxis of HCMV disease (1,7,20,22,39,50). However, long-term therapy is generally required due to recurrence of infection upon cessation of therapy, leading to the development of drug resistance and severe adverse effects (4,13,16,23,33,41). With the increased use of immunosuppression for cancer chemotherapy and organ transplantation, there is an increasing need for more effective and less toxic drugs to treat HCMV.We have demonstrated previously that cyclopropavir (CPV) (Fig. 1), a bis-hydroxymethyl methylenecyclopropane guanosine nucleoside analog, is approximately 10-fold more active in vitro (50% effective concentration [EC 50 ] of 0.46 M) than GCV (EC 50 of 4.1 M) (51, 52). In addition, CPV is active against several HCMV mutants that are resistant to GCV or PFA (29). Further experimentation in vivo with CPV demonstrated 2-to 5-log 10 reductions in titers of murine cytomegalovirus (MCMV), 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 (28).Previous studies have established that the mechanism of action of CPV involves inhibition of viral DNA synthesis (29). Furthermore, the activity of CPV was reduced approximately 20-fold against an HCMV UL97 deletion mutant (29), thereby indicating the importance of this gene product in the action of CPV. Taken together, these results suggest that the mechanism of action of CPV resembles that of GCV, in which the drug is first phosphorylated by viral pUL97, a protein kinase that can phosphorylate nucleoside analogs (32,36,46,47). Upon further phosphorylation by endogenous cellular kinases, the triphosp...

Section: Discussionmentioning

confidence: 80%

“…We suggest that because (R)-(Ϫ)-synguanol did not produce cytotoxicity (43), neither would the (Ϫ)-enantiomer of CPV triphosphate. However, it may be that the lack of cytotoxicity by (R)-(Ϫ)-synguanol is due to a lack of phosphorylation to its triphosphate (31).…”

Section: Discussionmentioning

confidence: 83%

Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Gentry

Kamil

Coen

et al. 2010

“…In both cases, the Z-and E-isomeric series of analogs were generated. We have reported previously that Z isomers of the first generation of methylenecyclopropane analogs are potent agents against some members of the herpesvirus family (6,10,(16)(17)(18)(19)(20)(21). These analogs had strong antiviral activity against HCMV, murine CMV (MCMV), human herpesvirus 6 (HHV-6), (10,20,24), rat CMV, rhesus monkey CMV, guinea pig CMV (20), and HHV-8 (10).…”

mentioning

confidence: 99%

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

Kern

Kushner

Hartline

et al. 2005

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 ...

“…Unlike cyclopropavir, MBX2168 is active against herpes simplex virus (8). Related synguanol 6-ether or -thioether derivatives retain activity against thymidine kinase-deficient strains highly resistant to acyclovir (8,18), suggesting that cellular kinases contribute to significant initial phosphorylation of these compounds. This may also explain the relatively lesser resistance of UL97 knockout mutants (9) and ganciclovir-resistant CMV UL97 mutants (Table 1) to MBX2168.…”

mentioning

confidence: 99%

Cytomegalovirus Mutants Resistant to Ganciclovir and Cidofovir Differ in Susceptibilities to Synguanol and Its 6-Ether and 6-Thioether Derivatives

Chou

Komazin-Meredith

Williams

et al. 2014

bThe methylenecyclopropane nucleoside (MCPN) analogs synguanol and its 6-alkoxy (MBX2168) and 6-alkylthio (MBX1616) derivatives retained good in vitro activities against several common ganciclovir-resistant UL97 kinase variants of human cytomegalovirus. Foscarnet-MCPN cross-resistance was observed among UL54 polymerase variants. UL54 exonuclease domain ganciclovir-cidofovir dual-resistant variants were remarkably more hypersensitive to these MCPNs than to cyclopropavir, with some 50% effective concentration ratios that were <0.1؋ the wild type. Different categories of MCPNs may have therapeutically exploitable mechanistic differences in viral DNA polymerase inhibition.