Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Abstract

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...

Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells

“…We have hypothesized that the mechanism of action of CPV involves phosphorylation to a triphosphate, the active compound that inhibits the viral DNA polymerase (14,17,20). However, the production of CPV-TP in HCMV-infected cells has not been demonstrated.…”

“…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.…”

“…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.…”

“…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.…”

Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection

“…Maribavir (MBV) is a selective pUL97 inhibitor that demonstrates high oral bioavailability and low toxicity (17)(18)(19)(20). It has undergone several clinical trials, been given orphan drug status, and could be useful for treating infections refractory to other antivirals (21).…”