Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells

Abstract

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...

Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase

“…Some of this increased potency may be due to greater accumulation in infected cells of CPV-TP than GCV-TP at equivalently effective concentrations, which is consistent with more extensive phosphorylation of CPV than GCV by UL97 (6,11). However, CPV is also more potent than GCV against HCMV lacking UL97 (3, 9, 13).…”

“…Previous work had established that CPV is phosphorylated to CPV-TP in HCMV-infected cells (11), that CPV inhibits HCMV DNA synthesis (3), and that mutations in the HCMV pol gene can confer CPV resistance (12). These results suggested that CPV-TP targets HCMV Pol to block viral DNA synthesis, but the action of CPV-TP on HCMV Pol had not been explored.…”

“…It has previously been shown that following treatment of HCMV-infected cells with 2.5 M CPV, which is 5 times the concentration that reduces viral replication by 50% (EC 50 ), CPV-TP accumulates linearly with time, starting at 24 h postinfection (hpi) and reaching a level of 121 pmol/10 6 cells at 120 hpi (11). To assess the levels of CPV-TP at the EC 50 , we treated infected cells with 0.5 M CPV (EC 50 ).…”

“…Accordingly, certain HCMV UL97 mutations confer CPV resistance (9,10). CPV inhibits viral DNA replication (3), and accumulation of CPV-TP has been detected during viral infection (11). As certain mutations affecting the catalytic subunit (UL54, Pol) of the HCMV DNA polymerase also confer CPV resistance, it has been hypothesized that CPV-TP targets HCMV Pol (12) and terminates viral DNA synthesis.…”

“…CPV-TP levels in human foreskin fibroblasts infected with HCMV strain Towne were measured at 120 h postinfection (hpi) using reverse-phase high-pressure liquid chromatography as described previously (11), except that the cells were treated with 0.5 M radiolabeled CPV (the 50% effective concentration [EC 50 ] rather than 2.5 M [5ϫ EC 50 ]).…”