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...
Abstract5′-O-D-and L-amino acid derivatives and 5′-O-(D-and L-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi-or more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was improved compared to the parent drug. KeywordsVidarabine; ara-A; Amino acid ester prodrug; Phosphoramidate prodrug; Antiviral activity; Selective protection of nucleoside; Levulinate ester; Oral bioavailability; Adenosine deaminase; Arahypoxanthine; ara-H; Caco-2 permeability There has been continued interest in the synthesis and biological evaluation of nucleoside analogs capable of delivering the corresponding nucleotides inside cells. 1 1-Beta-Darabinofuranosyladenine (vidarabine or ara-A) is an antiviral drug with activity against herpes viruses, poxviruses, and certain rhabdoviruses, hepadnarviruses, and RNA tumor viruses. [2][3][4] Vidarabine also is active against vaccinia virus both in vitro 5 and in vivo. 6 However, it is more toxic and less metabolically stable than other current antivirals such as acyclovir and ganciclovir; further it is poorly soluble with low oral bioavailability. It is readily deaminated by adenosine deaminase (ADA) to ara-hypoxanthine (ara-H), 7 which possesses some antiviral activity but is at least 10-fold less potent than vidarabine. 6-8 Adenosine deaminase (ADA) is a cytosolic enzyme that participates in purine metabolism where it degrades either adenosine or 2′-deoxyadenosine to inosine or 2′-deoxyinosine, respectively. Further metabolism of these deaminated nucleosides leads to hypoxanthine. ADA also degrades vidarabine to ara-H by same mechanism. 7 Our current interest in prodrugs of vidarabine was triggered by the report of the activity of vidarabine against cowpox virus 9 and by our discovery that vidarabine was 3-to 5-fold more active against vaccinia and cowpox viruses than cidofovir in plaque reduction assays. 10 Cidofovir is a broad spectrum antiviral agent, [11][12][13] that is, limited in its usage because of nephrotoxicity and poor oral bioavailability (~2% in humans) [14][15][16] and for which prodrugs have been developed. 17 Furthermore, we found that the activity of vidarabine against these viruses was enhanced approximately 10-fold when combined with 2′-deoxycoformycin (pentostatin, a potent inhibitor of ADA), thus providing significant superiority to cidofovir. Based on these results and earlier studies on 5′-substituted vidarabine analogs, we hypothesized that minimizing the conversion of vidarabine to its hypoxanthine analog could yield a significantly more potent anti-pox virus agent. With this goal in mind, we have developed a prodrug strategy that protects the vidarabine from metabolic conversion by making 5′-amino acid esters and 5′-phosphoramidates of the drug. Further, our rationale includes the design of prodrugs that increase aqueous solubility over that of the parent drug and also increase the potential for membrane transport by the dipeptide intest...
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