Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-␤-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-␤-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC 50 ) of 0.12 ؎ 0.01 M compared to a mean IC 50 for GCV of 0.53 ؎ 0.04 M, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.Human cytomegalovirus (HCMV) is a herpesvirus that causes a benign infection in an estimated 40 to 100% of populations in the United States (reviewed by Sia and Patel [23]). In most cases, HCMV infection is not associated with disease; however, in patients with an immature or compromised immune system, HCMV infection can be a serious or even lifethreatening disease. Four drugs-ganciclovir (GCV), its prodrug valganciclovir, cidofovir, and foscarnet-are currently used for the treatment of systemic HCMV infection; however, there are a number of disadvantages associated with each of these therapies. Cidofovir and foscarnet are available only as intravenous formulations, whereas GCV is given intravenously for initial treatment of systemic disease. With all anti-HCMV drugs currently available, there can be serious side effects associated with prolonged treatment. In addition, the drugs have similar mechanisms of action, all ultimately targeting the HCMV polymerase; therefore, selection of crossresistant HCMV mutants can occur. Thus, there is a need for other therapeutic agents that are safe, potent, and orally bioavailable, with a novel mechanism of action.As part of an ongoing program to develop novel anti-HCMV compounds that could potentially yield new therapeutic agents for treatmen...

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.

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The UL97 phosphotransferase coding sequences of clinical cytomegalovirus (CMV) isolates, 10 resistant and 11 sensitive to ganciclovir, were compared to define mutations associated with drug resistance. In each ganciclovir-resistant isolate, a mutation was found that resulted in an amino acid substitution at codon 460 (4 isolates), codon 594 (2 isolates), or codon 595 (4 isolates). No sensitive isolate carried any of these mutations. Marker transfer studies showed that each mutation was capable of conferring ganciclovir resistance to the laboratory CMV strain AD169. Rapid diagnostic tests based on DNA amplification and restriction enzyme analysis were developed for these mutations. Specific mutant DNAs were detected when they constituted at least 10% of the population in the specimen. Several mutations in UL97 appear to be common markers for ganciclovir resistance, and their detection may be a rapid alternative to conventional cell culture susceptibility testing.