Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action

Abstract

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-beta-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-beta-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 microM compared to a mean IC(50) for GCV of 0.53 +/- 0.04 microM, 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.

FIG. 1.

Chemical structures of 1263W94 and BDCRB.

FIG. 2.

Effects of 1263W94, GCV, and BDCRB on HCMV DNA synthesis during a single-cycle infection of MRC-5 cells.

FIG. 3.

Synthesis and processing of high-molecular-weight HCMV DNA in HCMV-infected cells in the presence or absence of 1263W94, BDCRB, and GCV. Calculations from integrated band intensities are shown below the figure. Abbreviations: M, monomeric viral DNA; C, concatemeric viral DNA.

FIG. 4.

“BDCRB washout” experiment for determining the effects of 1263W94, BDCRB, and GCV on the processing of concatemeric HCMV DNA that had accumulated during 96 h in the presence of BDCRB. Calculations from integrated band intensities are shown below the figure. Abbreviations: M, monomeric viral DNA; C, concatemeric viral DNA.