Identification of Acetylated, Tetrahalogenated Benzimidazole d -Ribonucleosides with Enhanced Activity against Human Cytomegalovirus

Abstract

DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminases. We previously demonstrated that the human cytomegalovirus (HCMV) terminase is composed of the large subunit pUL56 and the small subunit pUL89. While the large subunit mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required only for duplex nicking. An excellent inhibitor targeting HCMV terminase is 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB), but it was not developed as an antiviral drug due to its metabolic cleavage in experimental animals. We now have tested several new benzimidazole d-ribonucleosides in order to determine whether these compounds represent new, potent inhibitors. Analysis by bioluminometric ATPase activity assays identified two of the new compounds with a high inhibitory effect, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl benzimidazole (Cl(4)RB). By using viral plaque formation, viral yield, and viral growth kinetics, we demonstrated that the two compounds BTCRB and Cl(4)RB had antiviral activities similar to that of BDCRB. Interestingly, BTCRB retained its inhibitory activity after preincubation with HFF cells. By use of electron microscopy, we observed an increase of B capsids and a lack of cytoplasmic capsids in the presence of the compounds that correlated with the virus yield. Furthermore, cleavage of concatenated DNA was inhibited by both compounds, and inhibition by BTCRB was shown to be dose dependent. These results demonstrate that the new compounds are highly active against HCMV and act by mechanisms similar but not identical to those of BDCRB.