Susceptibilities of Human Cytomegalovirus Clinical Isolates and Other Herpesviruses to New Acetylated, Tetrahalogenated Benzimidazole d -Ribonucleosides

Abstract

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-␤-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-␤-D-ribofuranosyl) benzimidazole (Cl 4 RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl 4 RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl 4 RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV-and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCVsensitive clinical isolates and slightly active against other herpesviruses.Human cytomegalovirus (HCMV) is one of eight human herpesviruses and is a serious, life-threatening, opportunistic pathogen in immunocompromised patients (organ recipients or AIDS patients) (8,19). HCMV is widespread, with a seroprevalence throughout the world of up to 100% in adults. To date, nearly all anti-HCMV drugs for systemic treatment are inhibitors of the viral DNA polymerase (2,13,21,22). Due to the low bioavailability, number of side effects, dose-dependent toxicity, and appearance of resistances caused by the current available drugs, development of new antiviral compounds which have a different mode of action is needed. Consequently, to broaden therapy of HCMV infections and to circumvent current mechanisms of drug resistance, an inhibitor of HCMV terminase would be of great value, because it would act subsequently to DNA synthesis and block the first steps in viral maturation.Viral replication includes cleavage of newly synthesized, concatemeric DNA into unit-length genomes and packaging into preformed procaspids. These processes occur in or in close proximity to replication centers in the nucleus. Enzymes involved in the packaging process are responsible for duplex nicking and insertion of the DNA into the procapsids (1, 4, 9, 10), t...

DSTP-27 Prevents Entry of Human Cytomegalovirus

“…), the supernatants were harvested and frozen at Ϫ80°C. After collection at all time points, the supernatants were thawed and transferred to a 12-well plate, and titers were determined by plaque reduction assay (20).…”

“…All other HCMVs were propagated and studied in HELF. Virus titers were determined by plaque assay as described previously (20).…”

“…HELFs (5 ϫ 10 4 ) were seeded in 96-well plates and incubated with different concentrations of DSTP-27 (50 to 500 M) in a final volume of 100 l for 8 days at 37°C. The 50% cytotoxic concentration (CC 50 ) of DSTP-27 was determined using the cell proliferation kit II (XTT; Roche Diagnostic, Mannheim, Germany) as described previously (20).…”

Characteristics and functional roles of VP5 protein of herpesviruses

“…Meanwhile, the VP22a is released from the procapsid and the overall structure angularizes itself to create the icosahedral shape of the mature capsid. As it does not involve cleavage of linear DNA by a terminase as in DNA replication in mammalian cells, terminase inhibitors might be discovered and display similar antiviral as found with human cytomegalovirus [60,61]. DNA-filled capsids are released into the extracellular environment after undergoing envelopment and de-envelopment [62,63].…”

Cytomegalovirus antivirals and development of improved animal models

“…However, further studies are necessary to fully identify the mode of action. Two new benzimidazole D-ribonucleosides compounds have also been identified as effective inhibitors targeting the viral terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl) benzimidazole (Cl4RB) and encouragingly they are effective against murine and rat CMV [87, 88]. C14RB would appear to be more effective than BTCRB, which is more equivalent to BDCRB in HCMV plaque reduction assays.…”