In vitro activity and mechanism of action of methylenecyclopropane analogs of nucleosides against herpesvirus replication

Abstract

We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC(50)) levels of < or =50 microM. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC(50)s of 0.5 to 44 microM, and all were active against MCMV by PR (0.3 to 54 microM). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 microM). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 microM), and three compounds were active against HHV-8 (5.5 to 16 microM). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC(50)s of 0.7 to 8 microM. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.

FIG. 1.

Structures of second-generation methylenecyclopropane analogs.

FIG. 2.

Inhibition of HCMV DNA synthesis and expression of IE1, ppUL44, and pp28. Viral DNA synthesis was determined by a dot blot assay and is represented by filled squares. Expression of IE1 (open squares), ppUL44 (open circles), and pp28 (filled circles) was determined by western blotting. Images were captured, quantified, and expressed as percentages of untreated infected cells.