Mechanisms of action of drugs with dual or multiple antiviral activities

Viral enzymes that catalyze replication of the viral genome remain the main subject of currently available anti-viral therapies. This work focuses on three antiviral agents that target viral DNA polymerases: foscarnet, acyclovir and entecavir. Though the broad spectrum of antiviral activities of foscarnet has been recognized for decades, only recently has it been shown that the anti-herpesvirus drug acyclovir and the anti-hepatitis B virus drug entecavir are also active against the human immunodeficiency virus (HIV) 1. The clinical benefits of the multiple antiviral activities of foscarnet have been demonstrated in treating HIV/herpesvirus co-infections. Foscarnet is currently approved for treating human cytomegalovirus infections. But the precise molecular mechanism of foscarnet action and resistance remains poorly understood. The study of foscarnet's mode of action against the HCMV DNA polymerase (UL54) is complicated in part by the difficulty in expressing and purifying the viral enzyme. To address the first issue, I conducted biochemical studies of foscarnet/UL54 interactions using an unpurified viral enzyme. I then proposed a model based on these studies for the presumptive foscarnet binding site within UL54. To address the second issue I generated the RB69 bacteriophage/HCMV polymerase chimera, which is easily purifiable and displays the UL54 phenotype with respect to foscarnet and acyclovir. The recent discovery of the anti-HIV activity of acyclovir has been followed by a report showing the selection of the V75I mutation within HIV-1 reverse transcriptase (RT) under the selective pressure of acyclovir. My own biochemical studies have revealed that the major effect of V75I substitution involves reducing the catalytic rate of acyclovir incorporation. The recent report of entecavir anti-HIV-1 activity has demonstrated the selection of M184V-containing HIV-1. Subsequent biochemical studies have revealed that M184V-containing RT discriminates against entecavir at the level of incorporation. My own studies show that the major effect of entecavir incorporation has been the delayed chain termination (DCT) of the DNA synthesis. DCT protects the incorporated drug from excision. The research project described in this thesis provides novel tools and reveals new concepts for future drug design and development.