Cidofovir and ( S )-9-[3-Hydroxy-(2-Phosphonomethoxy)Propyl]Adenine Are Highly Effective Inhibitors of Vaccinia Virus DNA Polymerase When Incorporated into the Template Strand

Abstract

ABSTRACT The acyclic nucleoside phosphonate drug ( S )-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine [( S )-HPMPA], is a broad-spectrum antiviral and antiparasitic agent. Previous work has shown that the active intracellular metabolite of this compound, ( S )-HPMPA diphosphate [( S )-HPMPApp], is an analog of dATP and targets DNA polymerases. However, the mechanism by which ( S )-HPMPA inhibits DNA polymerases remains elusive. Using vaccinia virus as a model system, we have previously shown that cidofovir diphosphate (CDVpp), an analog of dCTP and a related antiviral agent, is a poor substrate for the vaccinia virus DNA polymerase and acts to inhibit primer extension and block 3′-to-5′ proofreading exonuclease activity. Based on structural similarities and the greater antiviral efficacy of ( S )-HPMPA, we predicted that ( S )-HPMPApp would have a similar, but more pronounced effect on vaccinia polymerase than CDVpp. Interestingly, we found that ( S )-HPMPApp is a good substrate for the viral enzyme, exhibiting K m and V max parameters comparable to those of dATP, and certainly not behaving like CDVpp as a functional chain terminator. Metabolic experiments indicated that ( S )-HPMPA is converted to ( S )-HPMPApp to a much greater extent than CDV is converted to CDVpp, although both drugs cause identical effects on virus DNA replication at their 50% effective concentration. Subsequent studies showed that both compounds can be faithfully incorporated into DNA, but when CDV and ( S )-HPMPA are incorporated into the template strand, both strongly inhibit trans -lesion DNA synthesis. It thus appears that nucleoside phosphonate drugs exhibit at least two different effects on DNA polymerases depending upon in what form the enzyme encounters the drug.