Kinetic analysis of the interaction of cidofovir diphosphate with human cytomegalovirus DNA polymerase

Abstract

Cidofovir [CDV, ( S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic cytosine nucleoside phosphonate analog with potent in vitro and in vivo activity against a broad spectrum of herpesviruses. CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is a competitive inhibitor of dCTP and an alternate substrate for human cytomegalovirus (HCMV) DNA polymerase. HCMV DNA polymerase used a synthetic DNA primer-template with a K m value of 90 ± 8 nM and incorporated dCTP approximately 42 times more efficiently than CDVpp. HCMV DNA polymerase also utilized a synthetic DNA primer containing a single molecule of CDV at the 3′-terminus. The K m value for this DNA primer-template was 165 ± 42 nM and incorporation of dCTP was approximately 17 times more efficient than that of CDVpp. The slower rate of incorporation of CDVpp was due mostly to the higher K m value of CDVpp toward the enzyme-primer-template complexes. These data demonstrate that incorporation of a single CDV into DNA by HCMV DNA polymerase does not lead to chain termination.