Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5′-phosphates by mammalian phosphotransferases

Abstract

Amdoxovir [(−)-β-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high K m 5′-nucleotidase (5′-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high K m (7 mM). Human 5′-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG–MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG–DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG–DP. In CEM cells incubated with 5 μM DXG for 24 h, 0.015 pmole/10 6 cells (~7.5 nM) of DXG–TP was detected as the primary metabolite. Our study demonstrated that 5′-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.