Incorporation of Selected Nucleoside Phosphonates and Anti-Human Immunodeficiency Virus Nucleotide Analogues into DNA by Human DNA Polymerases α, β and γ

Abstract

SummaryIncorporation of selected diphosphates of nucleoside phosphonates and friphosphotes of currently approved anti-human immunodeficiency virus nucleoside analogues into DNA by human DNA polymerases a,~and y was studied. All three polymerases were able to incorporate diphosphates of 9-(2-phosphonomethoxyethyl)adenine (PMEApp), 9-(2-phosphonomethoxyethyl)guanine (PMEGpp), (R)-9-(2-phosphonomethoxypropyl)adenine (PMPApp), (R)-9-(2-phosphononomethoxypropyl)-2,6-diaminopurine (PMPDAPpp) and (2R,5R)-9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4APpp) into primer/template DNA of defined sequence. After incorporation, these nucleoside phosphonates acted as terminators of primer extension. Kinetic constants of their incorporation were determined and compared with those for incorporation of ddATP, ddCTP, (-)-2'-deoxy-3'-thiacytidine triphosphate (3TC-TP),~',3'-dide hydro-3'-deoxythymidine triphosphate (d4T-TP) and 3'-azido-3'-deoxythymidine triphosphate (AZT-TP).Relative efficiencies of incorporation (percentage of the incorporation efficiency for the corresponding natural deoxynucleoside triphosphate) by DNA polymerase a ranged from 0.05% for 3TC-TP to 51% for PMEGpp. DNA polymerase~catalysed the incorporation with relative efficiencies ranging from 0.014% for AZT-TP to 125% for ddCTP, and efficiencies of incorporation by DNA polymerase y varied between 0.13% for 3TC-TP and 25% for ddCTP. Generally, the lowest incorporation efficiencies with all three polymerases were found for PMPApp (0.06-1 .4%) and PMPDAPpp (0.075-2.2%).

Assessment of Mitochondrial Toxicity in Human Cells Treated with Tenofovir: Comparison with Other Nucleoside Reverse Transcriptase Inhibitors

“…In contrast, HIV-1 reverse transcriptase is efficiently inhibited by tenofovir-DP, with K i values of 1.55 and 0.022 M when the values are determined with DNA and RNA templates, respectively (9). Incorporation of tenofovir into a DNA primer-template by DNA pol ␥ is also less efficient than that of other NRTIs (11), suggesting a weak inhibition of mtDNA synthesis in cells treated with tenofovir. In order to confirm this assumption in various cell types, the effects of tenofovir and six clinically used NRTIs on mtDNA synthesis were evaluated in HepG2 cells, normal SkMCs, and normal human RPTECs.…”

“…While 10 M d4T reduced the mtDNA content by 50% in different T-lymphoblastoid cell lines (28,31), HepG2 cells and SkMCs exhibited similar decreases in mtDNA synthesis following exposure to 300 M d4T. Since d4T triphosphate is a potent inhibitor of DNA pol ␥ (K i ϭ 0.05 M) (28) and also undergoes efficient incorporation into DNA primer-template by this enzyme (11), differences in the levels of drug accumulation and/or phosphorylation in the mitochondria of various cell types presumably account for the cell typedependent effects of d4T on mtDNA synthesis.…”

GS-9219—A Novel Acyclic Nucleotide Analogue with Potent Antineoplastic Activity in Dogs with Spontaneous Non–Hodgkin's Lymphoma

“…Its active metabolite, PMEGpp, is a potent inhibitor of DNA polymerases a, y, and q (21, 22), enzymes known to participate in eukaryotic DNA replication and/or DNA repair. PMEGpp is efficiently recognized as an alternative substrate by these polymerases (2,23), resulting in its incorporation into nascent DNA chains and termination of DNA synthesis. This leads to the arrest of cell division in S phase with subsequent induction of apoptosis.…”

Targeting human DNA polymerase α for the inhibition of keratinocyte proliferation. Part 1. Homology model, active site architecture and ligand binding

“…For the docking studies the dTTP ligand from the model complex was changed against a set of known nucleoside triphosphate inhibitors [13,14] and the non-nucleoside inhibitor aphidicolin [15]. The inhibitor molecules (all nucleosides contained a thymine base in order to get a correct Watson-Crick base pairing with the given template) were docked manually into the active site by superimposing the structures with the natural substrate in a conformation close to the dTTP template.…”