期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：3475-3480
DOI：10.1073/pnas.1420233112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe polymerization of nucleotides by DNA polymerases occurs through a common mechanism based on similar highly conserved amino acid motifs and the universal role of the coordination of Mg2+ by three dNTP phosphate oxygens. Based on these universal principles, we aimed at designing a dNTP mimic that could interact with a broad variety of DNA polymerases and should consist of three major indispensable entities: a nucleobase for Watson-Crick base-pairing, an enzymatically and chemically stable triphosphate replacement that can efficiently coordinate the Mg2+ cation, and a variable linker moiety between the nucleobase and the modified phosphate. The resulting -carboxy nucleoside phosphonates (-CNPs) were structurally, kinetically, and biochemically investigated, and the novel dNTP mimics were successfully validated in several DNA polymerase models. Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg2+ cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, -carboxy nucleoside phosphonate (-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that -CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the -CNP acts as the potential equivalent of the -phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the -CNP chelate Mg2+, mimicking the chelation by the {beta}- and {gamma}-phosphate oxygens of dNTPs. -CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg2+ ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, -CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg2+-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. -CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.
关键词：(deoxy)nucleoside triphosphate mimic ; HIV reverse transcriptase ; herpes virus DNA polymerase ; allosteric inhibition ; alpha-carboxy nucleoside phosphonate
