Alpha-Carboxy Nucleoside Phosphonates As Universal Nucleoside Triphosphate Mimics

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, alpha-carboxy nucleoside phosphonate (alpha-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that alpha-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 alpha-CNP acts as the potential equivalent of the alpha-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the alpha-CNP chelate Mg2+, mimicking the chelation by the beta- and gamma-phosphate oxygens of dNTPs. alpha-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, alpha-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. alpha-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.