3-Phosphono-l-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase (original) (raw)
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Antiviral Chemistry and Chemotherapy
Background The replacement of β,γ-pyrophosphate by β,γ-phosphonate moieties within the triphosphate chain of 5′-triphosphate nucleoside analogues was previously studied for various antiviral nucleoside analogues such as AZT and 2′,3′-dideoxynucleosides. Thus, it has been shown that these chemical modifications could preserve, in some cases, the terminating substrate properties of the triphosphate analogue for HIV-RT. Herein, we aimed to study such 5′-triphosphate mimics based on the scaffold of the well-known antiviral agent 9-[(2-phosphonomethoxy)ethyl]adenine (PMEA, Adefovir). Methods Synthesis involved coupling of a morpholidate derivative of PMEA with appropriate pyrophosphoryl analogues. The relative efficiencies of incorporation of the studied diphosphate phosphonates were measured using subtype B WT HIV-1 RT in an in vitro susceptibility assay, in comparison to the parent nucleotide analogue (PMEApp). Results Searching for nucleoside 5′-triphosphate mimics, we have synthesize...
2006
A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP i) analogs on PP i-and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP i-and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT⅐template/primer complex was hindered by the presence of a chain terminator at the 3-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZTtriphosphate in the presence of PP i or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.
Nucleic acids …, 2007
Some selected amino acids, in particular L-aspartic acid (L-Asp) and L-histidine (L-His), can function as leaving group during polymerase-catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. Although L-Asp-dAMP and L-His-dAMP bind, most probably, in a different way in the active site of the enzyme, aspartic acid and histidine can be considered as mimics of the pyrophosphate moiety of deoxyadenosine triphosphate. L-Aspartic acid is more efficient than D-aspartic acid as leaving group. Such P-N conjugates of amino acids and deoxynucleotides provide a novel experimental ground for diversifying nucleic acid metabolism in the field of synthetic biology.
Biochemical and Biophysical Research Communications, 2005
Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the b,c-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the b,c-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3 0 N 3 ), and ddTMP(3 0 NH 2 ) from the 3 0 -end of primers with an equal efficacy.
Organic & Biomolecular Chemistry, 2010
A polymer-bound α, β-methylene-β-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5′-O-α, β-methylene-β-triphosphates. Among all the compounds, cytidine 5′-O-α, β-methylene-β-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a K i value of 225 μM. Modified nucleoside triphosphates have received much attention as mimics of naturally occurring deoxyribo-and ribonucleoside triphosphates, as probes in several biochemical pathways involving DNA and RNA synthesis, and as potential diagnostic and therapeutic agents. 1,2 The structural similarity of modified nucleotides to natural nucleoside triphosphates make them useful reagents as substrates or inhibitors for DNA or RNA polymerases. 3,4 Although most natural polymerase enzymes incorporate natural nucleoside triphosphates into nucleic acids, there are certain polymerases that are capable of incorporating unnatural nucleoside triphosphates into nucleic acids. 5-7 A number of approaches have been focused on modifications and/or substitution on the base, 8-9 carbohydrate, 10-13 and linear triphosphate moieties 14-17 to design modified nucleotides for diverse applications in nucleic acid and antiviral research. Early in the life cycle of human immunodeficiency virus type 1 (HIV-1), viral RNA is reverse transcribed into double stranded DNA for integration into the genome of the infected cell. 18 This process is catalyzed by reverse transcriptase (RT), a virus encoded heterodimeric enzyme composed of 66 and 51 kD subunits (p66 and p51), possessing DNA polymerase and ribonuclease H (RNase H) activities. 19 DNA polymerase activity is required for the synthesis of RNA: DNA heteroduplex from the single stranded viral RNA. Whereas, RNase H activity † Electronic supplementary information (ESI) available: Experimental procedures, characterization of resins with IR and final compounds with NMR, high-resolution mass spectrometry, and quantitative phosphorus analysis, DNA polymerase assay results.
Nucleic Acids Research, 1998
Replacement of α-, βand γ-phosphate groups in 2′-deoxynucleoside 5′-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at α-, βand γ-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that γ-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the γ-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase α. For dUTP analogues substituted at both γ-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at γ-phosphonate or C5 position. Meanwhile, C5-substituted β,γ-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2′-deoxythymidine 5′-[β,γ-(methylphosphinyl)methylphosphonyl]-α-phosphate and its 3′-azido-3′-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.
Amino Acid Phosphoramidate Nucleotides as Alternative Substrates
… Chemie (International ed.), 2007
There has been significant progress in the design and synthesis of numerous nucleotide analogues bearing a modified nucleobase moiety or unnatural sugar and that are substrates for polymerases. Modifications at the phosphate moiety are introduced to increase the stability of a nucleotide toward enzymatic degradation or to mask the phosphate negative charge and facilitate its penetration into a cell. A common strategy in nucleotide prodrug design is protecting a phosphate moiety with a labile masking group. Removal of a masking group liberates a nucleoside monophosphate entity to be transformed to a nucleoside triphosphate, a substrate for intracellular enzymes. However, even after removal of the masking group, phosphorylation and activation of nucleoside monophosphates remain a problem owing to substrate specificity of cellular kinases. Therefore, design of a nucleotide analogue that would allow bypassing of the kinase activation pathway while behaving as a direct polymerase substrate/inhibitor would be a considerable challenge.
FEBS Letters, 1995
HIV-I lit is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA Ly*'3 to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating lit are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-I RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.
Nucleic Acids Research, 1998
4′-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3′-hydroxyl group. Here, we describe the action of the 4′-acetyl-(MeTTP) and 4′-propanoylthymidine 5′-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA-and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases α, β, δ and ε. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4′-substituent of the nucleoside 5′-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4′-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptase.