Cellular Phosphorylation of Anti-HIV Nucleosides (original) (raw)
Related papers
Cellular phosphorylation of anti-HIV nucleosides. Role of nucleoside diphosphate kinase
Journal of Biological Chemistry, 1996
Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy-or deoxynucleotides as substrates.
Febs Letters, 1993
Nucleoside analogues previously found to be inactive against the human immunodeficiency virus (HIV) may be activated by simple chemical derivatisation. As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of inactive nucleoside analogues become inhibitors of HIV replication. This discovery underlies the importance of the masked phosphate approach, and has significant implications for the future design of chemotherapeutic nucleoside analogues. If highly modified nucleoside analogues may be active without the intervention of nucleoside kinase enzymes, major advantage may accrue in terms of low toxicity and enhanced selectivity. Moreover, the increased structural freedom may have implications for dealing with the emergence of resistance. The concept herein described as 'kinase bypass' may thus stimulate the discovery of a new generation of antiviral agents.
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.
Inhibition of multi-drug resistant HIV-1 reverse transcriptase by nucleoside β-triphosphates
Bioorganic & Medicinal Chemistry Letters, 2011
Despite the success of potent reverse transcriptase (RT) inhibitors against human immunodeficiency virus type 1 (HIV-1) in combination regimens, the development of drug resistant RTs constitutes a major hurdle for the long-term efficacy of current antiretroviral therapy. Nucleoside β-triphosphate analogs of adenosine and nucleoside reverse transcriptase inhibitors (NRTIs) (3′-azido-2′,3′-dideoxythymidine (AZT), 3′-fluoro-2′,3′-dideoxythymidine (FLT), and 2′, 3′-didehydro-2′,3′-dideoxythymidine (d4T)) were synthesized and their inhibitory activities were evaluated against wild-type and multidrug resistant HIV-1 RTs. Adenosine β-triphosphate (1) and AZT β-triphosphate (2) completely inhibited the DNA polymerase activity of wild type, the NRTI multi resistant, and non-nucleoside RT inhibitors (NNRTI) resistant HIV-1 RT at 10 nM, 10 μM, and 100 μM, respectively. During HIV-1 replication, the viral RNA genome is reverse transcribed into an integrated competent double stranded DNA by the virally encoded multifunctional enzyme reverse transcriptase (RT). 1 HIV-1 RT remains a prime target for continued development of antagonists to inhibit virus replication and stem the devastating consequences of AIDS. HIV-1 RT is a heterodimeric enzyme composed of 66 and 51 kD subunits (p66 and p51) possessing RNA-and DNA-dependent DNA polymerase and RNase H activities. 2 DNA polymerase activity is essential for the synthesis of a RNA:DNA heteroduplex from the single stranded viral RNA genome. RNase H hydrolyzes the RNA strand of the RNA:DNA heteroduplex generated during reverse transcription and creates the primer for plus strand
Improving Nucleoside Diphosphate Kinase for Antiviral Nucleotide Analogs Activation
Journal of Biological Chemistry, 2002
Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3deoxy-3-azidothymidine (AZT), 2,3-didehydro-2,3dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10 4 lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-Å resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R p -␣-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3-azido group with the engineered serine.
KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis
Antiviral Research, 2005
We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis.
Antiviral Research, 2006
Nucleoside antiretroviral agents are chiral small molecules that have distinct advantages compared to other classes including long intracellular half-lives, low protein binding, sustained antiviral response when a dose is missed, and ease of chemical manufacture. They mimic natural nucleosides and target a unique but complex viral polymerase that is essential for viral replication. They remain the cornerstone of highly active antiretroviral therapy (HAART) and are usually combined with non-nucleoside reverese transcriptase and protease inhibitors to provide powerful antiviral responses to prevent or delay the emergence of drug-resistant human immunodeficiency virus (HIV). The pharmacological and virological properties of a selected group of nucleoside analogs are described. Some of the newer nucleoside analogs have a high genetic barrier to resistance development. The lessons learned are that each nucleoside analog should be treated as a unique molecule since any structural modification, including a change in the enantiomeric form, can affect metabolism, pharmacokinetics, efficacy, toxicity and resistance profile.
Antimicrobial Agents and Chemotherapy, 2011
To investigate the mechanism of the antiviral activity, the active metabolites of HPMPC and HPMPA were studied for their effects on reactions catalyzed by HIV-1 RT. Incorporation of HPMPC and HPMPA into a DNA primer strand resulted in multiple inhibitory effects exerted on the enzyme and showed that neither compound acts as an absolute chain terminator. Further, inhibition of HIV-1 RT also occurred when these drugs were located in the template strand. These results indicate that HPMPC and HPMPA inhibit HIV-1 by a complex mechanism and suggest that this class of drugs has a broader spectrum of activity than previously shown.
Inhibition of HIV-1 reverse transcriptase by 5′-triphosphates of 5-substituted uridine analogs
Antiviral Research, 1989
The 5'-triphosphates of some 5-substituted 2'-deoxyuridine analogs were investigated for their effects on purified recombinant reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) as well as cellular DNA polymerase et. The triphosphates were competitive inhibitors of the viral enzyme with dTrP as the variable substrate and poly(rA)oligo(dT) as template, and preferentially inhibited the viral polymerase. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the reverse transcriptase, gave nPrearaUTP > nPrdUTP > EtdUTP > nPredUTP > HMdUTP > CEdUTP. Although nPredUTP was less inhibitory than nPrearaUTP under conditions of competitive inhibition, nPredUTP caused a time-and concentration-dependent inactivation of reverse transcriptase activity when preincubated with template. This inactivation was not reversed by excess dTTP. The decrease in template-primer activity did not occur with nPrearaUTP, but was shown with the chain-terminating 5'-triphosphates of 3'-fluoro-and 3'-azidothymidine. As nPredUTP, but not nPrearaUTP, was an alternative substrate, shown by the ability to support DNA synthesis in absence of competing substrate, the incorporation of nPredUTP into the primer-template apparently leads to increased inhibition of the enzyme. Human immunodeficiency virus; Reverse transcriptase; Nucleoside analog; Chemotherapy