5',6'-nucleoside phosphonate analogues architecture: synthesis and comparative evaluation towards metabolic enzymes.

Nucleoside phosphonates have been designed as stable 5'-mononucleotide mimics and are nowadays considered a potent class of antiviral agents. Within cells, they must be metabolised to the corresponding diphosphate to exert their biological activity. In this process, the first phosphorylation step, catalysed by nucleoside monophosphate kinases (NMP kinases), has been proposed as a bottleneck.

Novel antiviral C5-substituted pyrimidine acyclic nucleoside phosphonates selected as human thymidylate kinase substrates.

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase.

Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: structural and catalytic properties.

Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement.

3'-(1,2,3-Triazol-1-yl)-3'-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure-activity investigations.

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3 -azido-3'-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3'-alpha-[1,2,3]triazol-3'-deoxythymidine analogs from AZT under Huisgen conditions are described.

Nucleoside diphosphate kinase and the activation of antiviral phosphonate analogs of nucleotides: binding mode and phosphorylation of tenofovir derivatives.

Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creatine kinase is significantly more active. The 1.

Structural basis for the specificity of thymidylate kinases from human pathogens: implications for nucleotide analogues activation.

Several human pathogens possess nucleoside or nucleotide kinases with large substrate specificity compared to their human counterparts. This phenomenon has been successfully exploited for the specific targeting of prodrugs such as Acyclovir against herpes virus. Combined structural and biochemical studies of these enzymes can thus provide essential information for the rational design of specific antimicrobial agents.

Mutational analysis of the CG recognizing DNA methyltransferase SssI: insight into enzyme-DNA interactions.

To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)-methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5'-CG-3'), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.

Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase.

Vaccinia virus thymidylate kinase, although similar in sequence to human TMP kinase, has broader substrate specificity and phosphorylates (E)-5-(2-bromovinyl)-dUMP and dGMP. Modified guanines such as glyoxal-dG, 8-oxo-dG, O(6)-methyl-dG, N(2)-ethyl-dG and N(7)-methyl-dG were found present in cancer cell DNA. Alkylated and oxidized dGMP analogs were examined as potential substrates for vaccinia TMP kinase and also for human TMP and GMP kinases.

Crystal structure of poxvirus thymidylate kinase: an unexpected dimerization has implications for antiviral therapy.

Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity.

Selective adenosine-5'-monophosphate uptake by water-compatible molecularly imprinted polymer.

Molecularly imprinted polymers (MIPs) were prepared for adenosine-5'-monophosphate (AMP), a substrate of AMP-activated protein kinase. The template molecule was formed by the vinylphenylboronate diester of adenosine on which 5'-free hydroxide was protected by tert-butyldimethylsilyl group in order to mimic the steric hindrance of the phosphate moiety of AMP. Molecular imprinting was performed by complexing acrylamide and the template in a highly cross-linked polymer.

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2.

Cross-metathesis mediated synthesis of new acyclic nucleoside phosphonates.

With the commercial availability of well-defined ruthenium metathesis catalysts which combine high stability and broad functional group compatibility, olefin metathesis is now routinely integrated in various syntheses. We will report here the overwhelming power and scope of cross-metathesis in the area of new acyclic nucleoside phosphonates. Scope and limitations of this approach, and especially the E/Z stereocontrol, are discussed on selected examples from our drug discovery group.

Alkyne-azide click chemistry mediated carbanucleosides synthesis.

Hitherto unknown 1,4-disubstituted-[1,2,3]-triazolo-4',4'-dihydroxymethyl-3'-deoxy carbanucleosides were synthesized based on a "click approach." Various alkynes were introduced on a key azido intermediate by the "click" 1,3-dipolar Huisgen cycloaddition. Their antiviral activities and cellular toxicities were evaluated on vaccinia virus.

Enantio-selectivity of human nucleoside monophosphate kinases.

Over recent years, there has been a renewed interest in the development of L-nucleosides as safe and efficacious drugs for the treatment of viral infections. Biological activity of these compounds requires phosphorylation to their triphosphate form, involving nucleoside monophosphate kinases in the second step. In order to characterize the activation pathway of L-nucleosides of the pyrimidine series, we studied the enantio-selectivity of human uridylate-cytidylate and thymidylate kinases.

Looking for new pyrimidine acyclic nucleotide analogues designed for phosphorylation by human UMP-CMP kinase.

Human UMP-CMP kinase is involved in the phosphorylation of nucleic acid precursors and also in the activation of antiviral analogues including cidofovir, an acyclic phosphonate compound that mimicks dCMP and shows a broad antiviral spectrum. The binding of ligands to the enzyme was here investigated using a fluorescent probe and a competitive titration assay. At the acceptor site, the enzyme was found to accommodate any base, purine and pyrimidine, including thymidine.

Enantioselectivity of human AMP, dTMP and UMP-CMP kinases.

L-nucleoside analogues such as lamivudine are active for treating viral infections. Like D-nucleosides, the biological activity of the L-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases.

Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the UMP-CMP binding site.

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.

Substrate specificity of vaccinia virus thymidylate kinase.

Anti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition.

Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases.

Amdoxovir [(-)-beta-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells.

Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs.

Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine L-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents.

Antiviral nucleoside analogs phosphorylation by nucleoside diphosphate kinase.

The reaction of NDP kinase was studied in vitro with several antiviral derivatives, using kinetic steady state and presteady state analysis. The enzyme is highly efficient with natural nucleotides but most of the analogs are slow substrates. The catalytic efficiency, also related to the affinity of the analog, is mainly dependent on the presence of a 3'-OH group on the ribose moiety.

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases.

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose.

Reaction of human UMP-CMP kinase with natural and analog substrates.

UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined.

Structural analysis of the activation of ribavirin analogs by NDP kinase: comparison with other ribavirin targets.

Ribavirin used in therapies against hepatitis C virus (HCV) is potentially efficient against other viruses but presents a high cytotoxicity. Several ribavirin triphosphate analogs modified on the ribose moiety were synthesized and tested in vitro on the RNA polymerases of HCV, phage T7, and HIV-1 reverse transcriptase. Modified nucleotides with 2'-deoxy, 3'-deoxy, 2',3'-dideoxy, 2',3'-dideoxy-2',3'-dehydro, and 2',3'-epoxy-ribose inhibited the HCV enzyme but not the other two polymerases.