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. Herein, we report the synthesis of a series of ribonucleoside phosphonate derivatives isosteric to 5'-mononucleotides, with different degrees of flexibility within the 5',6'-C-C bond, as well as different polarities, through the introduction of hydroxy groups. The influence of these modifications on the capacity of the compounds to act as substrates for appropriate human NMP kinases, involved in nucleic acids metabolism, has been investigated. Low flexibility, as well as an absence of hydroxy groups within the ribose-phosphorus architecture, is critical for efficient phosphotransfer. Among the series of pyrimidine analogues, one derivative was shown to be phosphorylated by human UMP-CMP kinase, with rates similar to those of dUMP and even better than dCMP.

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. Several derivatives in the but-2-enyl- series 9d and 9e, with (E) but not with (Z) configuration, were efficient substrates for human thymidine monophosphate (TMP) kinase, but not for uridine monophosphate-cytosine monophosphate (UMP-CMP) kinase, which is in contrast to cidofovir. Human TMP kinase was successfully crystallized in a complex with phosphorylated (E)-thymidine-but-2-enyl phosphonate 9e and ADP. The bis-pivaloyloxymethyl (POM) esters of (E)-9d and (E)-9e were synthesized and shown to exert activity against herpes virus in vitro (IC(50) = 3 µM) and against varicella zoster virus in vitro (IC(50) = 0.19 µM), in contrast to the corresponding inactive (Z) derivatives. Thus, their antiviral activity correlates with their ability to act as thymidylate kinase substrates.

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. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.

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. Thirteen 3'-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K(m)/V(max) values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.

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. Here we studied the structural basis for the specificity of a thymidylate kinase from the poxvirus family. Poxvirus thymidylate kinase has unusual substrate specificity and can accept bulky analogues such as 5-bromo-vinyl-dUMP (BVdUMP). The 2 A crystal structure of the thymidylate kinase bound to this compound now gives the structural basis for its specific molecular recognition.

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.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved S145 (motif IV), R232 (motif VIII, QxRxR), and T313 (variable region, conserved TL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, ENV) and R230 (motif VIII, QxRxR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and R230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases.

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. Molecular models obtained from structure-based docking rationalized the enzymatic data. All tested nucleotides are found surprisingly substrates of vaccinia TMP kinase and also of human GMP kinase. Interestingly, O(6)-methyl-dGMP is the only analog specific for the vaccinia enzyme. Thus, O(6)-Me-dGMP could be useful for designing new compounds of medical interest either in antipoxvirus therapy or in experimental combined gene/chemotherapy of cancer. These results also provide new insights regarding dGMP analog reaction with human GMP kinase and their slow recycling by salvage pathway nucleotide kinases.

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 1.65 A x-ray structure of NDPK in complex with tenofovir mono- and diphosphate shows that the analogs bind at the same site as natural nucleotides, but in a different conformation, and make only a subset of the Van der Waals and polar interactions made by natural substrates, consistent with their comparatively low affinity for the enzyme.

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. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer.

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. MIPs were tested in batch experiments with aqueous samples of nucleotides and a number of parameters were investigated. The use of tetrabutylammonium hydroxide (TBAH) was necessary to obtain a rebinding of nucleotides on MIP. The adsorption of AMP was optimal in 5 mM ammonium acetate buffer solution pH 9.5 for 30 min, with 30 mM of TBAH. The imprinted polymer was selective for AMP towards others nucleotides or deoxi analogues.

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. A method for screening analogues based on their affinity for the UMP binding site was developed. The affinities of uracil vinylphosphonate derivatives modified in the 5 position were found similar to (d)UMP and (d)CMP and improved when compared to cidofovir.

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. None of the synthesized compounds exhibited a significant antiviral activity.

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.

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. We have therefore investigated the capacity of L-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only D-(d)AMP. L-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than D-dTMP. Both L-dUMP and L-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2'-azido derivatives of dUMP and dUMP while, unexpectedly, the 2'-azido-D-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of D-(d)NMP to their respective kinases could account for the differences in interactions of the L-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for L-dT.

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. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.

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. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.

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. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both D- and L-configuration. Human PGK demonstrated catalytic efficiencies in the 10(4)-10(5)M(-1)s(-1) range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in D- or L-configuration. In contrast, it was poorly active with natural pyrimidine D-nucleotides (less than 10(3)M(-1)s(-1)). Pyrimidine L-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their D-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, L-2'-deoxycytosine and L-2'-deoxythymidine.

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. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.

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. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.

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. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.