Cellular phosphorylation of anti-HIV nucleosides. Role of nucleoside diphosphate kinase (original) (raw)
Related papers
Cellular phosphorylation of anti-HIV nucleosides
Journal of Biological …, 1996
Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho-and triphospho-derivatives of azidothymidine ...
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.
Pre-steady State of Reaction of Nucleoside Diphosphate Kinase with Anti-HIV Nucleotides
Journal of Biological Chemistry, 1998
The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s ؊1 and a K S of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3-OH in ddNTP causes a 10-fold increase in K D . Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in k cat values.
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.
Biochemistry, 1999
Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2′-position of the ribose, but the 3′-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3′-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, k cat decreases 15-200-fold from 1100 s -1 with TDP, and (k cat /K m ) NDP decreases from 12 × 10 6 to 10 3 to 5 × 10 4 M -1 s -1 , depending on the substrate. The poorest substrates are 3′-deoxyTDP and 3′-azido-3′-deoxyTDP, while the best modified substrates are 2′,3′-dehydro-3′-deoxyTDP and 3′-fluoro-3′-deoxyTDP. In a similar way, 3′-fluoro-2′,3′-dideoxyUDP was found to be a better substrate than 2′,3′-dideoxyUDP, but a much poorer substrate than 2′-deoxyUDP. (k cat /K m ) NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3′-fluoro-2′,3′-dideoxyUDP at 2.7 Å resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3′-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the -phosphate, which is the leaving group when a γ-phosphate is transferred to His122, hydrogen bonds to the 3′-hydroxyl group of the sugar; with 3′-fluoro-2′,3′-dideoxyUDP, the -phosphate hydrogen bonds to Asn119 and moves away from the attacking Nδ of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3′-position, these results are relevant to the role of NDP kinase in their cellular metabolism. a HA, human NDP kinase A; HB, human NDP kinase B; DR, human DR-nm23; H4, human Nm23-H4; Dd, Dictyostelium NDP kinase; Myxo, Myxococcus NDP kinase. kcat/Km values are expressed in M -1 s -1 . ∆∆G q values were calculated as described in the footnote of
3'-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity
Journal of Biological Chemistry, 1998
Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo-and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3-OH and the -O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3-phosphate, 5phosphoadenosine 3-phosphate (PAP) and adenosine 3-phosphate 5-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The K D of PAPS (10 M) is three times lower than the K D of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a K I in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-Å resolution. It shows a new mode of nucleotide binding at the active site with the 3-phosphate of PAPS located near the catalytic histidine, at the same position as the ␥-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.
Antiviral research, 2015
The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to de...
Nucleic Acids Research, 2006
L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-b-2 0 ,3 0 -dideoxy-3 0 -thiacytidine (3TC)-an approved anti-human immunodeficiency virus (HIV) agent-and troxacitabine (TRO)-an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D-and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L-and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.
Journal of Medicinal Chemistry, 2008
Biological molecules are predominantly enantioselective. Yet, currently two nucleoside analog prodrugs (3TC and FTC) with opposite chirality to physiological nucleosides are clinically approved for the treatment of HIV infections. These prodrugs require conversion to their tri-phosphorylated forms to achieve pharmacological activity. The first step in the activation of these agents is catalyzed by human deoxycytidine kinase (dCK). This enzyme possesses the ability to phosphorylate nucleosides of the unnatural L-chirality. To understand the molecular basis for the nonenantioselectivity of dCK we solved the crystal structures of the enzyme in complex with the Lenantiomer of its physiological substrate deoxycytidine and with the L-nucleoside analog FTC. These were compared to a structure solved with D-dC. Our results highlight structural adjustments imposed on the L-nucleosides, and properties of the enzyme endowing it with the ability to phosphorylate substrates with non-physiological chirality. This work reveals the molecular basis for the activation of L-nucleosides by dCK.