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Nucleic Acids Research Advance Access originally published online on July 10, 2007
Nucleic Acids Research 2007 35(14):4895-4904; doi:10.1093/nar/gkm479
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Nucleic Acids Research, 2007, Vol. 35, No. 14 4895-4904
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Enantioselectivity of human AMP, dTMP and UMP-CMP kinases

Julie A.C. Alexandre1, Béatrice Roy2, Dimitri Topalis1, Sylvie Pochet3, Christian Périgaud2 and Dominique Deville-Bonne1,*

1Laboratoire d’Enzymologie Moléculaire, FRE 2852-CNRS-Université Paris 6, 4, place Jussieu, 75005 Paris 2Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Universités Montpellier 1 et 2, case courrier 1705, Bâtiment Chimie 17, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5 and 3Unité de Chimie Organique, URA CNRS 2128, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex15, France

*To whom correspondence should be addressed. Tel: +33 1 44 27 59 93, Fax: +33 1 44 27 59 94; Email: ddeville{at}ccr.jussieu.fr

Received April 27, 2007. Revised May 30, 2007. Accepted May 31, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Recent developments indicate that L-nucleosides and L-nucleoside analogues are potent anti-viral, anti-tumour and even anti-malarial agents (1–4). The L-derivative, lamivudine (ß-L-2',3'-dideoxy-3'-thiacytidine, 3TC), has been approved for treating both HIV and HBV, while emtricitabine (ß-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine, FTC) is used in HIV therapy. Telbivudine (ß-L-thymidine, L-dT) was recently approved for treating hepatitis B by the US Food and Drug Administration. Many L-nucleosides are currently in advanced clinical trials for the treatment of a variety of virus diseases. These include clevudine [1-(2-fluoro-5-methyl-ß-L-arabinofuranosyl)uracil, L-FMAU], elvucitabine (2',3'-didehydro-2',3'-dideoxy-ß-L-5-fluorocytidine, ß-L-d4FC), valtorcitabine (val-ß-L-2'-deoxycytidine, val-L-dC), pentacept (2',3'-didehydro-2',3'-dideoxy-3'-fluoro-ß-L-cytidine, ß-L-3'-Fd4C) and ß-L-2'-Fd4C (2',3'-didehydro-2',3'-dideoxy-2'-fluoro-ß-L-cytidine). Most L-enantiomers considered for treating virus diseases have similar activities to their D-counterparts, but are less sensitive to degrading enzymes and have better safety profiles (5,6). These properties are due mainly to the enantioselectivity of the enzymes that interact with these substrates in vivo (7). L-nucleosides must be phosphorylated by cellular or viral kinases before they can reach the targeted virus enzymes. The enantioselectivity of the enzymes involved in nucleoside de novo synthesis and salvage pathway is governed by no general rule. Each individual metabolic enzyme must therefore be studied. Among the four deoxyribonucleoside kinases in human cells, cytosolic thymidine kinase 1 (TK1) is strictly enantioselective while mitochondrial thymidine kinase 2 (TK2) is less specific (8–10). Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are poorly enantioselective in vitro (9,11–12). The phosphorylation of nucleoside monophosphates and their analogues to their diphosphate derivatives is then carried out by NMP kinases in both de novo and salvage pathways. The NMP kinases in human cells include one dTMP kinase, one UMP-CMP kinase, six isoenzymes of adenylate kinase and several guanylate kinases (13). Only the abilities of hUMP-CMP and dTMP kinases to phosphorylate some L-deoxynucleoside monophosphate analogues has been studied to date (14–15). Finally, NDPK is strictly enantioselective (16). The final step can be carried out by phosphoglycerate kinase, which has a broad substrate specificity (17–18). Other ATP-synthesizing enzymes such as creatine kinase can also be involved in this step (19). Triphosphorylated L-derivatives can interact with viral polymerases, acting as competitive inhibitors or alternate substrates, usually leading to chain termination. Viral polymerases incorporate L-derivatives more readily than do the human ones (20–21). Nucleoside degrading enzymes, such as nucleoside deaminases and phosphorylases, tend to be strictly selective for D-nucleotides although only limited data is available (11,22). The lack of degradation of L-nucleosides has however been demonstrated in mice, where L-nucleosides were gradually excreted in the unchanged form following intraperitoneal administration (5). 5'-Nucleotidases also seem to be moderately to highly enantioselective (7,23). The ribonucleotide reductases, which catalyze the de novo conversion of ribonucleotides to 2'-deoxyribonucleotides, are also key enzymes in DNA replication and repair. In vitro, these enzymes are inhibited by 2'-azido-2'-deoxynucleoside 5'-diphosphates (24–27). However, N3-D-dUrd is not cytotoxic due to its poor intracellular phosphorylation. The mechanism of enzyme inactivation and the enzymatic monophosphorylation of the parent nucleosides has been thoroughly studied (24–25). More recently, we have shown that ribonucleotide reductase is enantiospecific with respect to the natural configuration of the sugar moiety (26–27). This report examines the phosphorylation of the ß-D and ß-L stereoisomers of 2'-azido pyrimidine monophosphates by human UCK, TMPK and AKs and characterizes the enantioselectivity and the possible cross-activities of these three human kinases for natural (d)NMP (Figure 1).


Figure 1
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  		Figure 1. Structures of ß-L and ß-D (deoxy)ribonucleoside monophosphates (Base = adenine, thymine, uracil, cytosine).

 
Part of this work was presented during the XVIIth Round Table for Nucleosides, Nucleotides and Nucleic Acids in Bern, in September 2006.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Natural D-nucleotides were purchased from Sigma chemicals (St Louis, MO, USA). MABA-dTDP was synthesized as described (28).

Synthesis of L-nucleoside 5'-monophosphates
L-dA, L-dU, L-dC and L-dT were a generous gift from Idenix Pharmaceuticals (http://www.idenix.com). L-ribonucleosides were synthesized starting from L-ribose following usual procedures. The azidonucleosides were synthesized as previously described (26). L-Nucleoside 5'-monophosphates (L-NMP) were prepared by selective 5'-phosphorylation of the corresponding L-nucleosides with POCl3 in triethylphosphate (29). L-NMPs were purified on DEAE-Sephadex A-25 (elution: linear gradient of TEAB pH 7.6 from 10 to 300 mM) followed by RP18 chromatography (elution: water to methanol 50%). The triethylammonium counter ions were exchanged for sodium by passing the nucleotide solution through a DOWEX-AG 50WX2-400 column. Yields were 43–80%. The structures and purities of L-NMP were assessed by nuclear magnetic resonance (1H, 13C, 31P), fast-atom-bombardment MS, UV spectroscopy, HPLC and polarimetry. In some cases, it was necessary to separate the nucleoside 5'-monophosphate from traces of nucleoside 3'-monophosphate. This was done by semi-preparative HPLC using Hypercarb®, a porous graphite carbon stationary phase (30).

The physico-chemical properties of the 2'-azido-2'-deoxy D-nucleotides were identical, except for the Formula value, to those of the corresponding 2'-azido-2'-deoxy L-nucleotides.

N3-L-dUMP: Formula (c 1.1, MeOH); 1H NMR (D2O, 300 MHz) {delta} 7.96 ({delta}, 1H, J = 8.1 Hz, H–6), 5.93 (d, 1H, J = 5.3 Hz, H–1'), 5.87 (d, 1H, J = 8.1 Hz, H5), 4.48 (pt, 1H, J = 5.1 Hz, H3'), 4.30 (pt, 1H, J = 5.4 Hz, H2'), 4.16 (sl, 1H, H4'), 4.04–3.90 (m, 2H, H5', H5''); 13C NMR (D2O, 300 MHz) {delta} 166.2, 151.6, 141.6, 102.7, 86.9, 84.4, 70.3, 65.4, 63.2; 31P NMR (D2O, 300 MHz) {delta} + 2.47; UV (H2O) {lambda}max 260 nm ({varepsilon} 9400); MS FAB + m/z 372 (M + H)+, 350 (M – Na + 2H)+. N3-D-dUMP: Formula – 29 (c 1.1, MeOH). N3-L-dCMP: Formula (c 1, H2O); 1H NMR (D2O, 300 MHz) {delta} 8.07 (d, 1H, J = 7.6 Hz, H–6), 6.14 (d, 1H, J = 7.6 Hz, H5), 6.06 (d, 1H, J = 4.5 Hz, H–1'), 4.54 (pt, 1H, J = 5.4 Hz, H3'), 4.34 (pt, 1H, J = 5.1 Hz, H2'), 4.26–4.22 (m, 1H, H4'), 4.16–4.04 (m, 2H, H5', H5''); 13C NMR (D2O, 300 MHz) {delta} 166.1, 157.3, 141.3, 96.6, 87.7, 83.5, 69.8, 66.0, 62.9; 31P NMR (D2O, 300 MHz) {delta} + 2.15; UV (H2O) {lambda}max 269 nm ({varepsilon} 10 500); MS FAB + m/z 371 (M + H)+, 349 (M – Na + 2H)+. N3-D-dCMP: Formula (c 1.1, H2O).

Bacterial production and purification of His-tagged NMP kinases
Human UCK, TMPK, AK1 and AK2 were produced in Escherichia coli as recombinant proteins as previously reported (15,31, Topalis, personal communication). E. coli Rosetta (DE3)pLysS cells transformed with the appropriate expression plasmid were grown at 37°C in LB medium supplemented with 34 µg·ml–1 chloramphenicol and 50 µg·ml–1 kanamycin. Gene expression was induced by adding 0.5 mM IPTG when the absorbance at 600 nm reached 0.8, and cells were grown at 30°C for a further 3 h. The cells were harvested, lysed by sonication and centrifuged at 5000 r.p.m., 8°C, for 30 min. The supernatant was loaded onto a Ni-NTA column (Qiagen, Germany) equilibrated with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH = 8.0). The column was washed with lysis buffer and the proteins eluted with a linear gradient of imidazole, (0–250 mM). The fractions containing the enzyme were pooled and dialysed against dialysis buffer (50 mM Tris-HCl pH = 7.4, 20 mM NaCl, 1 mM DTT, 50% glycerol). SDS-PAGE indicated that the protein was over 95% pure.

Enzymatic assays
The activities of the NMP kinases were followed by a coupled spectrophotometric assay (32). Assays were carried out at 37°C in the following reaction mixture (total volume: 140 µl): 50 mM Tris-HCl, pH = 7.4, 50 mM KCl, 5 mM MgCl2, 1 mM ATP, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 1 mM DTT, 4 U·µl–1 pyruvate kinase, 4 U·µl–1 lactate dehydrogenase. As the NDP kinase from Dictyostelium discoideum is strictly enantioselective, this enzyme was only added (4 U·µl–1) for assays with a D-enantiomer substrate (32). The reaction was started by adding the (d)NMP or (d)NMP analogue and the decrease in absorbance at 340 nm was measured. The kinase concentrations were 4 nM to 8 µM in order to measure initial rates below 0.2 {Delta}A/min. For inhibition studies, the inhibitor was added after the enzyme, the reaction was then started by adding the substrate(s). The results were analysed using the KALEIDAGRAPH software. Assays were carried out in duplicate or triplicate.

Only 0.2 mM ATP was added in the reaction mixture for studies on the inhibition of hAK1 and hAK2. Inhibition was studied at three inhibitor concentrations: 0, 400 and 600 µM L-AMP for inhibiting the phosphorylation of D-AMP by hAK1, and 0, 38 and 134 µM L-AMP for inhibiting the phosphorylation of D-dAMP by hAK2. The inhibitor was added immediately after the enzyme, and adding the substrate started the reaction. Data were plotted on double reciprocal plots and the KI values were determined by plotting the slopes against the concentrations of inhibitor.

Fluorescence assays
The dissociation constants of hTMPK for both dTMP enantiomers were measured using a fluorescence assay (28). Briefly, the binding of the fluorescent probe MABA-dTDP to hTMPK in buffer T (50 mM Tris-HCl, pH = 7.5, 5 mM MgCl2, 50 mM KCl and 5% glycerol) resulted in a 190% increase in fluorescence intensity (excitation at 340 nm and emission at 430 nm with emission and excitation slits at 2 and 4 nm, respectively). The binding constant was determined by titration of the fluorophore with the enzyme (KD = 6 µM). Competition titrations with D- or L-d(T/U)MP were done by mixing MABA-dTDP (6 µM) with the enzyme (6 µM), so that, as recommended, about half of the fluorophore was enzyme-bound at the start of the experiment (33). Upon addition of increasing amounts of ligand, the fluorescence probe was gradually displaced from the enzyme active site and the resulting decreases in fluorescence were monitored. The total specific signal was determined at the end of each experiment by adding excess dTDP. The data were corrected for dilution and plotted: IC50 values were obtained at half-displacement. The IC50 values are related to the dissociation constants for the ligand, KD, and for the fluorophore MABA-dTDP, Formula , by the following equation (33):

Formula 1 (1)
where B is the initial concentration of bound MABA-TDP, A is the total concentration of MABA-dTDP and P is the total concentration of kinase (considered to be monomers).

Modelling studies
L-nucleotides and nucleotides analogues were drawn on Molsoft (http://www.molsoft.com, 2D to 3D converter) and Smiles Translator (http://cactus.nci.nih.gov/services/translate/) to produce the PDB files. The conformation of the sugar ring was checked by visualizing the molecules within the PyMOL graphic system and, if necessary, modified to the ß-L or ß-D configuration, as desired (34). Docking of the L-nucleotides and nucleotide analogues was performed using ArgusLab software (35). The binding site was defined from the coordinates of the ligand in the original PDB files 2UKD for hUCK and 1E2D for hTMPK. Docking precision was set to ‘high’ and the ‘flexible ligand docking’ mode was used for each docking run. Resulting complexes were visualized with the PyMOL graphic system and the diagrams for each nucleotide analogue/NMP kinase model obtained were drawn using Chemdraw CambridgeSoft (ChemDraw, CambridgeSoft Corporation, USA, http://www.camsoft.com).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Strict stereospecificity of recombinant human AMP kinases 1 and 2
The activities of the major adenylate kinases hAK1 (cytosolic) and hAK2 (in the inter-membrane space of mitochondria) were measured in vitro as a function of the D- and L-(d)AMP concentrations as well as of pyrimidine nucleoside monophosphates. Human AK1 and AK2 were both specific for the D-enantiomers. They did not phosphorylate L-(d)NMP, except for hAK2 having very little action on L-dAMP corresponding to a ‘catalytic efficiency’ about 104 smaller than for the D-stereoisomer (Table 1). The rate of D-(d)AMP phosphorylation increased with the substrate concentration, as expected for Michaelis curves. The maximum rate Vmax of D-AMP phosphorylation by hAK2 was not reached due to substrate inhibition at concentrations above 0.3 mM (data not shown). This inhibition is generally attributed to unproductive binding of the ligand at the same site or at a secondary site. The reaction rates for hAK2 were slightly below those for hAK1 but were almost compensated for by the KM values, resulting in somewhat similar catalytic efficiencies as might be expected from the similarities of their active site sequences. D-dAMP was a slightly poorer substrate than D-AMP for both hAK1 and hAK2. The deoxyribonucleotides, D-dCMP and D-dUMP, were not substrates for the AKs although these enzymes phosphorylated the pyrimidine nucleotides, D-CMP and D-UMP, to a minor extent (Table 1) (15).

L-AMP was bound by both enzymes. It competitively inhibited the phosphorylation of D-AMP and D-dAMP by hAK1 and hAK2, with an estimated KI of 200 µM for hAK1 and 18 µM for hAK2 (data not shown). D-dAMP was used because the mitochondrial AK2 was inhibited by excess D-AMP. The binding of L-nucleotides to the AMP site of hAK1 and hAK2 was thus unproductive. Additionally, L-(d)AMP was also not a substrate for hUCK or hTMPK (data not shown).


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  		Table 1. Catalytic parameters for the (d)AMP enantiomers with human AMP kinases 1 and 2

 
Despite this poor phosphorylation, L-dA reduced the virus load in vivo in the woodchuck model of chronic B hepatitis although less efficiently than L-dC or L-dT (6). The other isoenzymes of AK, i.e. hAK3, 4, 5 and 6 could contribute to the phosphorylation of L-(d)AMP and explain this in vivo activity (36–37).

Relaxed stereospecificity of recombinant human dTMP kinase
The recent approval of L-dT for treating hepatitis B has prompted us to study the phosphorylation of L-dTMP by hTMPK. The catalytic efficiency of the enzyme for L-dTMP was around 0.7% that D-dTMP (Table 2). The catalytic turnover number kcat was 10 times smaller than that for D-dTMP, while the KM was 20-fold higher, resulting in a lower kcat/KM (about 900 M–1s–1) (Figure 2A, Table 2). L-dTMP also gave rise to substrate inhibition at 1 mM and the KI value was estimated to be 5 mM (Table 2). D-dUMP was a good substrate with an efficiency in the 104 M–1s–1 range, as already reported (38), but the phosphorylation of L-dUMP very slow (kcat/KM = 60 M–1 s–1).


Figure 2
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  		Figure 2. Reaction of human dTMP kinase with the natural nucleoside monophosphates dTMP and dUMP and their corresponding L-enantiomers. (A) Saturation curves of dTMP kinase with (open circle) D-dTMP, (filled circle) L-dTMP, (open triangle) D-dUMP and (filled triangle) L-dUMP. The experiments were carried out in the presence of 2 mM ATP and 2 mM Mg2+ using the standard coupled assay. The quantity of enzyme used in each experiment was: 0.18 µM for D-dTMP; 3 µM for L-dTMP; 0.12 µM for D-dUMP and 5 µM for L-dUMP. The KM and kcat values obtained by fitting to a hyperbole are shown in Table 1. The reaction rates v for D-dUMP and L-dTMP as substrates [S] were best fitted with Equation (2). v = (V • [S])/(KM + [S] + ([S]2 / KI)) (2) (B) Fluorescence competition assays of different substrates with MABA-dTDP bound to human dTMP kinase. The fluorescence of MABA-dTDP (6 µM) was monitored at 430 nm (excitation wavelength at 340 nm, excitation slit = 2 nm, emission slit = 4 nm) in the presence of 6 µM enzyme in T buffer resulting in the binding of 40% of the fluorophore. The complex was titrated with (open circle) D-dTMP, (filled circle) L-dTMP and (open triangle) D-dUMP. The IC50 values were 4.6, 82 and 260 µM respectively. The dissociation constants KD calculated according to Equation (1) were 2 µM, 45 µM and 0.15 mM, respectively.

 

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  		Table 2. Catalytic parameters for the dTMP and dUMP enantiomers with human TMP kinase

 
The relative binding constants for the L- and D-isomers of dTMP were measured using the fluorescent competition assay based on the fluorescent probe MABA-dTDP (28). The dissociation constants for D-dTMP and L-dTMP from hTMPK were 2 µM and 45 µM, respectively (Figure 2B). The KD ratio was 22, comparable to the KM ratio, i.e. 19 (Table 2). Using the same assay, the nucleosides, D- and L-dT, also competed with MABA-dTDP and had KD values of 40 and 450 µM, respectively, indicating that the L-nucleoside also binds to the active site less efficiently than its D-counterpart. The affinity of the enzyme for L-dTMP (KD = 45 µM), was greater than its affinity for D-dUMP (KD = 150 µM), which is a good substrate of the enzyme (Figure 2B).

Activity of recombinant human UMP-CMP kinase with D- and L-nucleoside monophosphates
L-dUMP and L-dCMP were substrates of hUCK, but they were phosphorylated much more slowly than were the natural nucleotides, despite the KM of the enzyme being slightly more favourable for the L-compounds. The KM values were 1.3 mM for D-dUMP and 1.0 mM for D-dCMP, compared to 0.70 mM for L-dUMP and 0.73 mM for L-dCMP (Figure 3, Table 3). But the reaction rate was 20 times slower for L-dUMP than for D-dUMP and 100 times slower for L-dCMP than for its D-counterpart. L-CMP was also phosphorylated by hUCK but it was a very poor substrate for this enzyme, with a catalytic efficiency of 100 M–1 s–1, 10 times lower than that for L-dCMP and 104 times lower than for D-CMP. This decreased efficiency is due to a much lower kcat, as the KM was similar to that for L-dCMP (0.75 mM). Excess of L-CMP was inhibitory, unlike the natural substrate. The L-derivate that was the best substrate for hUCK was L-dCMP, but it was far less efficient than the monophosphate form of the antiviral analogue L-3TC. L-3TCMP is indeed phosphorylated to L-3TCDP by hUCK with a catalytic efficiency kcat/KM of 2.8 x 105 M–1 s–1 (15).


Figure 3
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  		Figure 3. Reaction of human UMP-CMP kinase with the enantiomers of (d)CMP and (d)UMP and their 2'-azido-derivatives. (A) Saturation curves of hUMP-CMP kinase as a function L-derivatives as substrates: (filled circle) L-dCMP, (filled triangle) L-dUMP and (cross mark) L-CMP obtained with respectively 0.4, 1 and 7.6 µM of hUCK in the reaction mixture. (B) Saturation curves of hUMP-CMP kinase with the D-enantiomers of (open circle) dCMP and (inverted triangle) N3-D-dCMP. The quantity of enzyme used was 4 nM for dCMP and 0.8 nM for N3-D-dCMP. The dash line represents the saturation curve with D-CMP obtained with 4 nM hUCK as shown in (15) and fitted to Equation (2). (C) Saturation curves of hUMP-CMP kinase with the D-enantiomers of (open triangle) dUMP and (filled diamond) N3-D-dUMP. The quantity of enzyme used was 4 nM for dUMP and N3-D-dUMP. The dash line represents the saturation curve with D-UMP obtained with 4 nM hUCK as shown in (15) and fitted to Equation (2).

 

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  		Table 3. Catalytic parameters for the (d)CMP, (d)UMP enantiomers and their 2'azido analogues with human UMP-CMP kinase

 
Activity of recombinant human UMP-CMP kinase with 2'-azido-2'-deoxynucleotides
The activity of recombinant hUCK was evaluated using the nucleoside analogues, N3-D-dCMP and N3-D-dUMP (Figure 3). Both compounds were substrates of the enzyme and followed Michaelis–Menten kinetics. High concentrations did not give rise to substrate inhibition of hUCK, unlike the natural substrates, D-CMP and D-UMP. The KM values were 0.36 mM for N3-D-dCMP and 0.9 mM for N3-D-dUMP compared to 20 µM for D-CMP and 50 µM for D-UMP (Table 3). Despite the higher KM values, the catalytic efficiencies for phosphorylation of the 2'-azido derivatives were in the same order of magnitude (105–106) as those for the natural substrates of the enzyme. The reaction rate with N3-D-dUMP was similar to that with D-UMP (Figure 3, Table 3). The reaction rate of hUCK with N3-D-dCMP was almost 4 times faster than that with D-CMP, demonstrating that N3-D-dCMP is currently the best-known substrate for hUCK (Figure 3B). Other enzymes hAK1, hAK2 and hTMPK did not significantly phosphorylate these substrates.

The enantioselectivity of hUCK for 2'-azido-2'-deoxynucleoside monophosphates was also evaluated. The KM value for N3-L-dCMP was increased (1.2 mM) while the Vmax was lower (1.1 U·mg–1), leading to a 4000-fold lower catalytic efficiency than for the D-enantiomer (Table 3). N3-L-dUMP was also a poor substrate of the enzyme, with a KM of 1.3 mM and a kcat of 0.34 s–1(Table 3). The 2'-azido group was thus only favourable for the D-analogues. Overall, in the L-series, the pyrimidine 2'-deoxyribonucleotides were better substrates than the pyrimidine ribonucleotides and the 2'-azido-2'-deoxyribonucleotides, indicating that 2'-substitutions may cause steric hindrance for L-NMP binding to hUCK.

Structural analysis of the enantioselectivity of human AMP, TMP and UMP-CMP kinases
The NMP kinases all have a highly conserved structure with a central CORE domain that contains an ATP binding loop (P-loop) and two mobile domains: an NMP binding domain and an LID domain, which provide the catalytic residues for the reaction (39). Both the NMP and LID domains are extremely mobile and undergo large ‘hinge bending’ motions (40). The same conformational changes are believed to occur in all NMP kinases when they switch from their opened to closed conformation upon substrate binding. The X-ray structure of hAK1 complexed with the bisubstrate inhibitor Ap5A was recently solved: it showed the interactions of AMP with the active site in the closed conformation (41). The structure of hTMPK complexed with various ligands has been thoroughly explored (42), but the structure of the free apoenzyme is not known. In contrast, the structure of hUCK is only known in its open conformation (43). Substrate-free hTMPK probably also exists in an opened conformation and substrate-bound hUCK probably adopts a closed conformation as does the homologous enzyme from Dictyostelium (44).

L-AMP and L-dTMP were tentatively docked in the closed conformation of hAK1 and hTMPK, and L-dCMP was docked in an enzyme model based on the Dictyostelium UCK (44). The docking of L-(d)AMP in hAK1 failed to provide a model (data not shown): this could be due to the rather specific interactions of D-AMP with the protein, which are not mediated by water molecules, thus limiting its capacity to accommodate other substrates. The docking of L-deoxypyrimidine monophosphates in hTMPK and UCK was more successful (Figures 4 and 5). The dTMP and CMP binding sites are represented as four interacting motifs: (i) the LID domain, (ii) the P-loop, (iii) the mobile part of NMP binding domain and (iv) the stable part of NMP binding domain. The binding of L-dTMP differed from that of D-dTMP, especially at the deoxyribose moiety (Figure 4). The H-bonds between the first layer residues and the thymine ring involved several water molecules, providing a ‘flexible’ NMP binding domain that readily accommodates modified substrates. The thymine ring was stacked onto Phe72 in L-dTMP binding, but it was not optimal, as the base was shifted through 15°. The interaction of the phosphate group with Mg2+ion and Asp15 in the P-loop, via a water molecule, was conserved, as was its interaction with Arg97 from the mobile NMP domain. However, the deoxyribose 3'OH, which interacts with the LID Gln157 via a water molecule in the D-dTMP/hTMPK complex, was modelled as being H-bonded to the NH2 of the highly conserved Arg45 of the immobile NMP domain in the L-dTMP/hTMPK model.


Figure 4
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  		Figure 4. Scheme of the acceptor-binding site of human dTMP kinase in the closed form: (A) with D-dTMP bound to the acceptor-binding site; (B) with L-dTMP modelled in the acceptor-binding site. LID (red), P-loop (pink), immobile NMP domain (black) and mobile NMP domain (blue).

 
The major interactions responsible for the binding of D-CMP to hUCK involved the base and phosphate (Figure 5). The interaction of Asn100 with the 4-amino group of the cytidine base played an important role in the base specificity and explains why D-(d)CMP is a better substrate than D-(d)UMP (43). The phosphate does not interact with the P loop as in hTMPK but strongly connects the LID domain (Arg134 and Arg140) to the immobile NMP domain (Arg39, Arg96 and Glu36) (Figure 5A). These residues still interacted with L-dCMP phosphate group in the L-dCMP/UCK model, except for Arg134 (Figure 5B). The cytidine moiety for both D-CMP and L-dCMP was H-bonded to Val63 and Asn100 from the mobile NMP domain, resulting in similar positioning of the base for both enantiomers. The L-nucleotide did not have the major interaction of the D-CMP 2'OH with the main chain Lys61 carbonyl, contained within the NMP mobile domain. L-dCMP was a better substrate for hUCK than was L-CMP, probably due to 2'-OH steric hindrance. However, the ring oxygen in L-dCMP interacted with Arg96, anchoring the sugar to the immobile NMP domain (Figure 5B).


Figure 5
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  		Figure 5. Scheme of the acceptor-binding site of human UMP-CMP kinase in the closed form. (A) with D-CMP bound to the acceptor-binding site. (B) with L-dCMP modelled in acceptor-binding site. (C) N3-D-dCMP bound to acceptor-binding site.

 
The model obtained with N3-D-dCMP bound to hUCK had favourable interactions as the azido group interacts with both Asp142 in the LID domain and the {alpha}-carbonyl of Gly60 in the mobile NMP domain (Figure 5C). The 2'OH of the ribonucleotide has been shown to contribute to the LID closure of hUCK by its interaction with the carbonyl of Lys61 located in the mobile NMP domain (43). This interaction also explains the higher affinity of the enzyme for D-ribonucleotides compared to D-deoxyribonucleotides. The replacement of 2'-hydroxyl by a bulkier azido group prevents complete closure of the LID, which results in higher KM values. However, the opening of the LID, which promotes product release, is proposed to be the rate-limiting step of the reaction catalyzed by hUCK. A recent NMR experiment on hyperthermophilic and mesophilic homologues of AK also suggested that the opening of the AMP binding and/or LID domains upon product release was the rate-limiting step influencing the catalytic turnover (45,46). The azido group might thus facilitate the LID opening or could connect the LID and mobile-NMP domains and synchronize their opening, explaining the higher rate of phosphorylation of N3-D-dCMP compared to D-CMP. Dynamic studies should help to understand these higher rates.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
We have compared the activities of the kinases on L- and D-nucleotides and shown that both hTMPK and hUCK have relaxed enantioselectivities for dNMP, while hAK 1 and 2 are strictly devoted to D-(d)NMP. The pyrimidine kinases phosphorylated only L-derivatives of the deoxy series. UCK was active with L-dCMP but far less efficiently than with L-3TCMP (15). hTMPK, hUCK, hAK1 and hAK2 all phosphorylated their respective D-(d)NMP with some minor cross-reactivity. For example, D-CMP was phosphorylated efficiently by hUCK (kcat/KM = 6.5 x 106 M–1 s–1) and more slowly by hAK1 and hAK2 (kcat/KM about 104 M–1 s–1). D-dUMP was a better substrate for hTMPK (kcat/KM = 2.8 x 104 M–1 s–1) than for hUCK (kcat/KM = 6 x 103 M–1 s–1). N3-D-dCMP was the best D-series substrate for hUCK with a kcat 4-fold higher than that for the natural substrate (D-CMP). This could be due to a tighter interaction between the LID domain and the NMP domain as explained by the structural analysis.

RNAi studies have shown that hTMPK is implicated in the activation of L-FMAU (47). There was generally a good correlation between kinetic studies and the antiviral effect, emphasizing the need for efficient cellular activation of the antiviral drug to its triphosphate form, as shown for L-3TC (15). The present study demonstrates the activity of hTMPK on L-dTMP, even if the catalytic efficiency was relatively low (kcat/KM = 900 M–1 s–1). L-dTMP had the highest relative phosphorylation efficiency of all the L-(d)NMP tested. The phosphorylation of L-dTMP correlates with studies on the intracellular metabolism of L-dT in HepG2 cells and primary cultures of human hepatocytes. The conversion of L-dTMP to L-dTDP clearly appears to be the rate-limiting step in both cell types (48). The first phosphorylation of L-dT is believed to be carried out by either hdCK or hTK2 with catalytic efficiencies of 1.6 x 104 M–1 s–1 and 1 x 107 M–1 s–1, respectively (9). The recent crystallization of dCK with L-3TC and troxacitabine shows how the nucleoside binding site of dCK can conserve all essential interactions with these analogues or L-dC and thus maintain productive substrate positioning for phosphoryl-transfer (49). Given the great similarity between dCK and hTK2, we can assume that they are similarly flexible for the productive positioning of L-dT, explaining the high catalytic efficiency. The binding of L-dTMP to hTMPK is not optimal and may be the rate-limiting step in the pathway. NDPK does not recognize L-(d)NDP as substrates and phosphoglycerate kinase is probably involved in the conversion of L-dTDP into L-dTTP, with a catalytic efficiency of 500 M–1 s–1 (18). From these in vitro data, the second and third phosphorylation steps have low catalytic efficiencies and appear rate-limiting in the activation pathway. This study demonstrated the relaxed enantioselectivity of hTMPK, which is most likely essential to the formation of L-dTTP in vivo and thus to the antiviral properties of L-dT.


   ACKNOWLEDGEMENTS
 
These studies were supported by Université Pierre-et-Marie-Curie-Paris 6 and the French Centre National de Recherche Scientifique (FRE 2852 and ANR-05-BLAN-0368-02) and also by the Association pour la Recherche contre le Cancer. We thank Dr Gilles Gosselin and Idenix Pharmaceuticals for providing L-deoxynucleosides. We also thank Prof. Michèle Reboud (FRE 2852 CNRS- Université Paris 6) for laboratory facilities and Dr Laurent Chaloin (Montpellier) for helpful discussions. We are grateful to Laurence Dugué (Institut Pasteur) for synthesizing MABA-dTDP. The English text was edited by Owen Parkes. Funding to pay the Open Access publication charges for this article was provided by the French Centre de Recherche Scientifique (ANT-05-0368-02).

Conflict of Interest Statement. None declared.


   Footnotes
 
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 

  1. Nair V, Jahnke TS. Antiviral activities of isometric dideoxynucleosides of D- and L-related stereochemistry. Antimicrob. Agents Chemother (1995) 39:1017–1029.[Abstract]

  2. Zemlicka J. Enantioselectivity of the antiviral effects of nucleoside analogues. Pharmacol. Ther (2000) 85:251–266.[CrossRef][ISI][Medline]

  3. Cheng YC. Potential use of antiviral L(-)nucleoside analogues for the prevention or treatment of viral associated cancers. Cancer Lett (2001) 162(Suppl.):S33–S37.[CrossRef][Medline]

  4. Mathe C, Gosselin G. L-Nucleoside enantiomers as antivirals drugs: a mini-review. Antiviral Res (2006) 71:276–281.[CrossRef][ISI][Medline]

  5. Jurovcik M, Holy A. Metabolism of pyrimidine L-nucleosides. Nucleic Acids Res (1976) 3:2143–2154.[ISI][Medline]

  6. Bryant ML, Bridges EG, Placidi L, Faraj A, Loi AG, Pierra C, Dukhan D, Gosselin G, Imbach JL, et al. AntiviralL-nucleosides specific for hepatitis B virus infection. Antimicrob. Agents Chemother (2001) 45:229–235.[Abstract/Free Full Text]

  7. Maury G. The enantioselectivity of enzymes involved in current antiviral therapy using nucleoside analogues: a new strategy? Antivir. Chem. Chemother (2000) 11:165–189.[ISI][Medline]

  8. Munch-Petersen B, Cloos L, Tyrsted G, Eriksson S. Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides. J. Biol. Chem (1991) 266:9032–9038.[Abstract/Free Full Text]

  9. Wang J, Choudhury D, Chattopadhyaya J, Eriksson S. Stereoisomeric selectivity of human deoxyribonucleoside kinases. Biochemistry (1999) 38:16993–16999.[CrossRef][Medline]

  10. Verri A, Priori G, Spadari S, Tondelli L, Focher F. Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L-nucleoside analogues. Biochem. J (1997) 328(Pt 1):317–320.[ISI][Medline]

  11. Shafiee M, Griffon JF, Gosselin G, Cambi A, Vincenzetti S, Vita A, Eriksson S, Imbach JL, Maury G. A comparison of the enantioselectivities of human deoxycytidine kinase and human cytidine deaminase. Biochem. Pharmacol (1998) 56:1237–1242.[CrossRef][ISI][Medline]

  12. Van Rompay AR, Johansson M, Karlsson A. Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases. Pharmacol. Ther (2003) 100:119–139.[CrossRef][ISI][Medline]

  13. Yan H, Tsai MD. Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv. Enzymol. Relat. Areas Mol. Biol (1999) 73:103–134.[ISI][Medline]

  14. Liou JY, Dutschman GE, Lam W, Jiang Z, Cheng YC. Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates. Cancer Res (2002) 62:1624–1631.[Abstract/Free Full Text]

  15. Pasti C, Gallois-Montbrun S, Munier-Lehmann H, Veron M, Gilles AM, Deville-Bonne D. Reaction of human UMP-CMP kinase with natural and analog substrates. Eur. J. Biochem (2003) 270:1784–1790.[ISI][Medline]

  16. Kreimeyer A, Schneider B, Sarfati R, Faraj A, Sommadossi JP, Veron M, Deville-Bonne D. NDP kinase reactivity towards 3TC nucleotides. Antiviral Res (2001) 50:147–156.[CrossRef][ISI][Medline]

  17. Krishnan P, Liou JY, Cheng YC. Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase. J. Biol. Chem (2002) 277:31593–31600.[Abstract/Free Full Text]

  18. Gallois-Montbrun S, Faraj A, Seclaman E, Sommadossi JP, Deville-Bonne D, Veron M. Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs. Biochem. Pharmacol (2004) 68:1749–1756.[CrossRef][ISI][Medline]

  19. Cihlar T, Chen MS. Identification of enzymes catalyzing two-step phosphorylation of cidofovir and the effect of cytomegalovirus infection on their activities in host cells. Mol. Pharmacol (1996) 50:1502–1510.[Abstract]

  20. Feng JY, Anderson KS. Mechanistic studies comparing the incorporation of (+) and (–) isomers of 3TCTP by HIV-1 reverse transcriptase. Biochemistry (1999) 38:55–63.[CrossRef][Medline]

  21. Feng JY, Murakami E, Zorca SM, Johnson AA, Johnson KA, Schinazi RF, Furman PA, Anderson KS. Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2',3'-dideoxy-5-fluoro-3'-thiacytidine-triphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase. Antimicrob. Agents Chemother (2004) 48:1300–1306.[Abstract/Free Full Text]

  22. Liou JY, Krishnan P, Hsieh CC, Dutschman GE, Cheng YC. Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase. Mol. Pharmacol (2003) 63:105–110.[Abstract/Free Full Text]

  23. Mazzon C, Rampazzo C, Scaini MC, Gallinaro L, Karlsson A, Meier C, Balzarini J, Reichard P, Bianchi V. Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for therapy. Biochem. Pharmacol (2003) 66:471–479.[CrossRef][ISI][Medline]

  24. Thelander L, Larsson B. Active site of ribonucleoside diphosphate reductase from Escherichia coli. Inactivation of the enzyme by 2'-substituted ribonucleoside diphosphates. J. Biol. Chem (1976) 251:1398–1405.[Abstract/Free Full Text]

  25. Salowe S, Bollinger JM Jr, Ator M, Stubbe J, McCraken J, Peisach J, Samano MC, Robins MJ. Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate. Biochemistry (1993) 32:12749–12760.[CrossRef][Medline]

  26. Roy B, Verri A, Lossani A, Spadari S, Focher F, Aubertin AM, Gosselin G, Mathe C, Perigaud C. Enantioselectivity of ribonucleotide reductase: a first study using stereoisomers of pyrimidine 2'-azido-2'-deoxynucleosides. Biochem. Pharmacol (2004) 68:711–718.[CrossRef][ISI][Medline]

  27. He J, Roy B, Perigaud C, Kashlan OB, Cooperman BS. The enantioselectivities of the active and allosteric sites of mammalian ribonucleotide reductase. FEBS J (2005) 272:1236–1242.[CrossRef][Medline]

  28. Topalis D, Collinet B, Gasse C, Dugue L, Balzarini J, Pochet S, Deville-Bonne D. Substrate specificity of vaccinia virus thymidylate kinase. FEBS J (2005) 272:6254–6265.[CrossRef][Medline]

  29. Yoshikawa M, Kato T, Takenishi T. A novel method for phosphorylation of nucleosides to 5'-nucleotides. Tetrahedron Lett (1967) 50:5065–5068.[CrossRef][Medline]

  30. Xing J, Apedo A, Tymiak A, Zhao N. Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry. Rapid Commun. Mass Spectrom (2004) 18:1599–1606.[CrossRef][ISI][Medline]

  31. Pochet S, Dugue L, Labesse G, Delepierre M, Munier-Lehmann H. Comparative study of purine and pyrimidine nucleoside analogues acting on the thymidylate kinases of Mycobacterium tuberculosis and of humans. Chembiochem (2003) 4:742–747.[CrossRef][ISI][Medline]

  32. Blondin C, Serina L, Wiesmuller L, Gilles AM, Barzu O. Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Anal. Biochem (1994) 220:219–221.[CrossRef][ISI][Medline]

  33. Chen Y, Gallois-Montbrun S, Schneider B, Veron M, Morera S, Deville-Bonne D, Janin J. Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases. J. Mol. Biol (2003) 332:915–926.[CrossRef][ISI][Medline]

  34. DeLano W. The PyMOL Molecular Graphics System (2002) Palo Alto, CA, USA: DeLano Scientific.

  35. Thompson M. (2004) ArgusLab edn. Seattle, WA: Planaria Software LLC.

  36. Noma T, Fujisawa K, Yamashiro Y, Shinohara M, Nakazawa A, Gondo T, Ishihara T, Yoshinobu K. Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix. Biochem. J (2001) 358:225–232.[CrossRef][ISI][Medline]

  37. Ren H, Wang L, Bennett M, Liang Y, Zheng X, Lu F, Li L, Nan J, Luo M, et al. The crystal structure of human adenylate kinase 6: an adenylate kinase localized to the cell nucleus. Proc. Natl Acad. Sci. USA (2005) 102:303–308.[Abstract/Free Full Text]

  38. Arima T, Akiyoshi H, Fujii S. Characterization of pyrimidine nucleoside monophosphokinase in normal and malignant tissues. Cancer Res (1977) 37:1593–1597.[Abstract/Free Full Text]

  39. Schulz GE, Schiltz E, Tomaselli AG, Frank R, Brune M, Wittinghofer A, Schirmer RH. Structural relationships in the adenylate kinase family. FEBS J (1986) 161:127–132.[ISI][Medline]

  40. Vonrheim C, Schlauderer GJ, Schulz GE. Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases. Structure (1995) 3:483–490.[Medline]

  41. Filippakopoulos P, Bunkoczi G, Jansson A, Schreurs A, Knapp S, Edwards A, Von Delft F, Sundstrom M. Crystal structure of human AK1A in complex with AP5A. Structural Genomics Consortium Oxford (2005) PDB code 1Z83.

  42. Ostermann N, Segura-Pena D, Meier C, Veit T, Monnerjahn C, Konrad M, Lavie A. Structures of human thymidylate kinase in complex with prodrugs: implications for the structure-based design of novel compounds. Biochemistry (2003) 42:2568–2577.[CrossRef][Medline]

  43. Segura-Pena D, Sekulic N, Ort S, Konrad M, Lavie A. Substrate-induced conformational changes in human UMP/CMP kinase. J. Biol. Chem (2004) 279:33882–33889.[Abstract/Free Full Text]

  44. Scheffzek K, Kliche W, Wiesmuller L, Reinstein J. Crystal structure of the complex of UMP/CMP kinase from Dictyostelium discoideum and the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-uridyl) pentaphosphate (UP5A) and Mg2+ at 2.2 A: implications for water-mediated specificity. Biochemistry (1996) 35:9716–9727.[CrossRef][Medline]

  45. Wolf-Watz M, Thai V, Henzler-Wildman K, Hadjipavlou G, Eisenmesser EZ, Kern D. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol (2004) 11:945–949.[CrossRef][ISI][Medline]

  46. Bae E, Phillips GN Jr. Roles of static and dynamic domains in stability and catalysis of adenylate kinase. Proc. Natl. Acad. Sci. USA (2006) 103:2132–2137.[Abstract/Free Full Text]

  47. Hu R, Li L, Degreve B, Dutschman GE, Lam W, Cheng YC. Behavior of thymidylate kinase toward monophosphate metabolites and its role in the metabolism of 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (Clevudine) and 2',3'-didehydro-2',3'-dideoxythymidine in cells. Antimicrob. Agents Chemother (2005) 49:2044–2049.[Abstract/Free Full Text]

  48. Hernandez-Santiago B, Placidi L, Cretton-Scott E, Faraj A, Bridges EG, Bryant ML, Rodriguez-Orengo J, Imbach JL, Gosselin G, et al. Pharmacology of beta-L-thymidine and beta-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus. Antimicrob. Agents Chemother (2002) 46:1728–1733.[Abstract/Free Full Text]

  49. Sabini E, Hazra S, Konrad M, Burley SK, Lavie A. Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase. Nucleic Acids Res (2007) 35:186–192.[Abstract/Free Full Text]


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