Introduction

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Ribavirin is a broad-spectrum antiviral agent discovered in 1972 (Witkowski et al., 1972). It is used in therapy against chronic hepatitis C virus (HCV) infection in combination with interferon  (McHutchison et al., 1998; Poynard et al., 1998). It is also of potential interest against poliovirus, Lassa fever virus, respiratory syncytial virus, and emerging viruses, such as West Nile virus, but the exact mode of action of ribavirin remains uncertain (Patterson and Fernandez-Larsson, 1990). It may be indirect, through the inhibition of cellular IMP dehydrogenase, resulting in a decrease of the guanine nucleotide pool. Alternatively, ribavirin may directly target the replication of RNA viruses, acting as an analog of purine nucleosides. Ribavirin 5'-triphosphate (RTP) is an inhibitor of the replicative RNA polymerases of influenza virus (Wray et al., 1985), poliovirus (Crotty et al., 2000), and vesicular stomatitis virus (Patterson and Fernandez-Larsson, 1990). It is a substrate for the polymerases of poliovirus (Crotty et al., 2000) and HCV (Maag et al., 2001). These enzymes incorporate ribavirin in the viral RNA facing either cytosine or uracil. Although the efficiency of incorporation is decreased by a factor in the 10⁴ to 10⁵ range compared with natural purine nucleotides, the analog is a powerful mutagen and the accumulation of replicative errors may explain its antiviral effect (Crotty et al., 2000).

Ribavirin transport into cells is probably mediated by nucleoside transporters (Jarvis et al., 1998; Patil et al., 1998). Intracellular phosphorylation is required for antiviral activity and must reach the 5'-triphosphate level for incorporation by RNA polymerases. Phosphorylated anabolites (ribavirin mono-, di-, and triphosphates) are observed in erythrocytes because of the action of adenosine kinase (Page and Conner, 1990; Homma et al., 1999) and other kinases of the salvage nucleotide pathway. These include nucleoside diphosphate (NDP) kinase, which produces the triphosphate form by transferring the -phosphate of a nucleoside triphosphate (usually ATP) via a covalent phosphohistidine intermediate (Parks and Agarwal, 1973). NDP kinase has a high catalytic efficiency and a broad specificity, taking as substrates all natural diphosphate nucleosides and deoxynucleosides. It also phosphorylates the diphosphate derivatives of several antiviral nucleoside analogs such as 3'-azidothymidine and 2',3'-dideoxy-2',3'-didehydrothymidine (d4T), but with a much lower efficiency (Bourdais et al., 1996). These analogs, which are chain terminators in DNA synthesis, lack a 3'-OH group. Because the latter has been shown to play a major role in catalysis by NDP kinase (Schneider et al., 1998), ribavirin, which contains an unmodified ribose, should be more efficiently activated.

A major problem with ribavirin in therapy is its high cellular toxicity. This is possibly related to the inhibition by ribavirin monophosphate (RMP) of IMP dehydrogenase, which catalyzes the oxidation of IMP into xanthosine monophosphate, the rate-limiting step of the de novo synthesis of guanine nucleotides. The structural and functional properties of IMP dehydrogenase have been studied extensively (Sintchak and Nimmesgern, 2000), and X-ray structures are available (Colby et al., 1999). RMP probably mimics IMP. With the aim of decreasing its toxicity and improving the antiviral properties of ribavirin, we investigate here the action of NDP kinase on phosphorylated ribavirin derivatives. The enzymatic analysis is supported by an X-ray structure of NDP kinase in complex with ribavirin triphosphate. We also prepared ribavirin analogs bearing modifications on the 2'-OH and/or the 3'-OH of the ribose moiety, study their properties as NDP kinase substrates and their ability to inhibit viral polymerases, HIV-1 reverse transcriptase and T7 RNA polymerase, as well as the HCV polymerase, the RNA polymerase of the hepatitis C virus.

	Materials and Methods

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Synthesis of Ribavirin Triphosphate Analogs. Analytically pure ribavirin was either a kind gift from Pr. Chris Meier (Hamburg, Germany) or was purchased from ICN Pharmaceuticals. 2',3'-Dideoxy-2',3'-didehydroribavirin (d4R), 2',3'-dideoxyribavirin (ddR), and 2',3'-anhydro-ribavirin (epoxyR) (Fig. 1) were synthesized from ribavirin according to the procedures described by Upadhya et al. (1990). 2'-Deoxyribavirin (2'dR) (Pochet and Dugué, 1998) and 3'-deoxyribavirin (3'dR) (Upadhya et al., 1990) were obtained in four steps by radical deoxygenation of appropriately bis-silylated ribavirin at position 2' and 3', respectively, as described for the preparation of the 3'-deoxyadenosine (Meier and Huynh-Dinh, 1991). Conversion of ribavirin and analogs into their corresponding triphosphate was performed according to the method of Ludwig and Eckstein (1989). Spectra from electrospray ionization mass spectrometry were recorded in the negative-ion mode. HPLC analyses were performed on a PerkinElmer series 200 unit using a reverse-phase analytical column (Uptisphere UPS HDO, C18, 120 Å, 4.6 × 250 mm, 5 µm; Interchim, Montluçon, France) with detection at 230 nm. The eluants used were acetonitrile/10 mM triethylammonium acetate buffer, pH 6.6, 1/19 (A) and acetonitrile/10 mM triethylammonium acetate buffer, pH 6.6, 3/17 (B). A linear gradient from A to B over 20 min was used with a flow rate of 1 ml/min. Characteristic data for the target compounds are as follows: ribavirin 5'-triphosphate: HPLC, 4.91 min; C₈H₁₅N₄O₁₄P₃ (483.98) m/z 483 (M-H); 2'-deoxyribavirin 5'-triphosphate: HPLC, 5.56 min; C₈H₁₅N₄O₁₃P₃ (467.98) m/z 467 (M-H); 3'-deoxyribavirin 5'-triphosphate: HPLC, 4.87 min; C₈H₁₅N₄O₁₃P₃ (467.98) m/z 467 (M-H); 2',3'-dideoxy-2',3'-didehydroribavirin 5'-triphosphate: HPLC, 5.99 min; C₈H₁₁N₄O₁₂P₃ (449.97) m/z 449 (M-H); 2',3'-dideoxyribavirin 5'-triphosphate: HPLC, 6.01 min, C₈H₁₅N₄O₁₂P₃ (451.99) m/z 451 (M-H); and 2',3'-anhydroribavirin 5'-triphosphate: HPLC, 5.79 min; C₈H₁₃N₄O₁₃P₃ (465.97) m/z 465 (M-H).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structures of ribavirin and derivatives

Expression and Purification of NDP Kinases. Human NDP kinase A was purified as a recombinant protein overexpressed in Esherichia coli (BL21-DE3) using plasmid pJC20, a kind gift from M. Konrad (Max Planck Institute, Göttingen, Germany), according to the procedure described by Schneider et al. (2000). The protein, stored at 20°C in Tris-HCl, pH 7.5, buffer containing 1 mM DTT, 20 mM KCl, and 50% glycerol was >97% pure as determined by SDS-polyacrylamide gel electrophoresis. The H122G single-mutant and the F64W-H122G double-mutant NDP kinases from Dictyostelium discoideum were overexpressed in E. coli (XL1-Blue) and purified as described previously for F64W NDP kinase (Schneider et al., 1998). Neither mutant had measurable NDP kinase activity. Protein concentration is expressed as 17 kDa subunits. It was determined either colorimetrically or using (for a 1 mg/ml solution) an absorbance coefficient A₂₈₀ = 0.55 for H122G and A₂₈₀ = 0.85 for F64W-H122G.

Crystallization of the Complex Ribavirin Triphosphate with NDP Kinase and Data Collection. H122G D. discoideum NDP kinase in complex with RTP was crystallized by transferring tiny crystals obtained with a higher concentration of PEG, to a drop containing 13% PEG 550 monomethyl ester, 100 mM Tris-HCl, pH 7.5, 30 mM MgCl₂, 10 mg/ml protein, and 20 mM RTP, over wells containing 26% PEG 550 monomethyl ester in the same buffer. The crystals belong to the trigonal space group P3₁21 with unit cell a = b = 71.7 Å, c = 153.8 Å. The asymmetric unit contains a trimer.

Fluorometric Binding Studies on NDP Kinase. The affinity of RTP analogs for NDP kinase was determined by following the variation of the intrinsic fluorescence upon nucleotide binding as described previously (Schneider et al., 1998). The fluorescence of the F64W-H122G mutant in T₁ buffer (50 mM Tris-HCl, 75 mM KCl, and 5 mM MgCl₂, pH 7.5) was measured at 330 nm with excitation at 310 nm (2-nm excitation slit and 4-nm emission slit) (PTI, New Brunswick, NJ). Successive aliquots of nucleotide were added to a 1 µM enzyme solution. The inner filter effect was negligible. Experimental binding curves were fitted to a quadratic equation after correction for dilution.

Stopped-Flow Kinetic Experiments and Analysis of Kinetic Results. Stopped-flow kinetic experiments were performed with a Hi-Tech DX2 (Salisbury, UK) microvolume stopped-flow reaction analyzer equipped with a high-intensity xenon lamp as described previously (Schneider et al., 1998). The excitation wavelength was 304 nm, with a 2-nm excitation slit and a 320-nm cutoff filter at the emission. Mixing was achieved in less than 2 ms. After mixing NDPK (1 µM) and NTP (10-500 µM), the intrinsic protein fluorescence was recorded for 10-200 s. In each experiment, 400 pairs of data were recorded, and the data from three or four identical experiments were averaged and fitted to a number of nonlinear analytical equations using the software provided by Hi-Tech. All curves fitted to single exponentials.

1

Expression and Purification of HCV 1b Polymerase. The NS5B gene encoding HCV 1b polymerase was PCR-amplified using the primers HCV-1b-BamHI forward (5'-GGCCGGATCCTCAATGTCCTACACATGGACA-3') and HCV-1b-SalI reverse (5'-GGCCGTCGACCTAGAGTTTAAGTTTGGATTTTAC-3') from a clinical HCV isolate serotype 1b to yield 55 NS5B 1b cDNA, which specifies an NS5B protein truncated at its C terminus by 55 amino acids. The amplified fragment was digested by BamHI and SalI, cloned into PQE-30 (Amersham Biosciences, Orsay, France), which specifies an N-terminal His₆ tag, and the resulting vector was used to transform BL21pDNAy E. coli cells. Bacteria were cultured at 37°C in Luria broth medium supplemented with 100 µg/ml ampicillin and 25 µg/ml kanamycin until A_{600 nm} reached 0.6. The temperature of the culture was then switched to 27°C, and expression was induced by the addition of 0.3 mM isopropyl -D-thiogalactoside for 2 h. Bacteria were harvested by centrifugation, washed twice in phosphate-buffered saline, and the pellet was stored at 80°C until use. Bacteria were lysed for 30 min on ice in 3 ml of buffer 1 (50 mM sodium phosphate, pH 7.5, and 20% glycerol) per gram of bacteria supplemented with 1 mg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. Lysates were diluted with 3 ml of buffer 2 (50 mM sodium phosphate, pH 7.5, 20% glycerol, 0.6 M NaCl, 10 mM -mercaptoethanol, 1.6% Igepal, 20 mM imidazole, and 7 µg of DNase) per gram of bacteria, left on ice for 30 min, and sonicated on ice. Lysates were centrifuged for 30 min at 75,000g, and the cleared lysates were loaded on a 1-ml Hi-Trap heparin column (Amersham Biosciences). After extensive washes in buffer 3 (50 mM NaPO₄, pH 7.5, 10% glycerol, 0.3 M NaCl, 5 mM -mercaptoethanol, and 10 mM imidazole), bound proteins were eluted with 3 ml of buffer 3 adjusted to 900 mM NaCl. Eluted proteins were then diluted with buffer 3 without NaCl to an NaCl concentration of 0.3 mM and applied to 1.5 ml of a nickel-nitrilotriacetic acid agarose column (QIAGEN, Hilden, Germany). The column was washed extensively before elution of the His₆-tagged HCV polymerase with buffer 3 containing 350 mM imidazole. The fractions containing the HCV polymerase were dialyzed against buffer 3 without imidazole and stored at 20°C.

Polymerase Assays. Polymerase activity was assayed by monitoring the formation of radiolabeled nucleic acid product absorbed onto DE-81 ion exchange paper discs. All radioactive nucleotides come from Amersham Biosciences. For HIV reverse transcriptase experiments, reactions were performed in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl₂, and 0.05% Triton X-100) at 37°C with a bacteriophage MS2 template (Roche, Meylan, France) annealed to the primer (5'-CTCGGTCAGCTACCGAGGAGA-3'; 20 nM primer/template), dATP, dCTP, and dGTP (10 µM each), decreasing amount of ribavirin or analogs (4 mM, 0.8 mM, 160 µM, 32 µM, 6.4 µM, and 1.3 µM), and 5 µM [³H]dTTP (0.12 µCi of [³H]TTP at 1 µCi/µl). Reactions were initiated by the addition of recombinant RT (50 nM). After 30 min, aliquots were withdrawn and spotted onto DE-81 paper discs. Filter paper discs were washed three times for 10 min in 0.3 M ammonium formate, pH 8.0, washed two times in ethanol, and dried. The radioactivity bound to the filter was determined using liquid scintillation counting.

	Results

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X-Ray Structure of Ribavirin Triphosphate Bound to NDP Kinase. D. discoideum NDP kinase has 60 and 58% sequence identity, respectively, with the major A and B isoforms of the human enzyme, also called Nm23-H1 and Nm23-H2 (Lacombe et al., 2000). All eukaryotic NDP kinases are hexamers, including the D. discoideum and human proteins. Their subunit folds are very similar, and their active sites are essentially identical (Janin et al., 2000). Structural data show the same mode of binding for GDP to human NDP kinase B (Morera et al., 1995) and ADP or TDP to wild-type D. discoideum NDP kinase (Cherfils et al., 1994; Morera et al., 1994). Thus, the D. discoideum enzyme is a good model of human NDP kinase for functional studies. In addition, the H122G mutant is useful when studying nucleoside triphosphate binding, because the substitution of the active site histidine prevents transfer of the -phosphate to the protein. We previously used the mutant in the analysis of a complex with d4T triphosphate at 1.85-Å resolution (Meyer et al., 2000). With the wild-type protein, bound ATP can be mimicked by a complex of ADP and either aluminum or beryllium fluoride (Xu et al., 1997). The present X-ray structure of H122G in complex with ribavirin triphosphate has an R factor of 20.7% (R_free = 24.9%) at 2.9 Å resolution and good stereochemistry (Table 1). The protein has essentially the same conformation as in the d4T triphosphate complex, a superposition yielding a RMS distance of 0.26 Å for all equivalent C positions. The superposition with GDP-bound human NDP kinase B is almost as good, with an RMS distance of 0.41 Å, excluding nine C-terminal residues that differ in conformation between the D. discoideum and human proteins (Lacombe et al., 2000).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   RTP binding to NDP kinase. RTP bound to the H122G mutant of D. discoideum NDP kinase (yellow bonds) is superimposed in (A) to d4T triphosphate (green) bound to the same protein in the 1.85 Å resolution structure determined by (Meyer et al., 2000) in (B), to GDP bound to wild type human NDP kinase B (Morera et al., 1995). The active site histidine is His-122 in D. discoideum, His-118 in human. The spheres are Mg2+ ions.

View this table: [in this window] [in a new window]   TABLE 2 Polar interactions between RTP and NDP kinase Distance column shows mean and S.D. of distances (Å) observed in the three subunits present in the asymmetric unit of the H122G-RTP crystals. ADP-BeF3 column shows distances observed in the 2.3-Å crystal structure of the wild-type D. discoideum NDP kinase in complex with ADP and beryllium fluoride (Xu et al., 1997).

Catalytic Efficiencies of NDP Kinase for Ribavirin Triphosphate and Derivatives. We followed the time course of the reaction of RTP with human NDP kinase A in fast kinetic experiments by monitoring the intrinsic fluorescence of the protein. The fluorescence is quenched upon phosphorylation of the active site histidine by a NTP substrate and, reciprocally, enhanced upon its dephosphorylation by NDP (Deville-Bonne et al., 1996). The reaction with RTP yields a monoexponential fluorescence decay without a lag, as previously reported for other nucleoside triphosphates (Schaertl et al., 1998; Schneider et al., 1998). The pseudo-first-order constant k_obs of the exponential decay increased linearly with RTP concentrations in the range investigated (5-50 µM) (Fig. 3). The slope of the linear fit, which measures the catalytic efficiency of RTP as a substrate for NDP kinase, was k₂/K_S = 3 × 10⁵ M¹s¹. This makes RTP a good substrate, comparable with the natural substrate CTP, although inferior to GTP, which is the best known substrate of NDP kinase (Table 3).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Pre-steady-state kinetics of NDP kinase phosphorylation by ribavirin triphosphate and analogs. Concentration dependence of the first-order rate constant of NDP kinase phosphorylation by ribavirin triphosphate and analogs. NDP kinase A (1 µM) was rapidly mixed at 20°C with 5 to 50 µM ribavirin or natural substrates (A) or with 5 to 500 µM ribavirin triphosphate analogs (B), and the rates of phosphorylation were measured with a fluorescence stopped-flow device.

View this table: [in this window] [in a new window]   TABLE 3 NDP kinase binding and activation of nucleotide analogs The phosphotransfer reaction between human NDP kinase A and nucleotide analogs was studied in the direction NTP + E  E-P + NDP. Note that ribavirin is in the same range of activity as natural nucleotides, whereas 3'-deoxy derivatives lose most of their efficiency. The F64W-H122G variant of D. discoideum is designed to measure NTP binding in the absence of phosphotransfer. Protein fluorescence (exc = 310 nm, em = 330 nm) increases by 50% upon binding saturation amounts of NTP at 20°C in 50 mM Tris-HCl, 5 mM MgCl2, 75 mM KCl, 1 mM DTT, and 5% glycerol, pH 7.5. KD values are estimated by fitting the binding curve to a quadratic equation.

Binding Affinities of Ribavirin Triphosphate and Derivatives for NDP Kinase. Although the equilibrium dissociation constant K_S of the NDP kinase-NTP complex is not accessible in these kinetic experiments, it can be estimated by using a mutant NDP kinase, which lacks the catalytic histidine and where the phenylalanine (Phe-64 in D. discoideum NDP kinase) stacking on the base is replaced by a tryptophan. In the H122G-F64W double mutant, the protein fluorescence changes upon nucleotide binding although no phosphorylation takes place. Titration by RTP yielded K_S = 24 ± 5 µM, a 3-fold decrease in affinity relative to CTP and 160-fold relative to GTP (Table 3). RTP analogs with a modified sugar showed a further decrease in affinity by a factor of 2 for 2'dRTP, 10 for 3'dRTP and d4RTP, and 100 for ddRTP. Although smaller than for the catalytic efficiency k₂/K_S, these ratios indicate that all analogs bind NDP kinase with lower affinities than the natural substrates.

Inhibition of Viral Polymerase by RTP and Analogs. Ribavirin has been reported to exhibit some anti-HIV activity, but its combination with other nucleotide analogs leads to serious adverse effects (Sim et al., 1998). Indeed, although ribavirin is a ribonucleoside analog, RTP may also inhibit the DNA polymerase activity of HIV RT. Because 2',3'-dideoxynucleotides are efficient against HIV RT, it was of interest to test whether 2'- or 3'-deoxy-modified RTP is efficient against HIV RT and retains inhibitory power against HCV polymerase.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Binding affinity of ribavirin triphosphate and analogs for NDP kinase. Fluorescence titration curve of F64W-H122G mutant NDP kinase (1 µM) by RTP or analogs at 20°C. Solid lines represent the best fit of the data to a quadratic saturation curve. The apparent KD values are shown.

View larger version (70K): [in this window] [in a new window]   Fig. 5.   Inhibition of HCV NS5B polymerase by RTP and its ribose-modified derivatives. Assays were performed in the presence of 125 ng/µl of template/primer [poly(C)/GG] and 0.5 or 1.5 mM RTP or analogs. Relative activities were determined by calculating the slope of the incorporation linear phase occurring after the lag phase (see Inhibition of Viral Polymerase by RTP and Analogs). Mean values of relative activities for each compound are shown as percentage of NS5B activity without inhibitor. S.E. and mean values were calculated from two independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Inhibition of initiation or elongation of HCV polymerase by RTP and derivatives. Assays were performed in the presence of 125 ng/µl of template/primer [poly(C)/GG] and 0.5 mM of RTP or analogs (, no inhibitor; , RTP; , 2'dRTP;  3'dRTP; , epoxyRTP; , ddRTP; , d4RTP). For inhibition of initiation, polymerase activity was determined as incorporation of radiolabeled GTP nucleotide during the first 15 min. For inhibition of elongation, polymerase reaction was started without RTP derivatives. After 15 min, 0.5 mM RTP or derivatives were added, and the reaction continued for 80 min. Polymerase activity was determined as incorporation of radiolabeled GTP nucleotide over time.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Correlation of catalytic efficiencies of NDP kinase for several nucleotides triphosphate and their binding affinities. The equilibrium affinity constant KA is measured from fluorescence titrations curves on F64W-H122G mutant NDP kinase as shown on Fig. 4. Catalytic efficiencies are obtained in stopped-flow experiments with human NDP kinase A. The active site of both enzymes is highly conserved (Janin et al., 2000). AZTTP, 3'-azidothymidine triphosphate; d4TTP, 2',3'-dideoxy-2',3'-didehydrothymidine triphosphate; ddCTP, 2',3'-dideoxycytidine triphosphate; 2'dTTP, 2'-deoxythymidine triphosphate; 2'dRTP, 2'-deoxyribavirin triphosphate; 2'dCTP, 2'-deoxycytidine triphosphate; 3'dRTP, 3'-deoxyribavirin triphosphate; ddTTP, 2',3'-dideoxythymidine triphosphate; d4RTP, 2',3'-dideoxy-2',3'-didehydroribavirin triphosphate; ddRTP, 2',3'-dideoxyribavirin triphosphate.

	Discussion

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Unlike nucleoside analogs directed against HIV RT, which lack a 3'OH, ribavirin contains a normal ribose moiety and cannot act as a chain terminator. The derivatives tested here combine the base modification of ribavirin and some of the sugar modifications found in RT inhibitors. We tested the capacity of NDP kinase to produce the triphosphate form of these derivatives and the effect of the triphosphate form on several target viral polymerases.

Correlation between Binding Affinities and Catalytic Efficiencies of Nucleotides for NDP Kinase. RTP and its analogs were found to be excellent substrates for NDP kinase on the condition that the 3'hydroxyl group is present. This is in line with previous studies of natural substrates and analogs. Figure 7 shows a correlation between the catalytic efficiencies and the equilibrium association constant K_A for natural nucleotides and analogs. Substrates are found to belong to two classes: those carrying a 3'-OH and those lacking this hydroxyl group. The two lines obtained for the two classes indicate a change in catalytic efficiency by a factor 500 to 1000, illustrating the well-established contribution of the 3'-OH to substrate-assisted catalysis by NDP kinase. The lines themselves show that, within each class, the catalytic efficiency correlates linearly to the binding affinity.

The Use of Ribavirin Nucleotide as RNA Polymerization Inhibitors. Ribavirin is currently used as a therapeutical agent against HCV in combination with interferon (Lauer and Walker, 2001). Although it is a weak antiviral drug when used alone, ribavirin is phosphorylated by cellular kinases up to the triphosphate level, and ribavirin monophosphate is incorporated into the viral RNA by the recombinant HCV polymerase (Maag et al., 2001). Templates containing RMP can also block RNA elongation. This makes ribavirin an interesting candidate for chemical modification aiming to increase its antiviral effect.