Abstract
β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a kcat of 0.007 s−1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.
Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5, 6, 21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3′-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5′-triphosphate form by cellular kinases. 2′-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2′-C-Methyladenosine (2′-C-Me-A) and 2′-C-methylguanosine (2′-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2′-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the presence of 2′-C-Me-A or 2′-C-Me-G (20). Sequencing of the replicon identified the S282T mutation in NS5B RNA polymerase (20).
β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of HCV RNA replication in Huh-7 replicon cells (4), and its structure is shown in Fig. 1. PSI-6130 has been shown to be phosphorylated to the 5′-triphosphate (PSI-6130-TP), and a detailed study of the anabolism of PSI-6130 will be presented elsewhere (unpublished data). Here, we have studied the biochemical aspects of metabolism and the antiviral activity of PSI-6130 and have specifically addressed the following questions: (i) Which enzyme is responsible for phosphorylating PSI-6130 to the monophosphate (PSI-6130-MP)? (ii) Does UMP-CMP kinase (YMPK) phosphorylate PSI-6130-MP? (iii) Which enzyme phosphorylates PSI-6130 diphosphate (PSI-6130-DP)? (iv) Does PSI-6130-TP inhibit wild-type and mutant (S282T) HCV NS5B RNA polymerases? (v) If it does inhibit wild-type and mutant (S282T) HCV NS5B RNA polymerases, what is the molecular mechanism of the inhibition? These studies should help provide an understanding of the molecular mechanism of action of PSI-6130.
FIG. 1.
Chemical structures of the nucleoside analogs used in this study.
MATERIALS AND METHODS
Enzymes.
Human 2′-deoxycytidine kinase (dCK) was cloned from human hepatoma (HepG2) cells. Total RNA was isolated from HepG2 cells grown to 75 to 85% confluence in 75-cm2 tissue culture flasks. Poly(A) RNA was isolated from total RNA with a Poly(A) Purist MAG kit (Ambion, Austin, TX), and reverse transcription was performed with 1/10 of the poly(A) RNA by using a First Strand cDNA kit (Roche Applied Science, Indianapolis, IN) and the reverse primer from the first round of PCR. The target gene was amplified by a nested PCR procedure. The first round of PCR consisted of 25 cycles with the outer primer pair. A further 35 cycles used 2 μl of the PCR product from the first amplification as the template and the inner primer pair. The inner primer pair also introduced restriction enzyme sites that allowed directional cloning. The final PCR product was digested and cloned into the Escherichia coli expression vector pQE-60 (QIAGEN, Valencia, CA), which introduced arginine and serine residues followed by a 6-histidine tail at the carboxyl terminus of the protein for affinity purification. The resulting product was verified by sequencing, and the construct was cotransformed into XL1-Blue MRF′ competent E. coli cells (Stratagene, La Jolla, CA) along with plasmid pRep4, which codes for the Lac repressor protein. The protein was purified by a metal affinity column with Talon resin (Clontech, Mountain View, CA).
Human uridine-cytidine kinase 1 (UCK-1) was cloned from Huh-7 cells. We preformed RNA isolation; cDNA preparation; and amplification, cloning, and expression exactly as described above for dCK; however, UCK-1 was not expressed. We reasoned that this may have been due to codon usage problems and thought that an easily expressible leader peptide might solve the problem, and so a third amplification of the DNA was performed to allow directional cloning into the BamHI and HindIII sites of pTrcHis A. The subsequent plasmid codes for a 36-amino-acid leader peptide, including a 6-His tag at the amino terminus, while the carboxy terminus is identical to that of the wild-type enzyme. The amino acid sequence of the leader sequence is GGSHHHHHHGMASMTGGNNMGRDLYDDDDKDRWGS-GSAGG (the hyphen is where the wild-type protein sequence begins).
YMPK and nucleoside diphosphate kinase (NDPK) were cloned from Huh-7 and HepG2 cells, respectively. We followed the RNA isolation; cDNA preparation; and amplification, cloning, and expression procedures exactly as described above for dCK.
The 21-amino-acid C-terminal truncated HCV NS5B RNA polymerase was expressed and purified as described previously (27). The S282T mutant enzyme was made by using a Quikchange mutagenesis kit (Stratagene). Pyruvate kinase, lactate dehydrogenase, human creatine kinases (isoforms BB, MB, and MM), and yeast 3-phosphoglycerate kinase were purchased from Sigma (St. Louis, MO).
Other materials.
3′-Deoxycytidine triphosphate (3′-dCTP) was purchased from Trilink Biotechnologies (San Diego, CA). All other nucleoside/nucleotide analogs were synthesized by Pharmasset, Inc. (Princeton, NJ). The structures of the compounds used in this study are shown in Fig. 1. The negative-strand internal ribosomal entry site RNA template (referred to as minus IRES) was prepared by using an in vitro transcription kit from Ambion (22). Natural nucleoside triphosphates were purchased from Amersham Biosciences (Piscataway, NJ). [γ-32P]UTP was purchased from Perkin-Elmer (Boston, MA). RNA 21-mer (5′-CCU UUU CUA AUU CUC GUA UAC-3′) was synthesized and purified by New England Biolabs (Ipswich, MA).
dCK, UCK-1, YMPK, and NDPK assays.
The phosphorylation of nucleoside and nucleoside monophosphates by human dCK, UCK-1, YMPK, and NDPK was studied spectrophotometrically as described previously (13). The reaction was coupled with pyruvate kinase and lactate dehydrogenase, and oxidation of NADH was monitored at 340 nm by using a Lambda 35 UV/VIS spectrometer (Perkin-Elmer). The reaction temperature for dCK and UCK-1 was 25°C, and the reaction temperature for YMPK and NDPK was 37°C. A 1-ml reaction mixture contained 64 mM Tris-HCl (pH 7.5), 3.8 mM EDTA, 180 mM KCl, 12.8 mM MgCl2, 24 mM (NH4)2SO4, 1 mM ATP, 0.5 mM phosphoenolpyruvate, 0.1 mM NADH, 5 IU/ml pyruvate kinase, 13.8 IU/ml lactate dehydrogenase, nucleoside or nucleoside monophosphate substrate, and kinase. The final enzyme concentrations for the dCK and UCK-1 reactions were 240 nM and 41 nM, respectively, except for reactions with PSI-6130 and 2′-C-methylcytidine (2′-C-Me-C), in which a 10-fold higher concentration of UCK-1 (410 nM) was used. The enzyme concentrations in the YMPK reaction were 80 nM for natural nucleoside monophosphates and 200 nM for PSI-6130-MP. In the NDPK reaction, 216 nM enzyme was used in all the reactions. The reaction rates with different concentrations of the nucleoside substrate were determined, and steady-state parameters were determined by using the GraFit program (version 5; Erithacus Software, Horley, United Kingdom).
NS5B RNA polymerase assays.
NS5B RNA polymerase reaction was studied by monitoring the incorporation of 32P-labeled UMP into the newly synthesized RNA strand by using minus IRES as the template. A steady-state reaction was performed in a total volume of 140 μl containing 2.8 μg of minus IRES RNA template, 140 units of anti-RNase (Ambion), 1.4 μg of NS5B, an appropriate amount of [α-32P]UTP, various concentrations of natural and modified nucleotides, 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM dithiothreitol in 50 mM HEPES buffer (pH 7.5). The nucleotide concentration was changed depending on the inhibitor. The reaction temperature was 27°C. At the desired times, 20-μl aliquots were taken and the reaction was quenched by mixing the reaction mixture with 80 μl of stop solution containing 12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate. In order to determine steady-state parameters for a natural nucleotide triphosphate (NTP) substrate, one NTP concentration was varied and the concentrations of the other three NTPs were fixed at saturating concentrations. For determination of the Ki for 2′-C-Me-ATP, the concentrations of UTP, GTP, and CTP were fixed at 10, 100, and 100 μM, respectively, and the concentrations of ATP and 2′-C-Me-ATP were varied. For the 2′-C-Me-CTP and PSI-6130-TP experiments, UTP, ATP, and GTP concentrations were fixed at 10, 100, and 100 μM, respectively, and the CTP and inhibitor concentrations were varied.
The radioactive RNA products were separated from unreacted substrates by passing the quenched reaction mixture through a Hybond N+ membrane (Amersham Biosciences) by using a dot blot apparatus. The RNA products were retained on the membrane and the free nucleotides were washed out. The membrane was washed four times with a solution containing 0.6 M NaCl and 60 mM sodium citrate. After the membrane was rinsed with water followed by rinsing with ethanol, the dots were cut out and the radioactivity was counted in a Packard liquid scintillation counter.
The amount of product was calculated on the basis of the total radioactivity in the reaction mixture. The rate of the reaction was determined from the slope of the time course of product formation. To determine the inhibition constant (Ki), reaction rates were determined with different concentrations of the substrate and the inhibitor and were fit to a competitive inhibition equation: v = (Vmax · [s])/{Km · (1 + [I]/Ki) + [S]}, where v is the observed rate, [S] is the substrate concentration, [I] is the inhibitor concentration, and Vmax is the maximum rate. Km is the Michaelis constant, and Ki is the inhibition constant. A nonlinear fit was performed by using the GraFit program (version 5; Erithacus Software).
Chain-termination study.
An RNA polymerase reaction was performed in a total volume of 20 μl containing 2.7 μM RNA 21-mer (5′-CCU UUU CUA AUU CUC GUA UAC-3′); 10 μM UTP; 100 μM ATP and GTP; 5 μCi of [α-32P]UTP; 20 units of anti-RNase (Ambion); 100 μM either CTP, 3′-dCTP, 2′-C-Me-CTP, or PSI-6130-TP; 400 ng of NS5B; 1 mM MgCl2; 0.75 mM MnCl2; and 2 mM dithiothreitol in 50 mM HEPES buffer (pH 7.5). The reaction was allowed to proceed for 1 h at 27°C and was quenched by adding 1 μl of 0.5 M EDTA. After the reaction was quenched, 14 μl of a dye solution containing 95% formamide and 0.1% each of bromophenol blue and xylene cyanol was added for sequencing gel analyses. The samples (10 μl) were loaded onto a 20% polyacrylamide-12% formamide gel, and the electrophoresis was run at 60 W with a sequencing gel apparatus. The gel was then exposed to a phosphorscreen and visualized with a phosphorimager.
RESULTS
Phosphorylation of PSI-6130 by human dCK and UCK-1.
Other studies, presented elsewhere, have shown that incubation of cells with certain exogenous natural nucleosides can reduce the antiviral activity of a nucleoside analog (7, 15). It was shown that addition of exogenous 2′-deoxycytidine completely reversed the inhibition of HCV RNA replication by PSI-6130 in replicon cells, whereas the addition of cytidine resulted in only the partial reversal of antiviral activity (26). These findings suggest that PSI-6130 is phosphorylated by dCK and/or UCK. There are two isoforms of UCK (UCK-1 and UCK-2). Northern blot analyses have shown that UCK-1 is expressed in wide variety of tissues, including liver, kidney, skeletal muscle, and heart tissues, whereas UCK-2 is expressed only in the placenta (29). Therefore, UCK-1 was chosen for use for the investigation of the phosphorylation of PSI-6130.
Cell-free phosphorylation assays were performed with PSI-6130, 2′-deoxycytidine, cytidine, and other related compounds (see Fig. 1 for the structures). Steady-state kinetic assays were performed with an enzyme coupled system with pyruvate kinase and lactate dehydrogenase, and the reaction was followed by monitoring the oxidation of NADH at 340 nm (see the description of the experimental procedure above).
The steady-state parameters for dCK are summarized in Table 1. PSI-6130 and 2′-C-Me-C were phosphorylated by human dCK with significantly lower affinities than 2′-deoxycytidine, cytidine, 2′-deoxy-2′-fluorocytidine (2′-FdC), or gemcitabine, with Km values of 81 and 914 μM, respectively. Compared to the physiological substrate, 2′-deoxycytidine, PSI-6130 was phosphorylated approximately 300-fold less efficiently; however, it was a 15-fold better substrate than 2′-C-Me-cytidine. Addition of a methyl group at the 2′-arabinosyl (2′-up) position to 2′-deoxycytidine decreased the efficiency by 62-fold. Gemcitabine and 2′-FdC were phosphorylated at efficiencies similar to that for 2′-deoxycytidine.
TABLE 1.
Steady-state parameters for dCK reactionsa
| Substrate | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) | Relative substrate specificity |
|---|---|---|---|---|
| 2′-Deoxycytidine | 0.4 ± 0.1 | 0.025 | 6.3 × 10−2 | 1 |
| Gemcitabine | 1.4 ± 0.2 | 0.038 | 2.7 × 10−2 | 0.43 |
| 2′-FdC | 4.7 ± 1.2 | 0.050 | 1.1 × 10−2 | 0.17 |
| 2′-C-Me-dC | 46.2 ± 10.7 | 0.048 | 1.0 × 10−3 | 0.016 |
| Cytidine | 6.6 ± 1.0 | 0.009 | 1.4 × 10−3 | 0.022 |
| PSI-6130 | 81.2 ± 8.0 | 0.016 | 1.9 × 10−4 | 0.003 |
| 2′-C-Me-C | 914 ± 209 | 0.011 | 1.2 × 10−5 | 0.0002 |
Values are reported as means ± standard deviations.
We evaluated UCK-1 with cytidine, uridine, 2′-deoxycytidine, PSI-6130, and 2′-C-Me-C as phosphate acceptors. The steady-state kinetic parameters are shown in Table 2. No activity was observed for 2′-deoxycytidine up to 1 mM or for 2′-C-Me-C and PSI-6130 up to 3 mM when a 10-fold higher concentration of enzyme compared to those used in the cytidine and uridine experiments was used.
TABLE 2.
Steady-state parameters for UCK-1a
| Substrate | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) |
|---|---|---|---|
| Cytidine | 131 ± 27 | 0.50 ± 0.04 | 3.8 × 10−3 |
| Uridine | 407 ± 52 | 0.51 ± 0.03 | 1.3 × 10−3 |
| 2′-Deoxycytidine | NA, 1 mMb | NA, 1 mM | NA, 1 mM |
| 2′-C-Me-C | NA, 3 mMc | NA, 3 mM | NA, 3 mM |
| PSI-6130 | NA, 3 mM | NA, 3 mM | NA, 3 mM |
Values are reported as means ± standard deviations.
NA, 1 mM, no activity was observed up to 1 mM.
NA, 3 mM, no activity was observed up to 3 mM.
Phosphorylation of PSI-6130-MP by human YMPK.
The enzyme that catalyzes the phosphorylation of CMP, UMP, and dCMP to their corresponding diphosphates is known as YMPK. The enzyme also phosphorylates monophosphates of cytidine analogs. Since PSI-6130 is a cytidine analog, YMPK activity was examined with PSI-6130-MP. As shown in Table 3, the Km value for PSI-6130-MP was comparable to those of the natural nucleoside monophosphate substrates, with an approximately twofold higher value than that for UMP and a threefold lower value than that for dCMP. The kcat value for PSI-6130-MP was approximately 10-fold lower than those for CMP and dCMP. Overall, the catalytic efficiency (kcat/Km) for PSI-6130-MP was only threefold lower than that for dCMP, indicating that PSI-6130-MP is a good substrate for YMPK.
TABLE 3.
Steady-state parameters for YMPKa
| Substrate | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) |
|---|---|---|---|
| UMP | 151 ± 31 | 81 ± 5 | 0.54 |
| CMP | 56 ± 10 | 30 ± 2 | 0.54 |
| dCMP | 837 ± 69 | 30 ± 1 | 0.036 |
| PSI-6130-MP | 282 ± 88 | 3.3 ± 0.4 | 0.012 |
Values are reported as means ± standard deviations.
Phosphorylation of PSI-6130-DP by human NDPK.
In order to study the phosphorylation of PSI-6130-DP to PSI-6130-TP, human NDPK was cloned and purified. The steady-state parameters for CDP and PSI-6130-DP are summarized in Table 4. As described in the Materials and Methods section, our kinase assay was coupled with pyuvate kinase and lactate dehydrogenase. It is known that pyruvate kinase phosphorylates nucleoside diphosphates (9, 14). Therefore, the control reactions were performed in the absence of NDPK and the rate was subtracted from the overall reaction rate in the case of CDP phosphorylation. Typically, the rate of reaction of pyuvate kinase for CDP was less than 10% of the overall rate. In the reactions with PSI-6130, the pyruvate kinase reaction rate was negligible. Creatine kinase and 3-phosphoglycerate kinase are also candidates for the enzyme responsible for phosphorylating PSI-6130-DP, as these kinases are known to phosphorylate nucleoside diphosphates (9, 14). Phosphorylation of PSI-6130-DP was tested with different isoforms of creatine kinase (the MM, MB, and BB isoforms) and 3-phosphoglycerate kinase. No activity was observed with any of these enzymes up to 1 mM PSI-6130-DP (data not shown).
TABLE 4.
Steady-state parameters for NDPKa
| Substrate | Km (μM) | kcat (s−1) | kcat/Km (μM−1 s−1) |
|---|---|---|---|
| CDP | 307 ± 62 | 183 ± 14 | 0.60 |
| PSI-6130-DP | 1380 ± 310 | 21 ± 3 | 0.015 |
Values are reported means ± standard deviations.
Effects of PSI-6130-TP on HCV NS5B.
The inhibition of HCV NS5B RNA polymerase by PSI-6130-TP, 2′-C-Me-CTP, and 2′-C-Me-ATP was studied under steady-state kinetic conditions by using purified RNA polymerase, 32P-labeled UTP, and minus IRES as an RNA template. Since it has been shown that the S282T mutation in NS5B RNA polymerase causes resistance to 2′-C-Me-A and 2′-C-Me-G (20), experiments were performed with both wild-type and S282T mutant enzymes to see if the resistance is common to all 2′-C-Me-nucleosides. The Km and the maximum rate (kcat) were determined for natural nucleoside triphosphates (Table 5). Overall, the catalytic efficiencies (kcat/Km values) were similar for the wild type and the S282T mutant; however, the affinity for the natural nucleotides was tighter (lower Km) and the rate was slower (lower kcat) with the mutant than with the wild type. The Ki values for 2′-C-Me-nucleoside triphosphates (2′-C-Me-nucleoside-TPs) were also determined (Table 6). The inhibition constants for all the 2′-C-Me-nucleoside-TPs with the wild-type enzyme were very similar, with a Ki range of 1.5 to 4.3 μM. The Km values for natural nucleoside triphosphates were 5- to 10-fold lower with the S282T mutant than with the wild type. The drug resistance profile was analyzed by comparing Ki(drug)/Km(natural nucleotide) values between the wild-type enzymes and the S282T mutant enzymes. The greatest resistance was noted with 2′-C-Me-ATP (150-fold).
TABLE 5.
Steady-state parameters for wild-type and S282T mutant HCV NS5B RNA polymerasea
| Nucleotide | Wild type
|
S282T mutant
|
||||
|---|---|---|---|---|---|---|
| Km (μM) | kcat (min−1) | kcat/Km (min−1 μM−1) | Km (μM) | kcat (min−1) | kcat/Km (min−1 μM−1) | |
| UTP | 0.4 ± 0.15 | (12.8 ± 0.57) × 10−4 | 32 × 10−4 | 0.18 ± 0.05 | (1.25 ± 0.05) × 10−4 | 6.9 × 10−4 |
| CTP | 13.7 ± 2.59 | (15.2 ± 0.94) × 10−4 | 1.1 × 10−4 | 2.7 ± 0.92 | (2.14 ± 0.16) × 10−4 | 0.79 × 10−4 |
| ATP | 2.7 ± 0.66 | (20.0 ± 0.97) × 10−4 | 7.4 × 10−4 | 0.26 ± 0.14 | (1.67 ± 0.91) × 10−4 | 6.4 × 10−4 |
| GTP | 9.6 ± 1.73 | (16.9 ± 0.91) × 10−4 | 1.7 × 10−4 | 5.0 ± 1.5 | (1.98 ± 0.15) × 10−4 | 0.4 × 10−4 |
Values are reported means ± standard deviations.
TABLE 6.
Inhibition of wild-type and S282T mutant HCV NS5B RNA polymerase by the nucleoside triphosphate analogsa
| Nucleotide analog | Wild type
|
S282T mutant
|
Fold resistance (Ki/Km)mut/ (Ki/Km)WTc | ||||
|---|---|---|---|---|---|---|---|
| Km (μM)b | Ki (μM) | Ki/Km | Km (μM)c | Ki (μM) | Ki/Km | ||
| PSI-6130-TP | 13.7 ± 2.59 | 4.3 ± 0.7 | 0.31 | 2.7 ± 0.92 | 2.5 ± 0.9 | 0.93 | 3 |
| 2′-C-Me-CTP | 13.7 ± 2.59 | 1.6 ± 0.3 | 0.12 | 2.7 ± 0.92 | 3.8 ± 1.0 | 1.4 | 12 |
| 2′-C-Me-ATP | 2.7 ± 0.66 | 1.5 ± 0.4 | 0.56 | 0.26 ± 0.14 | 21.8 ± 4.7 | 84 | 150 |
Values are reported means ± standard deviations.
The Km for CTP is shown for PSI-6130-TP and 2′-C-Me-CTP, and the Km for ATP is shown for 2′-C-Me-ATP.
mut, mutant; WT, wild type.
Chain termination.
We have shown that PSI-6130-TP inhibits HCV NS5B RNA polymerase. In order to understand the molecular mechanism of inhibition, we examined whether or not PSI-6130-TP acts as a chain terminator. An RNA 21-mer oligonucleotide with only one guanine base in the strand, at the sixth position from the 3′ end, was used as a template. Thus, during RNA synthesis, the RNA polymerase needs to incorporate cytidine only once at the sixth position from the 5′ end. The polymerase experiments were performed in the absence of CTP but in the presence of CTP analogs, together with ATP, GTP, and radiolabeled UTP. Therefore, if these analogs are chain terminators, any products longer than 6-mers should not be observed. As shown in Fig. 2, except for the sample containing all four natural nucleoside triphosphates, the RNA product formation was stopped at the same position. The position of the band must be a 6-mer since 3′-dCTP is a known chain terminator (24) and it stopped the RNA synthesis at the same position (Fig. 2, lane 4). A minor band above the 6-mer band is very likely due to a misincorporated product, since the same band was seen with 3′-dCTP. These results indicate that both 2′-C-Me-C-TP and PSI-6130-TP are chain terminators.
FIG. 2.
Evidence for chain termination of RNA synthesis by incorporation of PSI-6130-MP and 2′-C-Me-CMP.
DISCUSSION
In order for a nucleoside analog to inhibit the viral polymerase, it must to be activated to the 5′-triphosphate form by host cell kinases. Typically, phosphorylation of a nucleoside to its monophosphate is a rate-limiting step for the activation of many cytidine analogs (1, 10). In order to understand the phosphorylation of PSI-6130 to PSI-6130-MP, human dCK and UCK-1 were cloned and purified. The kinetic results indicate that PSI-6130 is a substrate for dCK, although it was an approximately 300-fold poorer substrate than the physiological substrate, 2′-deoxycytidine. Similarly, some cytidine analogs, such as zalcitabine (25) and lamivudine (3), are known to be phosphorylated by dCK at low catalytic efficiencies; but this enzyme still serves as the enzyme responsible for the phosphorylation. On the other hand, UCK-1 was not able to phosphorylate PSI-6130 or 2′-C-Me-C. These UCK-1 results are consistent with the findings of previous structural and biochemical studies, which showed that UCK has a high specificity for the 2′-hydroxyl group (28, 29). Clearly, the presence of the 2′-methyl group also influences the affinities of these nucleosides analogs for UCK.
In previous cell-based studies, we have shown that the anti-HCV activity of PSI-6130 was prevented only by the addition of cytidine or 2′-deoxycytidine into the replicon cell culture (26). PSI-6130's anti-HCV replicon activity was completely reversed by exogenously added 2′-deoxycytidine, probably because phosphorylation of PSI-6130 by dCK was inhibited due to competition with the physiological substrate. Since PSI-6130 was a much poorer (300-fold) substrate than 2′-deoxycytidine, it is not surprising to see complete inhibition by deoxycytidine. Interestingly, cytidine inhibited the antiviral activity of PSI-6130 by 60% (26). Our enzyme studies have shown that the kcat/Km values for cytidine were similar for dCK (1.4 × 10−3 μM−1 s−1) and UCK-1 (3.8 × 10−3 μM−1 s−1); thus, cytidine could compete with PSI-6130 for the dCK active site. Furthermore, PSI-6130 was only a 7.4-fold poorer substrate for dCK than cytidine, whereas it was a 300-fold poorer substrate than 2′-deoxycytidine (Table 1). Therefore, there is a greater chance for PSI-6130 to be phosphorylated by dCK in the presence of exogenously added cytidine than in the presence of 2′-deoxycytidine. This could explain why cytidine partially inhibited the antiviral activity of PSI-6130. Taken together, the results of our enzyme and cell-based competition studies suggest that dCK is likely responsible for the phosphorylation of PSI-6130.
A structure-activity relationship (SAR) for 2′-modified nucleosides with dCK is summarized in Fig. 3. Addition of fluorine at the 2′ position of 2′-deoxycytidine (2′-FdC) caused an approximately 5-fold decrease in kcat/Km, whereas a hydroxyl group at this position (cytidine) led to a 50-fold decrease, indicating that the enzyme prefers fluorine over a hydroxyl group at the 2′-down position. A 2′-C-Me modification of 2′-deoxycytidine, 2′-FdC, and cytidine (2′-OH) significantly decreased the kcat/Km values (60- to 100-fold). Gemcitabine, which possesses two fluorines at the 2′ position, is a slightly better substrate than 2′-FdC. A fluoro or hydroxyl group at the 2′-arabinosyl (2′-up) position is proposed to form a hydrogen bond with the dCK active-site residue Arg 128, and the interaction could enhance the reaction rate (23).
FIG. 3.
Effect of 2′ substitutions on dCK activity SAR.
The enzyme responsible for phosphorylating CMP, UMP, and dCMP is YMPK. This enzyme also phosphorylates various cytidine analog monophosphates (12, 18). PSI-6130-MP was phosphorylated to the diphosphate with a catalytic efficiency only threefold lower than that of one of the natural substrates, dCMP. Previous SAR studies have shown that the most important determinant for the activity is the 3′-OH group (18). Our results are consistent with the findings of those studies, as PSI-6130 possesses a 3′-OH group.
The last step of the PSI-6130 activation pathway is the phosphorylation of PSI-6130-DP to PSI-6130-TP. Our kinetic study results suggest that the enzyme responsible for this reaction is likely to be NDPK, as creatine kinases and 3-phosphoglycerate kinase were not able to phosphorylate PSI-6130-DP. Therefore, we propose that the enzymes involved in PSI-6130 activation are dCK, YMPK, and NDPK. Our proposed scheme of PSI-6130 activation is shown in Fig. 4.
FIG. 4.
Proposed enzymatic phosphorylation pathway for PSI-6130.
Our steady-state parameters determined by using the 21-amino-acid C-terminus-truncated NS5B were comparable to previously reported values (19). The inhibition profile of wild-type NS5B enzyme by PSI-6130-TP was similar to those of other 2′-C-Me-nucleotides, with Ki values ranging from 1.5 to 4.3 μM and Ki/Km values ranging from 1.2 to 5.6 (Table 6). Interestingly, with the S282T mutant enzyme, the inhibition profiles were very similar for PSI-6130-TP and 2′-C-Me-CTP, but 2′-C-Me-ATP showed much less of an inhibitory effect. The Ki/Km value for 2′-C-Me-ATP was 60- and 90-fold higher than those for 2′-C-Me-CTP and PSI-6130-TP, respectively. When the fold resistances were compared, the value for 2′-C-Me-ATP (150-fold) was significantly higher than those for PSI-6130 (3-fold) and 2′-C-Me-CTP (12-fold). Previously, it was shown that the S282T mutation showed resistance to both 2′-C-Me-A and 2′-C-Me-G (20). Therefore, these results suggest that 2′-C-Me-nucleosides with purine bases show significantly greater drug resistance by S282T mutation than the cytidine derivatives. The Km value for ATP was 10-fold lower with the S282T mutant than with the wild type. This may also be contributing to the resistance with 2′-C-Me-ATP. A previous study showed that the Km for ATP increased by 3.8-fold due to the S282T mutation when the 55-amino-acid truncated enzyme was used (20). It is possible that ATP binding to the active site of the mutant enzyme may be affected by the different number of amino acids truncated.
It has been shown that 2′-C-Me-ATP and 2′-C-Me-GTP act as chain terminators once they are incorporated into an elongating RNA strand by NS5B RNA polymerase (2, 20). Here, we have examined if PSI-6130-TP is also a chain terminator. As presented in Fig. 2, RNA synthesis was inhibited when PSI-6130-MP was incorporated into the newly synthesized RNA strand. It is unclear how the 2′-C-Me-nucleoside monophosphates stop the RNA synthesis, as they possess the 3′-hydroxyl group, which is necessary to attack the alpha phosphate of the next incoming nucleoside triphosphate for further RNA synthesis. Migliaccio et al. (20) proposed for 2′-C-Me-A that the 2′-C-methyl group may sterically hinder the ribose ring of the next incoming nucleoside triphosphate. Since PSI-6130 also possesses a 2′-C-methyl group, it is possible that a similar interaction may occur such that PSI-6130 also acts as a nonobligate chain terminator.
In summary, we have investigated the mechanism of action of PSI-6130, which is a potent HCV RNA replication inhibitor, in a replicon system. Our enzyme kinetics and previous cell-based competition assays suggested that dCK is responsible for the first phosphorylation step. YMPK was able to phosphorylate PSI-6130-MP to the diphosphate efficiently. The enzyme that catalyzes the last step of the phosphorylation pathway is likely to be NDPK, since other host kinases that are known to phosphorylate nucleoside diphosphate were not capable of phosphorylating PSI-6130-DP. The extent of inhibition of wild-type NS5B RNA polymerase by PSI-6130-TP was similar to those by other 2′-C-Me-nucleoside-TPs. However, our studies with the S282T mutant enzyme suggested that this mutation causes high levels of resistance to 2′-C-Me-nucleosides with purine bases but not to the cytidine derivatives. Similar to other 2′-C-Me-nucleoside-TPs, PSI-6130-TP acted as a nonobligate chain terminator during RNA synthesis by NS5B RNA polymerase. These results suggest that PSI-6130 is worthy of further investigation as a treatment for HCV infection.
Acknowledgments
R. F. Schinazi is the principal founder and former director and consultant of Pharmasset, Inc. His laboratory received no funding for his participation in this work.
Footnotes
Published ahead of print on 13 November 2006.
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