Non-nucleoside Analogue Inhibitors Bind to an Allosteric Site on HCV NS5B Polymerase

CRYSTAL STRUCTURES AND MECHANISM OF INHIBITION*

Meitian WangDagger , Kenneth K.-S. NgDagger §, Maia M. CherneyDagger , Laval Chan, Constantin G. Yannopoulos, Jean Bedard, Nicolas Morin, Nghe Nguyen-Ba, Moulay H. Alaoui-Ismaili, Richard C. Bethell, and Michael N. G. JamesDagger ||

From the Dagger  Canadian Institutes for Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and  Shire BioChem Inc., Laval, Quebec H7V 4A7, Canada

Received for publication, September 13, 2002, and in revised form, December 20, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

X-ray crystal structures of two non-nucleoside analogue inhibitors bound to hepatitis C virus NS5B RNA-dependent RNA polymerase have been determined to 2.0 and 2.9 Å resolution. These noncompetitive inhibitors bind to the same site on the protein, ~35 Å from the active site. The common features of binding include a large hydrophobic region and two hydrogen bonds between both oxygen atoms of a carboxylate group on the inhibitor and two main chain amide nitrogen atoms of Ser476 and Tyr477 on NS5B. The inhibitor-binding site lies at the base of the thumb domain, near its interface with the C-terminal extension of NS5B. The location of this inhibitor-binding site suggests that the binding of these inhibitors interferes with a conformational change essential for the activity of the polymerase.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Hepatitis C virus (HCV)1 infects about 3% of the world's human population. HCV infection can develop into chronic hepatitis, which, in some cases, causes cirrhosis of the liver, eventually leading to hepatocellular carcinoma (1). There is no vaccine against HCV currently, and no generally effective therapy for all genotypes of HCV is available. At the present time, the use of recombinant interferon alpha -2a, alpha -2b, "consensus" interferon, and pegylated interferon alpha -2b either in monotherapy or in combination with ribavirin is the only approved therapy available (2). However, limited efficacy and some adverse side effects are associated with these therapies (3). Therefore, the development of HCV-specific antiviral agents is needed urgently.

Extensive studies have been done to understand the structures and functions of the individual components of the HCV-encoded polyprotein (structural proteins C, E1, and E2 and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4-6). Among them, NS2, NS3 protease and helicase, and NS5B RNA-dependent RNA polymerase are essential enzymes for the replication of HCV. The high resolution crystal structures of NS3 protease (7-9) and helicase domains (10, 11) and NS5B polymerase (12-14) have been determined by crystallographic methods in the past 5 years. These enzymes are potential targets for structure-based drug design. The inhibitors of NS3 protease and, in some cases, corresponding structures of NS3 protease/inhibitor complexes have been reported recently (15). In the case of HCV NS5B polymerase, both nucleoside and non-nucleoside inhibitors have been discovered in recent years (16). 3TC® (2'-deoxy-3'-thiacytidine proprietary compound lamivudine) triphosphate has been reported to have a weak inhibitory effect with a 50% inhibitory concentration (IC50) of 180 µM (17), whereas numerous non-nucleoside compounds have been documented to possess relatively potent anti-NS5B activity. Examples include specific rhodanines and barbituric acid derivatives, many of which were found to exhibit anti-NS5B activity with IC50 values below 1 µM (18, 19). Classes of dihydroxypyrimidine carboxylic acids and diketoacid derivatives were claimed as well with IC50 values within the submicromolar range for the latter class (20, 21). 2-Methylidenylbenzothiophene compounds were demonstrated to exert an in vitro anti-NS5B activity with IC50 values in the range of 0.2-30 µM (22), whereas two series of pyrrolidine and benzimidazole analogues were described as novel anti-NS5B inhibitors with the latter class having a range of IC50 values from <1 to 25 µM (23-25). However, no structure of an NS5B polymerase/inhibitor complex is available in the literature. In terms of compounds demonstrating effectiveness in surrogate cell-based assays for HCV replication, numerous nucleoside derivatives have been reported to inhibit self-replicating subgenomic HCV RNAs in the human hepatoma cell line (26).

In this work, we present the three-dimensional structures of two HCV NS5B polymerase/inhibitor complexes. These inhibitors were synthesized as part of structure-activity relationship optimization (SAR) program and are phenylalanine derivatives compound A ((2s)-2-[(2,4-dichloro-benzoyl)-(3-trifluoromethyl-benzyl)-amino]-3-phenyl-propionic acid) and compound B ((2s)-2-[(5-Benzofuran-2-yl-thiophen-2-ylmethyl)-(2,4-dichloro-benzoyl)-amino]-3-phenyl-propionic acid) (see Fig. 1a).2 Both occupy a common binding site in the thumb subdomain near to the C terminus of NS5B polymerase. The detailed interactions between NS5B and inhibitors, revealed by crystallographic studies, provide a starting point to improve currently known inhibitors and to design new inhibitors. Moreover, the structural information also suggests a possible inhibition mechanism, which would shed light on the enzymology of the NS5B polymerase itself.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Expression and Purification of HCV NS5B Protein-- For crystallographic studies, a recombinant soluble form representing a 21-amino acid C-terminally truncated HCV NS5B genotype 1b strain BK enzyme containing an N-terminal hexahistidine tag was expressed in Escherichia coli BL21 (DE3). Purification was done as described by Lesburg et al. (14) and Ferrari et al. (27) with minor modifications. Briefly, the initial step consisted of loading soluble bacterial lysates onto a HiTrap nickel chelating affinity column (Amersham Biosciences). The bound enzyme was eluted using an imidazole gradient. Imidazole was then removed from the buffer of the pooled active fractions using PD-10 desalting columns (Amersham Biosciences). Further purification was achieved by loading the protein preparation onto a cation exchange HiTrap SP-Sepharose column (Amersham Biosciences) using a NaCl gradient for elution. Thereafter, buffer was changed to 10 mM Tris, pH 7.5, 10% glycerol, 5 mM dithiothreitol, 600 mM NaCl using a PD-10 column. The NS5B enzyme was precipitated with ammonium sulfate at 50% saturation and conserved at 4 °C until use. For enzyme kinetic study purposes, a full-length version of the enzyme expressed from baculovirus-infected Sf9 insect cells was purified following the procedure of Alaoui-Ismaili et al. (28).

In Vitro NS5B Assay-- Measurement of the inhibitory effect of compounds on HCV NS5B polymerization activity was performed by evaluating the amount of radiolabeled UTP incorporated by the enzyme in a newly synthesized RNA using a homopolymeric RNA template/primer. Essentially, the compounds were tested at a variety of concentrations (0.75-3.75 µM) in a final volume of 50 µl of reaction mixture consisting of 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCl, 400 ng of purified NS5B enzyme, 500 ng of poly(rA)/oligo(dT)15 (Invitrogen), various concentrations (7.5-60 µM) of nonradioactive UTP, and 0.3-2.5 µCi of alpha -32P-labeled UTP (3000 Ci/mmol; Amersham Biosciences). RNA-dependent RNA polymerase reactions were allowed to proceed for 15, 30, and 45 min at 22 °C. The reactions were stopped by the addition of 10 µl of 0.5 mM EDTA. Thereafter, a volume of 50 µl (25 µg) of sonicated salmon sperm DNA and 100 µl of a solution of 20% trichloroacetic acid, 0.5% tetrasodium pyrophosphate at 4 °C were added to the mixture followed by an incubation on ice for 30 min to ensure complete precipitation of nucleic acids. The samples were then transferred onto 96-well MultiScreen filter plates (Millipore Corp., Bedford, MA). The filter plates were washed with 600 µl of 1% trichloroacetic acid, 0.1% tetrasodium pyrophosphate/well and dried 20 min at 37 °C. A 50-µl volume of liquid scintillation mixture (Wallac Oy, Turku, Finland) was added, and the incorporated radioactivity was quantified using a liquid scintillation counter (Wallac MicroBeta Trilux; PerkinElmer Life Sciences). The Ki values were calculated using the computer software GraphPad Prizm (version 2.0, GraphPad Software Inc.).

Crystallization and Data Collection-- The reported crystallization conditions were adopted with some modifications (14). HCV NS5B Delta C21 protein (20 mg/ml) in 5 mM 2-mercaptoethanol was crystallized by the hanging drop method. Three µl each of protein solution and precipitant solution (18% (w/v) PEG 4000, 0.3 M NaCl, 0.1 M sodium acetate buffer, pH 5.0, 5 mM 2-mercaptoethanol) were mixed on a coverslip and then equilibrated over a 1-ml reservoir of the precipitant solution. Seeding was used to improve the quality of crystals. Crystals adopt an orthorhombic lattice (space group P212121) with unit cell parameters a = ~86 Å, b = ~105 Å, and c = ~126 Å, which is consistent with published results (14). The protein/inhibitor complexes were prepared by soaking the crystals in the inhibitor solutions. The soaking solution contains 5 mM inhibitor, 20% (w/v) PEG 4000, 50 mM NaCl, 10 mM MgCl2, 5 mM mercaptoethanol, and 20 mM Tris buffer, pH 7.5. The cryo-protectant solution has a similar composition as the soaking buffer with 30% (v/v) glycerol added as cryo-protectant instead of 5 mM inhibitor. Diffraction data were collected at 105 K using an R-AXIS IV++ image plate detector with copper Kalpha radiation generated by a Rigaku RU-300 rotating anode x-ray generator. Indexing and integration of the raw image plate data were carried out with DENZO, and the data were merged and reduced with SCALEPACK (29). The data were complete to 2.0 and 2.9 Å resolution for complexes NS5B/A and NS5B/B, respectively. Crystallographic data and further details of the data collection and statistics are given in Table I.

                              
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Table I
Crystallographic data for HCV NS5B/inhibitor complexes

Structure Determination and Refinement-- The correct orientation and position of NS5B in the unit cell were determined by molecular replacement methods with the CNS package (30) using 1C2P as an initial model. The inhibitors were located unambiguously in sigma A-weighted |Fo- |Fc| and 2|Fo| - |Fc| electron density maps. Refinement of coordinates and atomic temperature factors was carried out using the CNS package and a maximum likelihood target. Bulk solvent corrections and anisotropic B-factor scaling were also applied in the refinement. Model rebuilding was performed using XtalView (31). Model geometry was checked using PROCHECK (32). Solvent molecules were added into the model for the NS5B/A complex. No attempt was made to include solvent for the NS5B/B complex because of the lower resolution limit (2.9 Å). The main refinement statistics are listed in Table I. The refined atomic coordinates and B-factors have been deposited in the Protein Data Bank (accession codes 1NHU and 1NHV for the complexes NS5B/A and NS5B/B, respectively).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Effects of Non-nucleoside Inhibitors on NS5B Activity-- Both compounds A and B were found to inhibit NS5B polymerization activity in a dose-dependent manner. In an assay using poly(rA)/oligo(dT)15 as a homopolymeric template/primer, both compounds were found to act as noncompetitive inhibitors with respect to the UTP substrate, with a common Ki value of 2.2 µM (Fig. 1b).


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Fig. 1.   a, chemical structures of non-nucleoside inhibitors A and B. b, Dixon plot of the inhibition of polymerase in the presence of A or B. NS5B activity was measured as described under "Experimental Procedures" using poly(rA)/oligo(dT)15 as template/primer. The reactions were performed in the presence of increasing concentrations of UTP substrate (7.5-60 µM) and in the absence or presence of increasing concentrations (0.75-3.75 µM) of inhibitors.

Binding Site-- As in other nucleic acid polymerases, the domain arrangement of NS5B can be described in terms of the fingers, palm, and thumb subdomains of a right hand (Fig. 2a) (33). One of the unique features of the HCV NS5B structure is that two loops of the fingers subdomain extend to pack against the thumb subdomain, which, together with the palm subdomain, encircles the polymerase active site (14). The NS5B polymerase active site is located in the palm subdomain, as seen in all known polymerases. Two conserved aspartic acid residues (Asp220 and Asp318 for HCV NS5B) in the active site coordinate two Mg2+ ions during the polymerization reaction. The non-nucleoside inhibitors of the present study are bound in a wedge-like fashion to a hydrophobic binding pocket located near the second last helix in the C-terminal region of the thumb subdomain (Fig. 2, a and b). This inhibitor-binding site is ~35 Å away from the polymerase active site. The omit electron density maps (Fig. 3) clearly reveal the orientation and conformation of regions I, II, and III of both inhibitors (Fig. 1a). Region IV of both inhibitors is poorly defined in the omit map, indicating the presence of multiple conformations for that part of the inhibitor.


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Fig. 2.   a, a ribbon diagram of HCV NS5B/compound A complex structure. The protein is colored according to subunit and domain. Two conserved aspartic acid residues at the polymerase active site are shown in ball and stick fashion. The inhibitor A is represented as a CPK model with carbon atoms in green. b, the binding pockets in thumb subdomain. The main chain nitrogen and alpha carbon atoms of Ser476 and Tyr477 are represented as stick models. The two hydrogen bonds between oxygen atoms of the carboxylate group on the inhibitor and main chain amide nitrogen atoms on NS5B (Ser476 and Tyr477) are indicated by dashed cyan lines. The side chains of residues Tyr477, Arg422, Met423 (carbon atoms in purple), and Trp528 (gray) of NS5B are shown as stick models, which form the primary bonding pocket interacting with the dichlorophenyl group of inhibitor A. c, a stereo view of the inhibitor-binding site of NS5B/A complex aligned with the native structure (1C2P) of NS5B. The carbon atoms of the inhibitor, the protein in complex, and the native protein are represented by green, gray, and brown, respectively. The dashed lines indicate hydrogen bonds. d, a schematic drawing showing the interactions between inhibitor A and NS5B polymerase. The red residues interact with inhibitor through main chain atoms primarily. The hydrogen bonds and van der Waals contacts are represented by cyan and dark dashed lines, respectively. The closest van der Waals contact distances between compound A and the enzyme are given in Ångstroms. e, conservation of residues in the inhibitor-binding pocket. a-c were generated using Molscript (45) and rendered using Raster3D (46).


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Fig. 3.   Stereo views of simulated annealed omit |Fobs- |Fcalc| electron density maps contoured at 3sigma level for inhibitor A complex (a) and inhibitor B complex (b). The maps were calculated to 2.0 and 2.9 Å resolution for A and B, respectively. The final refined inhibitor models are superimposed on the maps. The atoms of the inhibitors and the protein are indicated by thick (carbon atoms in green) and thin (carbon atoms in gray) lines, respectively. The main chain atoms of Trp528 have been omitted for clarity. The figures were generated using BobScript (47) and were rendered using Raster3D (46).

The inhibitor-binding site consists of two hydrophobic pockets and a pair of adjacent main chain amide groups. The primary binding pocket makes predominantly hydrophobic interactions with the 2,4-dichlorophenyl group (region I, Fig. 1a) of both inhibitors. The bottom of the pocket is formed by the side chain of Arg422, and the surrounding walls of the pocket are formed by the side chains of Trp528, Met423, Leu419, and Tyr477 and the main chain atoms of His475 and Leu474 (Fig. 2, b and c). This pocket exists in the native HCV NS5B structure and has the appropriate size to accommodate a phenyl group and its substituents. Adjacent to the primary binding pocket are two main chain amide nitrogen groups (Ser476 and Tyr477) that form hydrogen bonds with the carboxyl group (region II, Fig. 1a) of both inhibitors. These two binding features form the key components of the inhibitor-binding site. In addition, there is a second hydrophobic surface-binding pocket that accommodates the phenyl ring of region III (Fig. 1a). Region IV of the inhibitors makes only a few minor contacts with the enzyme. Both inhibitors bind to the enzyme at the same binding site and in the same manner as expected on the basis of their similar chemical structures. The following discussion will focus on the NS5B/compound A complex, because its structure was refined to higher resolution.

Inhibitor/Enzyme Interactions-- Both inhibitors make a series of hydrogen bonding, hydrophobic, and van der Waals interactions with a shallow binding pocket on HCV NS5B, as shown schematically for compound A in Fig. 2d. One of the most significant interactions is a pair of hydrogen bonds that are formed between the carboxyl group on the inhibitors and the main chain peptide nitrogen atoms of residues Ser476 and Tyr477. The three atoms of the carboxyl group and the two main chain nitrogen atoms roughly define a plane. The 2,4-dichlorophenyl group (region I) of each inhibitor has extensive favorable interactions with seven residues. Extensive van der Waals and hydrophobic interactions are made with the side chains of Leu419, Met423, Tyr477, and Trp528, as well as the Calpha and Cbeta atoms of His475. In addition, the positively charged guanidinium group of Arg422 forms favorable electrostatic interactions with the electronegative 4-chlorine atom of the dichlorophenyl group, and the electronegative carbonyl oxygen atom of Leu474 forms a short 3.6 Å contact with the positively charged edge of the aromatic ring.

Hydrophobic contacts of 3.9 to 4.2 Å exist between the phenyl ring (region III) of the inhibitor and the side chain of Leu497. The Cdelta atom of Ile482 makes a contact with the carboxylate group of the inhibitor with a distance of 4.2 Å. Although the side chain of Ile482 has the correct orientation to interact with the phenyl ring (region III) of the inhibitor, the closest approach is only 4.7 Å. The 3-trifluoromethylphenyl group (region IV) of compound A is located above the protein surface, with contact distances of 3.2 to 4.2 Å to Arg501. Region IV is not well defined in the electron density map. The inhibitor-binding site forms a basic patch in the region of the thumb subdomain (Fig. 4). Electrostatic interactions between the positively charged protein surface and the negatively charged inhibitor likely further enhance binding.


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Fig. 4.   A surface representation of the region of NS5B to which inhibitor A binds. The inhibitor is shown as a stick model and the atom color-coding is as follows: carbon, green; nitrogen, blue; oxygen, red; and fluorine, cyan. The surface electrostatic charge is depicted qualitatively. Negative and positive regions of the electrostatic potential of the surface are represented by red and blue coloring, respectively. The figure was prepared using GRASP (48).

The enzyme undergoes only minor conformational changes upon inhibitor binding, as shown when the inhibitor-bound complex structure is superimposed on the HCV NS5B native structure (Fig. 2c). The 2-chlorine atom of the 2,4-dichlorophenyl group of the inhibitor flips the Sdelta and Cepsilon of the Met423 side chain away from the primary binding pocket. There are multiple conformations of the Met423 side chain observed in NS5B native structure (1C2P). The side chain of Arg501 is pushed away by the 2,4-dichlorophenyl group of inhibitor. The guanidinium group of the side chain of Arg501 is not well defined in the electron density map, which may indicate the mobile nature of this residue. No clear main chain movement in NS5B is observed (The r.m.s. deviation between 557 Calpha atoms (NS5B/compound A complex versus NS5B native (1C2P)) is 0.3 Å.).

In addition to the chemical, hydrophobic, and physical interactions, shape recognition plays an important role in facilitating the specificity of protein/inhibitor binding as well (Fig. 4). For both inhibitors, the 2,4-dichlorophenyl group (region I) occupies the primary binding pocket of NS5B. Region I is connected to the carboxyl group (region II) of both inhibitors by a cis-amide group. The carboxylate group in turn forms two hydrogen bonds with main chain nitrogen atoms of Ser476 and Tyr477. This is the main topological feature of both inhibitors. Inspection of the binding site also reveals that region IV of inhibitors (trifluoromethylphenyl group of A and the thiophene moiety of B) is basically floating above the protein surface without any strong interactions to the protein except to Arg501. This results from the multiple rotamers of region IV of both inhibitors and explains the poorly defined electron density around that area. The interaction between region III of inhibitors and NS5B is also not optimal, suggesting that further chemical optimization efforts should be focused on region III. As indicated above, region IV of the inhibitors does not appear to contribute significantly to the binding, but its presence may be required to position the other regions in the proper conformation. Overall, regions I and II of the inhibitors are the key to NS5B/inhibitor recognition. The amide bridge connecting regions I and II has a cis-conformation in the observed binding state. The trans-isomer would not be expected to bind to the NS5B polymerase efficiently. The C-N bond of an amide group cannot rotate freely, which imposes some constraints on the stereochemistry of the inhibitor. Although this constraint may hinder the adoption of the preferred conformation of the inhibitor, it may also predispose a conformation that is roughly optimal prior to interaction with the protein. In our search for better inhibitors, it is therefore a worthwhile endeavor to design molecules that can adopt a conformation similar to the one depicted above and to characterize the structures of the new complexes.

Conservation of Residues in the Binding Site Pocket-- The hydrophobic pocket targeted by the inhibitors reported in the current study is highly conserved in all genotypes of HCV (Fig. 2e). Residues Leu419 and Ile482 are replaced by Ile and Leu, respectively, in some serotypes, which is a conservative substitution that is not expected to interfere with the hydrophobic interactions between protein and inhibitor. In HCV serotype 5a, residue 423 is Ile instead Met, which is present in all other serotypes. This is also a conservative substitution that may even favor hydrophobic interactions between this residue and the dichlorophenyl group of inhibitor. Residue Leu497 is conserved in all serotypes except for type 6a, where Ser is found instead. In this case, the interaction between inhibitor and protein may be decreased slightly, but this residue is located in the surface binding pocket where hydrophobic interactions may not be so critical. Exchange of Arg and Lys at residue 501 is not expected to have a significant impact because of the similar electrostatic properties and structure of these two residues. Although residue 476 is not well conserved across all serotype families, it should not be a problem for inhibitor binding because it is the backbone nitrogen of residue 476 that interacts with the inhibitor. Therefore, it is expected that both inhibitors will bind to the polymerases of all genotypes on the basis of our structural analysis. The high degree of conservation in these surface binding pockets also indicates that these residues are important for normal enzyme function.

Mechanism of Inhibition-- Because the inhibitors reported in this study bind remotely from the polymerase active site (~35 Å) and act kinetically in a noncompetitive manner with respect to the UTP substrate, they must exert an indirect mode of inhibition. Non-nucleoside inhibitors of other polymerases have previously been observed to bind remotely from the active site (34). Most notably, a large class of therapeutically efficacious HIV-1 reverse transcriptase non-nucleoside inhibitors bind in a hydrophobic pocket ~10 Å from the active site. The binding of an inhibitor in this pocket on the reverse transcriptase appears to stabilize an open, inactive conformation of the polymerase. This open conformation is unable to chelate divalent metal ions at the active site and is hence unable to catalyze the phosphoryl transferase reaction. In the uninhibited enzyme, this hydrophobic pocket closes when the reverse transcriptase adopts the closed conformation capable of catalyzing the phosphoryl transferase reaction.

The HCV polymerase inhibitors reported in the present study may exert an indirect mode of inhibition analogous to that previously documented for HIV-1 reverse transcriptase. Both of the HCV polymerase inhibitors bind to a conformation of the HCV polymerase that is similar to the open, inactive conformation observed in HIV-1 reverse transcriptase and other polymerases (35). In this conformation, the active site aspartic acid residues that normally coordinate to two divalent metal ions are too far apart to coordinate to metal ions. Both HCV inhibitors also bind to a hydrophobic pocket on the surface of the thumb domain and form intimate hydrogen bonding interactions with main chain amide nitrogen atoms at residues Ser476 and Tyr477 of the thumb domain. Although the structure of the closed, active conformation of HCV polymerase has yet to be determined, it is possible that the hydrophobic inhibitor-binding pocket may close and change orientation relative to residues Ser476 and Tyr477, because the thumb reorients to produce the closed, active conformation of the enzyme. If this is the case, then the binding of inhibitors to this pocket may prevent the polymerase from undergoing a conformational change essential for enzymatic activity.

An eight-degree rotation of the thumb domain relative to the palm and thumb domains has recently been observed in the RNA-dependent RNA polymerase from the rabbit hemorrhagic disease virus (36). In the "open" conformation, the thumb domain and the portion of the palm domain against which it packs is rotated away from the active site, leaving the active site in an inactive state unable to bind to divalent metal cations, nucleoside triphosphates, and RNA. In the "closed" conformation, the thumb domain and an adjacent strand of the palm domain rotate toward the active site, resulting in an active conformation that is able to catalyze the nucleotidyl transfer reaction. Because the HCV polymerase has a very similar arrangement of palm and thumb domains to that of the polymerase from rabbit hemorrhagic disease virus, it is possible that an analogous series of conformational changes occurs in the HCV enzyme. If this is the case, then the binding of inhibitors stabilizing the open conformation will likely prevent the enzyme from adopting an active, closed conformation.

The noncompetitive inhibitors of our study may also be inhibiting HCV polymerase by interfering with the binding of the allosteric activator riboguanosine triphosphate (rGTP). High concentrations of rGTP stimulate RNA synthesis by enhancing the initiation step (37, 38). Recent high resolution crystal structures of HCV polymerase bound to metal ions and rGTP show that the allosteric binding site of rGTP is located about 30 Å from the active site at the interface between the extension of the fingers and the thumb (39). Remarkably, this allosteric binding site is only 15 Å from the binding site of our inhibitors. Because there are no common residues to both binding sites, it does not appear that our inhibitors compete for the same binding site, which is also in agreement with our NMR-based competition experiment.3 However, the proximity of the two binding sites suggests that our inhibitors may exert a second mode of inhibition by interfering with the allosteric effect exerted by the binding of rGTP.

The oligomerization of several enzymes has been reported among the nucleic acid polymerase family with the palm, fingers, and thumb substructure. In the case of the RNA-dependent RNA polymerases, it has been suggested that the oligomer of poliovirus 3D polymerase is crucial for catalytic activity (40, 41). Similar behavior for HCV NS5B polymerase has also been reported recently (42, 43). Therefore, these polymerases may catalyze RNA synthesis in a cooperative manner. Although the structural details of polymerase oligomers have not been elucidated clearly yet, some interactions between polymerase molecules, in certain crystal forms of these enzymes, have been proposed to be responsible for cooperative RNA binding and polymerization activity. For poliovirus 3D polymerase, two interfaces (I and II) have been described (44). Interface I involves extensive interactions between the front of the thumb domain and the back of the palm domain. This interface is important for RNA binding based on mutagenesis studies (41). Two interfaces (A and B) are observed for HCV NS5B polymerase as well (43). Interface A contains extensive interactions between the fingers domain and thumb domain, specifically in the region of the last two helices and the C-terminal extension, which is adjacent to our inhibitor-binding site. Thus, there is also the possibility that the non-nucleoside inhibitors reported in this study may inhibit polymerase activity by disturbing the oligomerization of the NS5B polymerase.

    ACKNOWLEDGEMENTS

The Edmonton group thanks the Canadian Institutes for Health Research and the Alberta Heritage Foundation for Medical Research for generous funding in the purchase of area detector facility.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Support by a Canadian Institutes for Health Research Senior Research Fellowship and funds from the Alberta Heritage Foundation for Medical Research.

|| Canada Research Chair in Protein Structure and Function. To whom correspondence should be addressed. Tel.: 780-492-4550; Fax: 780-492-0886; E-mail: Michael.james@ualberta.ca.

Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M209397200

2 Chan, L., Reddy, T. J., Proulx, M., Das, S. K., Pereira, O., Wang, W., Siddiqui, A., Yannopoulos, C. G., Poisson, C., Turcotte, N., Drouin, A., Alaoui-Ismaili, M. H., Bethell, R. C., Hamel, M., Bilimoria, D., and Nguyen-Ba, N. (2003) J. Med. Chem., in press.

3 C. G. Yannopoulos, P. Xu, F. Ni, and R. C. Bethell, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HCV, hepatitis C virus; NS5B, nonstructural protein 5B; r.m.s., root mean square; rGTP, riboguanosine triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Rice, C. M. (1996) in Fields Virology (Fields, B. N. , Knipe, D. M. , Howley, P. M. , Chanock, R. M. , Melnick, J. L. , Monath, T. P. , and Roizman, B., eds), 3rd Ed., Vol. 1 , pp. 931-959, Lippincott-Raven Press, Philadelphia, PA
2. Ronald, B. (2001) www.hivandhepatitis.com/hep_c/hep_c_treat.html
3. Myles, D. C. (2001) Curr. Opin. Drug Discov. Dev. 4, 411-416[Medline] [Order article via Infotrieve]
4. Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Sugimura, T., and Shimotohno, K. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9524-9528[Abstract]
5. Takamizawa, A., Mori, C., Fuke, I., Manabe, S., Murakami, S., Fujita, J., Onishi, E., Andoh, T., Yoshida, I., and Okayama, H. (1991) J. Virol. 65, 1105-1113[Medline] [Order article via Infotrieve]
6. Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M., and Rice, C. M. (1993) J. Virol. 67, 1385-1395[Abstract]
7. Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., and Thomson, J. A. (1996) Cell 87, 343-355[Medline] [Order article via Infotrieve]
8. Love, R. A., Parge, H. E., Wickersham, J. A., Hostomsky, Z., Habuka, N., Moomaw, E. W., Adachi, T., and Hostomska, Z. (1996) Cell 87, 331-342[Medline] [Order article via Infotrieve]
9. Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuehler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847[Abstract/Free Full Text]
10. Yao, N., Hesson, T., Cable, M., Hong, Z., Kwong, A. D., Le, H. V., and Weber, P. C. (1997) Nat. Struct. Biol. 4, 463-467[Medline] [Order article via Infotrieve]
11. Yao, N., Reichert, P., Taremi, S. S., Prosise, W. W., and Weber, P. C. (1999) Structure 7, 1353-1363[Medline] [Order article via Infotrieve]
12. Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K., and Miyano, M. (1999) Structure 7, 1417-1426[Medline] [Order article via Infotrieve]
13. Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R., and Rey, F. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13034-13039[Abstract/Free Full Text]
14. Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F., and Weber, P. C. (1999) Nat. Struct. Biol. 6, 937-943[CrossRef][Medline] [Order article via Infotrieve]
15. Steinkuhler, C., Koch, U., Narjes, F., and Matassa, V. G. (2001) Curr. Med. Chem. 8, 919-932[Medline] [Order article via Infotrieve]
16. Lesburg, C. A., Radfar, R., and Weber, P. C. (2000) Curr. Opin. Invest. Drugs 1, 289-296[Medline] [Order article via Infotrieve]
17. Ishii, K., Tanaka, Y., Yap, C. C., Aizaki, H., Matsuura, Y., and Miyamura, T. (1999) Hepatology 29, 1227-1235[Medline] [Order article via Infotrieve]
18. Young, D. C., and Bailey, T. R. (March 2, 2000) World Intellectual Property Organization Patent WO-0010573
19. Bailey, T. R., and Young, D. C. (March 16, 2000) World Intellectual Property Organization Patent WO-0013708
20. Gardelli, C., Giuliano, C., Harper, S., Koch, U., Narjes, F., Ontoria, J. M., Poma, M., Ponzi, S., Stansfield, I., and Summa, V. (2002) WO-0206246
21. Koch, U., Altamura, S., Tomei, L., Neuner, P. J. S., and Summa, V. (2000) WO-0006529
22. Young, D. C., and Bailey, T. R. (2000) WO-0018231
23. Dhanak, D., and Carr, T. (2001) WO-0185720
24. Beaulieu, P. L., Fazal, G., Gillard, J., Kukolj, G., and Austel, V. (2002) WO-0204425
25. Yoshida, A., Mizutani, K., and Hashimoto, H. (2001) WO-0147883
26. Devos, R., Dymock, B. W., Hobbs, C., John, Jiang, W.-R., Martin, J., Armstrong, Merrett, J. H., Najera, I., Shimma, N., and Tsukuda, T. (2002) WO-0218404
27. Ferrari, E., Wright-Minogue, J., Fang, J. W., Baroudy, B. M., Lau, J. Y., and Hong, Z. (1999) J. Virol. 73, 1649-1654[Abstract/Free Full Text]
28. Alaoui-Ismaili, M. H., Hamel, M., L'Heureux, L., Nicolas, O., Bilimoria, D., Labonte, P., Mounir, S., and Rando, R. F. (2000) J. Hum. Virol. 3, 306-316[Medline] [Order article via Infotrieve]
29. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
30. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
31. McRee, D. E. (1999) J. Struct. Biol. 125, 156-165[CrossRef][Medline] [Order article via Infotrieve]
32. Laskowski, R. J., Macarthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
33. Steitz, T. A. (1999) J. Biol. Chem. 274, 17395-17398[Free Full Text]
34. Sarafianos, S. G., Das, K., Ding, J., Boyer, P. L., Hughes, S. H., and Arnold, E. (1999) Chem. Biol. 6, R137-146[CrossRef][Medline] [Order article via Infotrieve]
35. Doublie, S., Sawaya, M. R., and Ellenberger, T. (1999) Structure 7, R31-R35[CrossRef][Medline] [Order article via Infotrieve]
36. Ng, K. K., Cherney, M. M., Lopez Vazquez, A., Machin, A., Alonso, J. M., Parra, F., and James, M. N. (2002) J. Biol. Chem. 277, 1381-1387[Abstract/Free Full Text]
37. Lohmann, V., Overton, H., and Bartenschlager, R. (1999) J. Biol. Chem. 274, 10807-10815[Abstract/Free Full Text]
38. Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., Lemm, J., and Colonno, R. J. (2000) J. Virol. 74, 851-863[Abstract/Free Full Text]
39. Bressanelli, S., Tomei, L., Rey, F. A., and De Francesco, R. (2002) J. Virol. 76, 3482-3492[Abstract/Free Full Text]
40. Pata, J. D., Schultz, S. C., and Kirkegaard, K. (1995) RNA 1, 466-477[Abstract]
41. Hobson, S. D., Rosenblum, E. S., Richards, O. C., Richmond, K., Kirkegaard, K., and Schultz, S. C. (2001) EMBO J. 20, 1153-1163[Abstract/Free Full Text]
42. Qin, W., Luo, H., Nomura, T., Hayashi, N., Yamashita, T., and Murakami, S. (2002) J. Biol. Chem. 277, 2132-2137[Abstract/Free Full Text]
43. Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J., Parsons, S., Zhang, F., Rathnachalam, R., Kirkegaard, K., and Colacino, J. M. (2002) J. Virol. 76, 3865-3872[Abstract/Free Full Text]
44. Hansen, J. L., Long, A. M., and Schultz, S. C. (1997) Structure 5, 1109-1122[Medline] [Order article via Infotrieve]
45. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
46. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524
47. Esnouf, R. M. (1997) J. Mol. Graph. Model. 15, 132-134[CrossRef][Medline] [Order article via Infotrieve]
48. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve]


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