From the 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
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ABSTRACT |
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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.
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 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.
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
Crystallization and Data Collection--
The reported
crystallization conditions were adopted with some modifications (14).
HCV NS5B 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
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).
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.
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
C
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 C
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 S
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-2a,
-2b, "consensus" interferon, and
pegylated interferon
-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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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.).
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 K
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.
Crystallographic data for HCV NS5B/inhibitor complexes
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (18K):
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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.
View larger version (38K):
[in a new window]
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).
View larger version (60K):
[in a new window]
Fig. 3.
Stereo views of simulated annealed omit
|Fobs| |Fcalc| electron density maps contoured
at 3
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).
and C
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.
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.
View larger version (95K):
[in a new window]
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).
and C
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 C
atoms (NS5B/compound A complex
versus NS5B native (1C2P)) is 0.3 Å.).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are: HCV, hepatitis C virus; NS5B, nonstructural protein 5B; r.m.s., root mean square; rGTP, riboguanosine triphosphate.
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