ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2011, p. 4196–4203
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 9
Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel
Nonnucleoside Inhibitor of Hepatitis
C Virus NS5B Polymerase?
I-hung Shih,1Inge Vliegen,2Betty Peng,1Huiling Yang,1Christy Hebner,1Jan Paeshuyse,2
Gerhard Pu ¨rstinger,3Martijn Fenaux,1Yang Tian,1Eric Mabery,1Xiaoping Qi,1
Gina Bahador,1Matthew Paulson,1Laura S. Lehman,1Steven Bondy,1
Winston Tse,1Hans Reiser,1William A. Lee,1Uli Schmitz,1
Johan Neyts,2and Weidong Zhong1*
Gilead Sciences, Inc., Foster City, California1; Rega Institute, Leuven, Belgium2;
and University of Innsbruck, Innsbruck, Austria3
Received 4 March 2011/Returned for modification 10 April 2011/Accepted 9 June 2011
GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro
and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV.
GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious
virus, suggesting that the compound’s mechanism of action involves a genotype-specific viral component. To
further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b
and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural
proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric
replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing
GT2a NS5B. GT1b replicons in which the ?-hairpin region (amino acids 435 to 455) was replaced by the
corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the
thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several
mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype.
Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number
of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against
previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different
from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel
class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the
?-hairpin in the thumb subdomain.
Hepatitis C virus (HCV) is a major cause of morbidity,
affecting approximately 170 million people worldwide with an
estimated 3 to 4 million additional new infections occurring
each year (36). HCV is a positive-strand RNA virus with six
major genotypes that are further divided into multiple sub-
types. Due to the error-prone nature of its replication enzyme,
a myriad of different viral quasispecies exists within an infected
individual (32). With this high degree of viral variability, the
current treatment regimen, which consists of weekly injections
of pegylated alpha interferon (PEG-IFN) and twice-daily oral
doses of ribavirin (RBV), is of limited efficacy and, in addition,
carries significant side effects (8, 23). Although the HCV
NS3/4A protease inhibitors telaprevir and boceprevir for treat-
ment of chronic HCV infection will soon be available, these
compounds will still need to be combined with the current
standard of care (PEG-IFN/RBV) to be efficacious and will not
cure all infected individuals (10, 14, 30). Therefore, the devel-
opment of additional direct antiviral agents with diverse resis-
tance profiles is necessary, with the ultimate goal of developing
all-oral antiviral combinations that can achieve superior sus-
tained virologic response (SVR) without the use of IFN or
RBV. Thus, major efforts are under way to identify additional
novel inhibitors of HCV. In particular, much emphasis has
been placed on the viral polymerase NS5B as a target.
Viral polymerases are attractive targets for drug discovery
and have yielded approved drugs for HIV, HBV, herpes sim-
plex virus, and cytomegalovirus. The HCV NS5B polymerase is
an RNA-dependent RNA polymerase containing canonical
thumb, finger, and palm subdomains (2, 3, 19, 37, 40). Both
nucleoside inhibitors (NIs) and nonnucleoside inhibitors (NNIs)
of NS5B have been reported in the literature and are currently
in clinical trials (4, 9, 16, 18, 26, 31, 34). NIs act as chain
terminators and tend to show pan-genotypic activity compared
to NNIs. However, efficacies of some nucleoside inhibitors in
the clinic have been marred by significant adverse events (7).
NNIs in clinical development target one of the several alloste-
ric binding sites in the NS5B polymerase with compounds that
bind in a similar manner and that demonstrate overlapping
resistance profiles. Novel NNIs with resistance traits diverse
from those already in clinical trials will be essential in the
development of effective combination therapy and in overcom-
ing viral resistance.
Recently, a novel class of substituted imidazopyridine com-
* Corresponding author. Mailing address: Gilead Sciences, Inc., 333
Lakeside Dr., Foster City, CA 94404. Phone: (650) 522-5780. Fax:
(650) 522-5890. E-mail: Weidong.Zhong@gilead.com.
?Published ahead of print on 11 July 2011.
pounds showing selective inhibition of HCV was reported (35).
Here we report on the molecular target of the most promising
member of this class, GS-9190 (Tegobuvir), which has demon-
strated antiviral activity in HCV-infected patients (1, 39). By
using chimeric replicons, kinetic comparison, and resistance
selection, we demonstrate that GS-9190 inhibits viral replica-
tion by targeting the NS5B polymerase. Furthermore, by using
data gleaned from reverse genetics and molecular modeling,
we propose that GS-9190 exploits a unique pocket on NS5B
and utilizes a novel binding mechanism to inhibit HCV repli-
MATERIALS AND METHODS
Replicon cell lines. Huh-luc and Huh7-Lunet cells were obtained from
ReBlikon GmbH (Mainz, Germany). All Huh7-Lunet-based replicon cell lines were
grown in Dulbeccos’s modified Eagle’s medium (DMEM) with GlutaMAX-I
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS;
HyClone, Logan, UT), 1 U/ml penicillin (Invitrogen), 1 ?g/ml streptomycin
(Invitrogen), and 0.1 mM nonessential amino acids (Invitrogen). Stable replicon
cell lines were selected and maintained in medium containing 0.5 mg/ml G418
(Geneticin; Invitrogen). Creation of Huh7-Lunet stable genotype 1b (Con-1) and
1a (1a H77-51) subgenomic replicon cells has been reported previously (21, 29).
Analogous stable subgenomic genotype 2a (JFH1) replicon cells were created by
electroporating Huh7-Lunet cells with RNA transcribed from a linearized bicis-
tronic construct encoding an HCV JFH1 internal ribosome entry site (IRES)-
driven neomycin cassette and encephalomyocarditis virus-controlled coding
region containing the nonstructural NS3, NS4A, NS4B, NS5A, and NS5B poly-
protein sequences. Twenty-four hours after electroporation, replicon-containing
cell clones were selected using 0.5 mg/ml G418. Following selection and clonal
expansion, a single 2a replicon clone was chosen for further experimentation
based upon sequence fidelity, replicon levels as assessed in an NS3 protease assay
(38), and 50% effective concentrations (EC50s) when tested against a panel of
antiviral control compounds.
Compounds. GS-9190 was synthesized at Gilead Sciences, Inc. (Foster City,
CA). BILN-2061 and 2?-C-methyl adenosine (2?CMeA) were purchased from
Acme Bioscience (Belmont, CA). The ViroPharma/Wyeth HCV NS5B site IV
inhibitor HCV-796 (13) was synthesized by Curragh Chemistries (Cleveland,
OH). An Abbott benzothiadiazine NS5B polymerase inhibitor (A-782759) (24)
was synthesized by ChemALong Laboratories (Lemont, IL).
Construction of mutant and chimeric replicons. NS5B point mutations were
engineered into the pFK I341 PI-Luc/NS3-3?/ET replicon construct (ReBlikon
GmbH) using the Stratagene QuikChange XL mutagenesis kit (Stratagene) and
appropriate primers. Mutagenesis was confirmed via sequencing of the NS5B
region of the plasmid (Elim Biopharmaceuticals, Inc., Hayward, CA). The NS5B
region was then subcloned into a new pFK I341 PI-Luc/NS3-3?/ET replicon
backbone by using the BclI and SpeI sites, and the subcloned NS5B region was
A plasmid carrying the chimeric 1b NS4A4B/2a replicon DNA was constructed
by replacing NS4A and NS4B in the 2a JFH strain with the corresponding region
derived from the 1b Con1 strain. NsiI and BamHI restriction sites were intro-
duced into the PCR primer set 1b NS4A PCR NsiI F (5?-ACATCATGGCAT
GCATGTCGGCTGACCTGGAGGTCG-3?; NsiI site underlined) and 1b NS5A
PCR BamHI R (5?-CAAACATCTCTTAGCCAGGATCCGGAGCATGGCGT
GGAGC-3?; BamHI site underlined), which was used to amplify the HCV-1b
NS4A-4B fragment. After digestion of the 1b PCR fragment and pJFH1 DNA
with NsiI and BamHI, the 1b NS4A-4B fragment was ligated into pJFH1 (Toray,
Inc.). The junctions of the resulting chimeric replicon (1b NS4A4B/2a) were
confirmed by DNA sequencing. Overlapping PCR was employed to obtain the
fragment containing 1b protease and 2a helicase by using the PCR product of 1b
protease and 2a helicase as templates and primer sets 1b Protease PCR MluI
TAC-3?; MluI site underlined) and 2a Hel PCR NsiI R (5?-CCTCAAGGTCA
GCTTGCATGCATGTGGCGATG-3?; NsiI site underlined) to incorporate the
restriction sites MluI and NsiI. After digestion of the PCR fragment and 1b
NS4A4B/2a DNA with MluI and NsiI, the 1b protease plus 2a helicase fragment
was ligated into 1b NS4A4B/2a.
A 2a NS5B/1b chimeric replicon was created by amplifying the NS5B-3?-
untranslated region (UTR) via PCR from the pJFH plasmid using the primers
G-3?) and 2a3?UTRSpeIrev2 (5?-AGCTTAGACTAGTACATGATCTGCAGA
GAGACCAG-3?). PCR fragments were gel purified (Qiagen), digested with
SpeI and BclI (New England BioLabs [NEB]), and ligated into an analogously
digested and purified pFK I341 PI-Luc/NS3-3?/ET vector fragment. A ?-hairpin
chimera was created using multiple rounds of site-directed mutagenesis
(QuikChange XL; Stratagene) to change amino acids 434 to 455 of the NS5B
region in pFK I341 PI-Luc/NS3-3?/ET to that of genotype 2a (JFH1). The NS5B
region was then subcloned into a fresh vector backbone by using BclI and SpeI
restriction sites. All constructs were sequenced to confirm sequence fidelity.
Transient transfections. DNA plasmids carrying replicon sequences were lin-
earized in vitro using SpeI restriction endonuclease (NEB) followed by electro-
phoresis and gel purification of the linearized fragment (QIAquick gel extraction
kit; Qiagen). Replicon RNA was derived from the purified template by using T7
runoff transcription (MEGAscript T7 kit; Ambion). For transfection of RNA
into Huh7-Lunet cells, cells were trypsinized and washed three times with phos-
phate-buffered saline (PBS). A suspension of 4 ? 106cells in 400 ?l PBS was
mixed with 10 ?g RNA and subjected to electroporation at settings of 270 V and
950 ?F capacitance. Cells were then transferred into 20 ml of prewarmed culture
medium and seeded into appropriate plates for further analyses.
Antiviral EC50and CC50determinations. Replicon-containing cells were
trypsinized and seeded in cell culture medium without G418 in white or black
96-well plates for EC50or 50% cytotoxic concentration (CC50) analyses, respec-
tively. Stable replicon cell lines were seeded at a density of 5,000 cells per well,
while Huh7-Lunet cells transiently tranfected with replicon RNA were seeded at
20,000 cells per well postelectroporation. Ten-point dilutions of compounds were
performed in dimethyl sulfoxide (DMSO) followed by further dilution in cell
culture medium and subsequent addition to cell plates. Compound-treated cells
were incubated for 72 h at 37C in a 5% CO2incubator. Following incubation,
CC50values were determined by using a commercial reagent according to the
manufacturer’s specifications (Cell Titer Glo; Promega, Madison, WI). For lu-
ciferase-containing replicons, luciferase expression following compound treat-
ment was quantified using a commercially available assay system (Luciferase
assay system; Promega). For replicon cell lines not containing a luciferase
reporter, antireplicon efficacy was determined using an NS3 protease assay
described previously (38). Antiviral effects against a cell culture-adapted
genotype 2a (J6/JFH) infectious virus were determined as previously reported
(27). Curve fitting and EC50/CC50values were derived using nonlinear regression
analyses (GraphPad Prism). Curves were extrapolated from duplicate points, and
all experiments were individually performed at least twice.
NS5B enzymatic assays. GS-9190 inhibition of NS5B-dependent RNA poly-
merization activity was determined using two assay formats in vitro. The first
format utilized a heteropolymeric RNA template described previously (12). The
reaction was assembled in 50 mM Tris-HCl (pH 7.5), 10 mM KCl, 5 mM MgCl2,
1 mM dithiothreitol (DTT), 1 mM EDTA, 40 ng/?l RNA template, 0.5 ?Ci of
[?-33P]nucleoside triphosphate, and 500 ?M remaining nucleoside triphosphates
(NTPs). Purified NS5B enzyme was preincubated with compounds for 20 min at
34°C, after which the reaction was started by the addition of RNA template and
NTPs. The radiolabeled NTP was used at the Kmconcentration. The reaction
was allowed to proceed for 90 min and then the mixture was transferred onto
96-well DE81 filter membranes, washed three times with 100 mM Na2HPO4and
once with ethanol, and dried. Scintillation fluid was added to the wells, and
counts per minute (cpm) were measured in a TopCount apparatus.
The second assay format utilized a poly(C) RNA template and GTP as the
initiation nucleotide (20). The reaction mixture was assembled with 20 mM
HEPES (pH 7.3), 12 mM KCl, 50 mM NaCl, 1 mM DTT, and 10 mM MgCl2–5
mM MnCl2, 40 ng/?l RNA template, 1 ?Ci of [?-33P]GTP, and 50 ?M GTP.
Purified enzyme was preincubated with compounds for 15 min at 34°C, after
which the reaction was started by the addition of RNA template and GTP. The
reaction was allowed to proceed for 60 min, and then the reaction mixture was
transferred to 96-well DE81 filter membranes, washed three times with 100 mM
Na2HPO4and once with ethanol, and dried. Scintillation fluid was added to the
wells, and radioactivity was measured on a TopCount analyzer (Perkin-Elmer).
Resistance selection. Huh-Luc cells were seeded in 10-cm tissue culture dishes,
grown to subconfluency and then treated with Geneticin and GS-9190 at various
concentrations (G418, 0.1 to 1 mg/ml; GS-9190, 0.05 to 10 ?M). Medium was
changed twice weekly, and cells were passaged when necessary to maintain
subconfluent levels. After 2 to 3 weeks, cells began to die and small colonies
began to form. Colonies were further cultured in the presence of drugs for an
additional 8 to 10 weeks. Expanded colonies of resistant cells were characterized
by phenotypic evaluation to confirm reduced susceptibility to GS-9190. RNA was
extracted from clones using the RNeasy midikit (Qiagen). Amplification of
replicon DNA was achieved using Qiagen’s one-step reverse transcription-PCR
(RT-PCR) kit and combinations of HCV-specific primer pairs. Product forma-
VOL. 55, 2011 MECHANISTIC CHARACTERIZATION OF GS-91904197
tion was confirmed by agarose gel electrophoresis, and DNA products were
submitted for sequence analysis (ELIM Biopharmaceuticals, Hayward, CA).
Quantitative RT-PCR. Huh-9-13, a Huh7 cell line carrying the persistent
I377/NS3-3?/wild-type replicon (genotype 1b), was obtained from ReBLikon
GmbH (Mainz, Germany). The Huh-9-13 cells were plated at 1 ? 105cells/well
in 6-well plates in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% FBS
(HyClone, Logan, UT), 100 U/ml penicillin, 100 ?g/ml streptomycin, and 0.1 mM
nonessential amino acids (Invitrogen, Carlsbad, CA) including 1 mg/ml G-418.
At 24 h after plating, the medium was replaced with G418-free medium and the
cells were treated with 50 nM BILN-2061, 150 nM HCV-796, 30 nM GS-9190, or
the equivalent concentration of DMSO as a negative control. Cell samples were
collected at 1, 3, 6, 9, 12, 16, and 24 h posttreatment. Total RNA was extracted
with the RiboPure kit (AM1924; Life Technologies Corporation, Carlsbad, CA)
following the manufacturer’s protocol. Extracted RNA samples were stored at
?80°C until use. For the quantitative RT-PCR (QRT-PCR) assay, the Qiagen
one-step QRT-PCR kit was used according to the manufacturer’s protocol
(Qiagen, Valencia, CA). The genotype 1b HCV NS3 gene-specific primers
forward primer NS3_180FL (5?–CGGCGGACTGTCTATCATGGTGCG–6-
carboxyfluorescein [FAM]–3?) and reverse NS3_180 (5?–GGTCCTGGTCCAC
ATTGGTGT-3?) and 18S rRNA LUX (FAM) endogenous control primer set
(115HM-01) were produced by Invitrogen Corporation (Carlsbad, CA). For the
reverse transcriptase step, the reaction mixtures were incubated at 44°C for 30
min, and the reverse transcriptase enzyme was then degraded by heating the
sample to 94°C for 10 min. The QPCR step included 38 cycles at 94°C for 15 s
and 58°C for 30 s.
Modeling. Docking studies were carried out with Accelrys’ DiscoveryStudio
2.55 and 3.0 using the default settings of the CDOCKER and LigandFit algo-
rithms. Best results were obtained when the NS5B central cavity was partitioned
into 10 subsites. Figure 6, below, was prepared by aligning the backbone atoms
of crystal structures 3FKQ and 3HKK. All ligands are shown within the 3FKQ
Activity of GS-9190 in HCV replicon and infectious virus
assays. GS-9190 is a novel imidazopyridine analogue derived
from a previously described series of pestivirus replication in-
hibitors and optimized for selective inhibition of HCV repli-
cation (28, 35) (Fig. 1). To assess the anti-HCV activity of
GS-9190, the compound was evaluated for inhibitory effects on
the replication of genotype 1a and 1b subgenomic replicons
along with NS3 protease inhibitor BILN-2061 (17), the nucle-
oside inhibitor 2?CMeA (6), and the nonnucleoside inhibitor
HCV-796 (13) (Table 1). GS-9190 proved to be a highly potent
and selective inhibitor of genotype 1 replicons, displaying sub-
nanomolar to nanomolar potencies as measured by the EC50,
with minimal cytotoxicity in all cell lines tested (50% cytotoxic
concentration of ?50 ?M). Similar to BILN-2061 and HCV-
796, although GS-9190 was highly active against both the 1a
and 1b genotypes, potency against 1b was consistently 5- to
10-fold higher than that against genotype 1a, suggesting that
genotypic differences may influence GS-9190 antiviral activity.
To further gauge cross-genotype antiviral activity, GS-9190 was
also evaluated against a genotype 2a (JFH-1) replicon. Inter-
estingly, GS-9190 lost more than 1,500-fold activity over geno-
type 1a and over 20,000-fold over genotype 1b in the genotype
2a replicon assay. To further confirm the reduced effectiveness
of GS-9190 against genotype 2a, the compound was also tested
against a cell culture-adapted infectious virus (J6/JFH-1). As
was observed in the 2a replicon assay, GS-9190 displayed a loss
in potency against the genotype 2a virus with an EC50of 2.9
?M. Taken together, these results show that GS-9190 is a
potent inhibitor of HCV genotype 1 and suggest that differ-
ences in the nonstructural protein region between genotypes
may influence compound potency.
The NS5B genotype determines susceptibility to GS-9190.
We next attempted to identify which HCV nonstructural pro-
tein(s) may be involved in the GS-9190 mechanism of action.
In biochemical assays, no or poor activity was observed when
GS-9190 was tested against the NS3/4A serine protease, NS3
RNA helicase, NS5B polymerase (using both heterologous and
homopolymeric RNA as template), and HCV IRES-mediated
translation (Table 2 and data not shown). Although GS-9190
lacked activity in these in vitro assays, it is possible that GS-
9190 may possess an inhibitory function requiring the context
of viral replication complexes and/or cellular factors. We took
FIG. 1. Structures of GS-9190, HCV-796, and A-782759.
TABLE 1. Antiviral activities of GS-9190 and other known HCV inhibitors against HCV genotypes 1 and 2a
13.8 ? 9.6
0.8 ? 0.7
21,900 ? 19,000
7.8 ? 5.0
0.8 ? 0.7
73.0 ? 43.3
420.5 ? 306.5
217.5 ? 90.8
420.3 ? 94.2
15.0 ? 7.1
4.0 ? 2.7
144.0 ? 71.1
2,900 ? 1250
86.9 ? 34.0
527.5 ? 324.7
104.1 ? 15.1
aData shown are the CC50(in nM) rather than the EC50.
4198 SHIH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
advantage of the apparent activity differences between geno-
types 1b and 2a replicons for GS-9190 and constructed a series
of chimeric replicons incorporating mixtures of viral proteins
derived from the 1b and 2a genotypes (Fig. 2). GS-9190 was
tested against the chimeric and wild-type replicons in transient
assays along with appropriate control compounds. Interest-
ingly, the predictive determinant of GS-9190 activity in these
chimeric replicons appeared to be associated with the NS5B
polymerase gene. Introduction of the GT2a NS5B gene alone
into the 1b replicon drastically reduced the susceptibility of
GS-9190 (?6,000-fold) to a level similar to that of the GT2a
replicon. Hence, these findings imply that the NS5B genotype
is a main contributing determinant of GS-9190 susceptibility
and also that the compound may target the NS5B polymerase
to inhibit HCV replication.
To further explore the inhibitory nature of GS-9190, we
analyzed the kinetics of replicon RNA inhibition by using a
real-time RT-PCR detection method (33). The drop in repli-
con RNA copy number following GS-9190 treatment was mon-
itored over time compared to the protease inhibitor BILN-
2061 and NNI HCV-796 (Fig. 3). BILN-2061 is expected to
have slower inhibition kinetics, since it prevents assembly of
new replication complexes (RC) but has no effect on preexist-
ing RC. In contrast, a direct inhibitor of the NS5B polymerase
is expected to have faster kinetics since it can inhibit the ac-
tivity of existing RC. Using seven time points in a 24-hour
period, we found that the kinetics of replicon inhibition fol-
lowing GS-9190 treatment was nearly identical to that ob-
served following treatment with HCV-796, further suggesting
that GS-9190 does not function as a protease inhibitor and,
rather, may directly act on the RNA replication machinery.
NS5B mutations confer resistance to GS-9190. To further
confirm that NS5B polymerase is the target of inhibition by
GS-9190, we performed drug resistance selection using stable
genotype 1b replicon cells treated with various concentrations
of GS-9190. Individual colonies resulting from the selection
were expanded, followed by a phenotypic assay to assess resis-
tance to GS-9190. The nonstructural coding regions from re-
sistant replicons were then reverse transcribed and sequenced
to identify deviations from the wild-type sequence that may
confer compound resistance. Interestingly, although several
mutations were found in the NS3, NS4B, and NS5A genes, the
majority of mutations uncovered were clustered in the NS5B
region (Table 3). All found mutations were then individually
reengineered into the wild-type replicon, and the susceptibility
to GS-9190 was determined. None of the mutations detected in
NS3, NS4B, or NS5A individually conferred resistance to GS-
9190. However, several mutations in NS5B, C316Y, C445F,
and Y452H, significantly reduced susceptibility to GS-9190,
each resulting in a 7- to 10-fold shift over the wild-type geno-
type 1b replicon. Moreover, an independent resistance selec-
tion using a closely related GS-9190 analogue identified yet
TABLE 2. IC50values of GS-9190 in NS5B-dependent
polymerase assays in vitro
aND, not done.
FIG. 2. The NS5B genotype determines susceptibility to GS-9190.
Chimeric replicons containing mixed combinations of genotype 1b and
2a nonstructural gene sequences were tested transiently in Huh7-
Lunet cells. Chimeras were analyzed in a minimum of two independent
FIG. 3. The kinetics of RNA reduction following GS-9190 treat-
ment is similar to that of the NNI HCV-796. 1b replicon cells were
treated with inhibitors at 30 times the EC50, and total RNA was
extracted and subjected to real-time analyses at various time points
following treatment. Data shown are averages of samples run in trip-
TABLE 3. Mutations in NS5B genes confer resistance to GS-9190a
Mutation in GS-9190r
Fold shift in GS-9190
aWT, wild type. *, Y488H was the result of independent resistance selection
using a closely related GS-9190 analogue.
VOL. 55, 2011 MECHANISTIC CHARACTERIZATION OF GS-91904199
another NS5B mutation, Y448H (denoted by the * in Table 3).
When Y448H was engineered into the wild-type 1b replicon,
susceptibility to GS-9190 was reduced 36-fold, the most pro-
nounced effect imparted by any single mutation tested. In
addition to analyzing the effect of individual NS5B mutations
on GS-9190 resistance, mutations were also tested in combi-
nations (Fig. 4). Combining NS5B mutant residues led to an
even greater degree of resistance. Most strikingly, although
less-than-10-fold resistance was observed when C316Y, C445F,
and Y452H were individually engineered into the replicon,
combining these mutations led to a nearly 1,000-fold change in
EC50. Taken together, these results indicate that mutations in
the NS5B gene alone are sufficient to confer resistance to
GS-9190, and they further corroborate that the NS5B polymer-
ase is the target by which GS-9190 exhibits its antiviral effects.
The ?-loop region in the NS5B thumb subdomain is in-
volved in the GS-9190 interaction. In order to more precisely
define which region of NS5B might be involved in interacting
with GS-9190, we compared the amino acid sequences of ge-
notypes 1b, 1a, and 2a NS5B in the regions of resistance mu-
tations identified from resistance selection experiments. The
?-hairpin region between amino acids 435 and 455 was of
particular interest for further study since it is characterized by
sequence similarity between genotypes 1a and 1b yet a high
degree of divergence between genotypes 1b and 2a (Fig. 5A).
To test whether the ?-hairpin region played a role in the
differential susceptibility of genotype 1 and genotype 2a repli-
cons to GS-9190, we constructed and tested a chimeric replicon
in which all viral nonstructural protein sequences were derived
from genotype 1b except for the ?-hairpin region, which was
changed to that of genotype 2a (Fig. 5B). The ?-hairpin chi-
meric replicon was 97-fold less sensitive to GS-9190 than the
wild-type 1b replicon. In contrast, no change in susceptibility to
the nucleoside polymerase inhibitor 2?CMeA was observed.
With this intriguing finding, we then went on to test two known
NNIs that bind to the NS5B palm subdomain: HCV-796, which
binds to NNI site IV, and the benzothiadiazine inhibitor
A-782759, which binds to NNI site III (Fig. 1). HCV-796,
known to bind near the catalytic active site of NS5B, was
29-fold more potent against the wild-type 1b replicon than
against the 1b/2a NS5B chimeric replicon. However, consistent
with the reported binding mechanism, HCV-796 remained
equipotent against the ?-hairpin chimera. The benzothiadia-
zine A-782759 also displayed a significant decrease in potency
against the 1b/2a NS5B chimera compared to the wild-type 1b
replicon. However, like GS-9190, the potency of A-782759 was
significantly decreased against the ?-hairpin chimera. This re-
sult is in agreement with the resistance mutation data for
A-782759, showing that a mutation in the ?-hairpin (Y448H)
confers resistance (24). The notable parallel activities between
GS-9190 and A-782759 against the chimeric replicons suggest
that the antiviral activity of GS-9190 likely results from inhi-
bition of NS5B via interaction with a pocket that overlaps with
the binding site of A-782759 and involves the ?-hairpin.
Cross-resistance studies demonstrated a unique resistance
profile for GS-9190. We next wanted to test the cross-resis-
tance of GS-9190 against previously reported NS5B mutations
selected by other NNIs. Noting the possibility of an overlap-
ping binding site, insinuated by the ?-hairpin chimeric data, we
also tested the site III NNI A-782759 and the site IV NNI
HCV-796 (Fig. 1). The antiviral effects of these three com-
pounds were analyzed against replicons containing either
the NS5B site I mutation P495L, the site II mutation M423T,
the site III mutation M414T, the site IV mutation Y448H,
or the palm site mutation C316Y (Table 4). As previously
reported, resistance to HCV-796 only occurred in replicons
carrying the C316Y mutation, consistent with the binding re-
gion of the compound. The site III inhibitor A-782759 also
demonstrated a resistance profile consistent with the literature
in which the primary site of resistance is at M414, with Y448H
and C316Y also conferring lower levels of resistance as well.
Like HCV-796 and A-782759, GS-9190 showed some degree of
EC50shift in response to C316Y, albeit significantly less than
observed with the other NNIs. Like A-782759, significant re-
FIG. 4. The degree of GS-9190 resistance correlates with the num-
ber of NS5B mutations. NS5B mutations were engineered either alone
or in combination via site-directed mutagenesis into wild-type 1b rep-
licons. The antiviral phenotype of the constructs was evaluated in
Huh7-Lunet cells transiently transfected with the constructs to deter-
mine the effect on the GS-9190 EC50. Resistance was characterized as
the average fold change in the GS-9190 EC50versus the wild-type
genotype 1b replicon from multiple experiments. Data are mean
values ? standard deviations for at least two independent experi-
FIG. 5. Replacement of the genotype 1b ?-hairpin sequence in
NS5B with that of genotype 2a (amino acids 435 to 455) significantly
reduces replicon susceptibility to GS-9190. The antiviral phenotype of
chimeric replicons was assessed in transiently transfected Huh7-Lunet
cells and analyzed 72 h post-inhibitor addition. EC50s shown are av-
erages of at least two independent experiments, each carried out in
4200 SHIH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
sistance to GS-9190 occurred in response to the Y448H mu-
tant. However, unlike A-782759, replicons bearing the M414T
mutation were still susceptible to GS-9190, suggesting that
GS-9190 does not interact with NS5B in a manner identical to
A-782759. Taken together, the resistance profile of GS-9190
appears to be unique among NNIs, sharing only partial resis-
tance features with the two NNIs interacting with this region of
Model of the GS-9190 interaction site in NS5B. Based on the
above biological data and the apo-enzyme crystal structure of
NS5B, we conducted modeling to explore the possible inter-
actions between GS-9190 and NS5B. Despite the absence of
viral polymerase inhibition in biochemical assays, the overlap
in the resistant mutations with site III and IV inhibitors led us
to probe for potential GS-9190 interaction sites in the NS5B
cavity. Using various publicly available crystal structures (PDB
1C2P, 3FKQ, 3HKK, and 1GX5), we employed docking meth-
ods to identify a potential high-affinity site for GS-9190. The
only structure to produce an orientation with significant dock-
ing scores was 3FKQ (Fig. 6A). Here, GS-9190 is partially
inserted into the site IV inhibitor pocket and extends across
the site III inhibitor pocket along the ?-hairpin. This pose is
most consistent with the mutations observed and the general
structure-activity relationships exhibited by this compound se-
ries (unpublished data). In the absence of evidence of a direct
interaction of GS-9190 with NS5B, Fig. 6B illustrates how the
putative GS-9190 binding site would encompass parts of the
space occupied by both site III and site IV inhibitors. However,
we must give serious caution that the hypothetical interactions
between NS5B and GS-9190 might not reflect the actual inter-
action mode based on the fact that the NS5B crystal structure
was derived from bacterially expressed recombinant NS5B
proteins for which GS-9190 failed to demonstrate inhibitory
activity in biochemical assays. Nonetheless, these interac-
tion models may shed some light into the possible mode of
interaction between NS5B polymerase and GS-9190.
The combining of multiple direct-acting antiviral agents with
diverse resistance profiles will be the key to a truly effective
direct antiviral therapy. Here we have reported on a novel
HCV-specific inhibitor, GS-9190 (Tegobuvir), that is highly
potent against genotype 1 HCV, the most prevalent genotype
in the Western world and one that is significantly refractory to
the current standard of care. Although clearly possessing an-
tiviral activity as demonstrated in vitro and in HCV-infected
patients (1, 39), little was known about the exact mechanism of
action of GS-9190 and other imidazopyridine analogues. Like
most other anti-HCV compounds that have been optimized
specifically for activity against genotype 1, GS-9190 exhibits
reduced potency against genotype 2a. Here we were able to
take advantage of this genotype-dependent difference in po-
tency to explore the mechanism by which GS-9190 inhibits
As mentioned, previous analyses of GS-9190 in in vitro as-
says for effects on the NS3 serine protease, NS3 RNA helicase,
IRES-directed translation, and NS5B polymerase showed no
inhibition, suggesting that GS-9190 may be dependent on the
cellular context of HCV replication for activity. We therefore
created a series of chimeric replicons carrying various combi-
nations of nonstructural proteins derived from genotypes 1b
and 2a and assessed their antiviral phenotypes in a transient
cell-based replicon assay. Using the combined data from the
chimeras, we found that the NS5B genotype defined sensitivity
to GS-9190, identifying the NS5B polymerase as a likely target
TABLE 4. Cross-resistance of GS-9190 with common NS5B
Fold shift in EC50vs. WT
FIG. 6. Illustration of the NS5B cavity encompassing allosteric sites III and IV and the putative region targeted by GS-9190. (A) The right-hand
model of the NS5B structure (PDB 3FKQ) (color coding: the palm is red, thumb is green, fingers are blue, tail is gray, and ?-hairpin is mauve)
with GS-9190 modeled in a potential pocket. (B) Close-up of GS-9190 (blue) in the putative binding region along with site IV inhibitor HCV-796
(gray) and site III inhibitor A-782759 (black). Positions of mutations moderating GS-9190 antiviral activity are indicated by their ? carbon.
VOL. 55, 2011 MECHANISTIC CHARACTERIZATION OF GS-91904201
of activity. This conclusion was further corroborated by a ki-
netics experiment using real-time quantitative PCR to measure
replicon RNA inhibition, in which GS-9190 behaved in nearly
an identical manner to the NS5B NNI HCV-796 and faster
than the NS3 protease inhibitor BILN-2061, suggesting GS-
9190 inhibits replication.
To further identify the mechanism of action of GS-9190, we
performed resistance selections in genotype 1b replicon cells.
Replicon sequences were obtained from phenotypically resis-
tant colonies, and individual mutations were reengineered into
wild-type 1b replicons. Using this method, we uncovered that
the only mutations conferring GS-9190 resistance were those
found in NS5B. Specifically, C316Y, C445F, and Y452H indi-
vidually induced 7- to 10-fold reductions in susceptibility to
GS-9190. When a closely related analogue of GS-9190 was
used to select for drug resistance, an additional mutation,
Y448H, was identified. This particular mutation resulted in
36-fold resistance to GS-9190. Furthermore, combining these
mutations had an even greater effect on GS-9190 potency.
Taken together, these data pinpoint NS5B as the target by
which GS-9190 enacts antiviral activity. In addition, by using a
?-hairpin chimeric replicon, we found specifically that the 20-
amino-acid of the ?-hairpin region of the polymerase thumb
subdomain played a significant role in establishing GS-9190
potency. The involvement of this region as a site for inhibition
of HCV replication is not unprecedented, as a secondary re-
sistance mutation for the benzothiadiazine NNI A-782759 has
previously been reported to reside in the ?-hairpin (Y448H)
(24). We confirmed this finding in our study, as A-782759 was
markedly less active against the ?-hairpin chimeric replicon.
Interestingly, the amino acid Y448 was not changed in this
chimera, suggesting that resistance arising from sequence mod-
ification within the ?-hairpin may be due to gross conforma-
tional change versus alterations of compound interactions with
specific residues. Cross-resistance analyses of the site III in-
hibitor A-782759 and site IV inhibitor HCV-796 with GS-9190
revealed that GS-9190 has a different drug resistance profile
from that of the known site III and site IV inhibitors, further
implying that GS-9190 has an NS5B interaction mechanism
distinct from other NNIs.
Using the NS5B crystal structure, we were able to map these
mutations and found that their locations centered on the
?-hairpin and near the catalytic active site. Interestingly, al-
though the Y448H mutation was previously shown to be in-
volved in resistance to benzothiadiazines (NNI site III), the
primary site of resistance for that class is M414 (25). We
observed no M414 mutations in GS-9190-resistant replicons.
Furthermore, additional studies evaluating GS-9190 cross-re-
sistance showed no alteration of the GS-9190 EC50in replicons
harboring the NS5B M414T mutation. These data, along with
a model overlapping GS-9190, HCV-796, and A-782759 in a
putative NS5B pocket, suggest that the mode by which GS-
9190 interacts with NS5B is different from other palm-site
Given the preponderance of evidence obtained in cell-based
assays that NS5B is the target of GS-9190, it is bewildering that
the compound is inactive in in vitro NS5B polymerase assays.
There are several possible hypotheses that may explain this
apparent discrepancy: (i) GS-9190 may only interact with the
NS5B protein in the context of the intact replication com-
plexes. It is conceivable that the conformation of NS5B in the
context of the replication complexes is different from that of
the C-terminally truncated recombinant NS5B protein used in
the polymerase assays and that the precise conformation
needed to allow binding with GS-9190 may not be adopted in
the purified recombinant enzyme preparation. (ii) To address
this possibility, we evaluated the effect of GS-9190 on the
polymerization activity of endogenous replication complexes
by employing membrane fractions isolated from replicon cells
(15). However, GS-9190 failed to show inhibitory activity in
this assay (data not shown), suggesting that it is unlikely that
the lack of direct interaction between GS-9190 and purified
NS5B protein can be ascribed to the absence of other viral or
cellular proteins in the replication complexes or due to certain
conformational differences. (iii) The interaction of GS-9190
and NS5B may occur during protein translation and before
the enzyme adopts its final conformation. It is possible
that the binding pocket for GS-9190 is formed transiently
during the translation process. GS-9190 may require metabolic
activation before it can interact with the enzyme and exert
antiviral activity. To explore this hypothesis, we tested the
effects of several known cytochrome P450 (CYP) inhibitors on
the antiviral activity of GS-9190 in a replicon assay. These
inhibitors include 1-benzylimidazole, ?-naphthoflavone, and
tetramethoxystilbene, which have been shown to be either non-
selective CYP inhibitors or selective for certain CYP isoforms
(5, 11, 22). To our surprise, GS-9190 showed significantly re-
duced antiviral activity (up to 50- to 90-fold) in the replicon
assay when tested in combination with the CYP inhibitors
(Table 5). Note that the concentrations of the CYP inhibitors
used in this experiment were chosen based on their lack of
cytotoxic or antireplicon effect when tested individually in the
replicon assay. Furthermore, these CYP inhibitors had no ef-
fect on the antiviral activity of 2?CMeA, suggesting that this
detrimental effect is specific for GS-9190. These data suggest
that GS-9190 may undergo an intracellular activation step,
likely through a CYP-mediated oxidative reaction, to produce
the active metabolite and subsequently interact with and in-
hibit the NS5B polymerase function. This hypothesis could
explain the lack of inhibitory activity of GS-9190 in NS5B
biochemical and biophysical assays in vitro. Follow-up studies
are under way to further investigate the molecular mechanism
of this effect and how it relates to the GS-9190 antiviral mech-
anism of action. In conclusion, given the data from this current
study, it is clear that GS-9190 exerts its antiviral activity
through targeting the NS5B polymerase via a mechanism dif-
ferent from other NNIs. The locations of the primary resis-
TABLE 5. GS-9190 replicon EC50s in the presence of
cytochrome P450 inhibitors
GS-9190 EC50in the presence of CYP inhibitor at
indicated concn (?M)
0 44400 0.0440.440.444.4
aND, not done.
4202SHIH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
tance mutations further suggest that inhibition of NS5B by Download full-text
GS-9190, or most likely by an intracellular metabolite from
GS-9190, is via a potentially new mode of binding.
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