Identification of ap80978, a novel small-molecule inhibitor of hepatitis C virus replication that targets NS4b

Article abstract of DOI:10.1128/AAC.00113-14

A small-molecule inhibitor of hepatitis C virus (HCV) designated AP89652 was identified by screening a compound library with an HCV genotype 1b subgenomic replicon assay. AP89652 contains two chiral centers, and testing of two syn enantiomers revealed that activity in the replicon assay resided with only one, AP80978, whose 50% effective concentration (EC₅₀) (the concentration at which a 50% reduction in Renilla luciferase levels was observed relative to an untreated control) was 630 nM. AP80978 was inhibitory against HCV genotypes 1a and 1b but not genotype 2a. In a replicon clearance assay, the potency and clearance rate of AP80978 were similar to those of telaprevir (VX950) and cyclosporine (CsA). AP80978 was nontoxic when tested against a panel of human cell lines, and inhibitory activity was HCV specific in that there was limited activity against negative-strand viruses, an alphavirus, and flaviviruses. By selection of resistant replicons and assessment of activity in genotype 1b/2a intergenotypic replicons, the viral protein target of this compound was identified as NS4B. NS4B F98V/L substitutions were confirmed by site-directed mutagenesis as AP80978 resistance-Associated mutations. When tested against HCV produced in cell culture, the compound was significantly more potent than other HCV inhibitors, including VX950, CsA, and 2'-C-methyladenosine (2'CmeA). In addition, AP80977, the enantiomer that was inactive in the replicon assay, had activity against the virus, although it was lower than the activity of AP80978. These results suggest that AP80978 has the potential to be optimized into an effective antiviral drug and is a useful tool to further study the role of NS4B in HCV replication.

Full text of DOI:10.1128/AAC.00113-14

AAC Accepts, published online ahead of print on 7 April 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.00113-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Identification of AP80978, a Novel Small Molecule Inhibitor of Hepatitis C Virus
Replication that Targets NS4B
Jodi Dufner-Beattie1,3, Andrew O’Guin1,4, Stephanie O’Guin1,5, Aaron Briley1,6, Bin
Wang1,7, Jennifer Balsarotti1,8, Robert Roth1, Gale Starkey1,9, Urszula Slomczynska1,3,
Amine Noueiry1,10, Paul D. Olivo1,11, Charles M. Rice2#
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1Apath LLC, 760 Parkside Avenue, Suite 310, Brooklyn, NY 11226
2Laboratory of Virology and Infectious Disease, The Rockefeller University, 1270 York
Avenue, New York, NY 10065
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3Present address: Monsanto Company, Chesterfield, MO
4Present address: Fluidigm Corp., South San Francisco, CA
5Present address: Genome Technology Access Center, Washington University in St.
Louis, St. Louis, MO
6Present address: American Type Culture Collection, Manassas, VA
7Present address: Dept. of Pathology and Immunology, Washington University in St.
Louis, St. Louis, MO
8Present address: Dept. of Neurology, Washington University in St. Louis, St. Louis,
MO
9Present address: Global Patent Group, St. Louis, MO
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10Present address: Polsinelli PC, St. Louis, MO
11Present address: CPH, LLC., St. Louis, MO

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#corresponding author: ricec@mail.rockefeller.edu
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running title: A novel hepatitis C virus NS4B inhibitor

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Abstract
A small molecule inhibitor of hepatitis C virus (HCV) designated AP89652 was identified
by screening a compound library with an HCV genotype 1b subgenomic replicon assay.
AP89652 contains two chiral centers, and testing of two syn enantiomers revealed that
activity in the replicon assay resided with only one, AP80978, whose EC50 was 630 nM.
AP80978 was inhibitory against HCV genotypes 1a and 1b but not genotype 2a. In a
replicon clearance assay, potency and clearance rate of AP80978 were similar to
telaprevir (VX950) and cyclosporine A (CsA). AP80978 was non-toxic when tested
against a panel of human cell lines, and inhibitory activity was HCV-specific in that there
was limited activity against negative-strand viruses, an alphavirus, and flaviviruses. By
selection of resistant replicons and assessment of activity in 1b/2a intergenotypic
replicons, the viral protein target of this compound was identified as NS4B. NS4B
F98V/L substitutions were confirmed by site-directed mutagenesis as AP80978
resistance-associated mutations. When tested against HCV produced in cell culture,
the compound was significantly more potent than other HCV inhibitors, including VX950,
CsA, and 2’-C-methyl-adenosine (2’C-meA). In addition, AP80977, the enantiomer that
was inactive in the replicon assay, had activity against the virus, although it was lower
than AP80978. These results suggest that AP80978 has the potential to be optimized
into an effective antiviral drug and is a useful tool to further study the role of NS4B in
HCV replication.
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Introduction
HCV is a positive-strand RNA virus belonging to the Flaviviridae family. Within the viral
genome, the internal ribosome entry site (IRES) drives translation of a single
polypeptide that is cleaved by both cellular peptidases and viral proteases to produce
viral structural and non-structural proteins (1). The virus-encoded RNA-dependent RNA
polymerase NS5B is exceptionally error-prone, resulting in significant genome
sequence variability. Based on sequence differences, HCV can be categorized into
seven distinct genotypes, which differ both in global distribution and response to therapy
(2, 3).
Worldwide, over 170 million people are infected with this virus (4). HCV infection
is a major cause of chronic liver disease such as cirrhosis and hepatocellular
carcinoma, and is a leading cause of liver transplantation (5, 6). Until recently, the
standard of care (SOC) was a combination of pegylated interferon and ribavirin, which is
commonly associated with severe side effects and low sustained virological response
rates for patients infected with genotype 1, the most prevalent genotype in North
America and Europe (7, 8). Direct-acting antivirals (DAA) have been the focus of
intensive drug discovery efforts, particularly the viral NS3-4A protease, the NS5A
phosphoprotein, and the NS5B polymerase. A triple combination composed of the SOC
with one of two protease inhibitors, VX950 or boceprevir enhances cure rates and is
now approved for treatment of patients with chronic genotype 1 HCV infection (9, 10).
However, resistance develops quickly to these as well as other antiviral compounds,
and severe side effects and drug interactions complicate treatment (11). New protease
and polymerase inhibitors have recently been approved but the development of

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additional classes of antiviral compounds against novel viral targets will broaden
treatment options and provide multiple options for interferon-free HCV therapy (1, 3, 12-
14).
To this end, we carried out a high throughput, cell-based genotype 1b
subgenomic replicon screen to identify novel compounds with antiviral activity against
HCV. One compound that was selected for further study was a molecule with two chiral
centers, designated AP89652. After separation of enantiomers, antiviral activity was
found to be associated with AP80978, one of the two tested isomers. The active
enantiomer was genotype 1-specific, non-cytotoxic, and inactive against numerous
other virus replication systems, which included other flaviviruses. Two approaches
were taken to study the molecular target of the compound, including selection of
resistant replicons and generating intergenotypic 1b/2a replicons with differential
susceptibility to the compound. Both approaches indicated a novel target of this
compound, HCV NS4B. When tested against HCV produced in cell culture, the
compound was significantly more potent than other HCV inhibitors, including VX950,
CsA, and 2’C-meA.
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Materials and Methods
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Maintenance of Huh-7.5 cells
Huh-7.5 cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM)
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS)
(Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen)
at 37°C in a humidified 5% CO2 incubator. Cells were subcultured by washing once

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with phosphate buffered saline (PBS) (Invitrogen), followed by incubating for up to 5 min
in 0.05% Trypsin-EDTA (Invitrogen) at 37°C until cells detached from the vessel. Upon
detachment, complete medium was added to inactivate trypsin, and cells were counted
and seeded at the desired density into T-flasks (TPP, Midwest Scientific, St Louis, MO).
Cells were grown to 80% maximum confluence, and were seeded at a density no less
than 13,000 cells/cm2.
Preparation of CA32 replicon cells
The replicon utilized in the primary and secondary screens, CA32, was created at
Apath, LLC, and is a transient genotype 1b subgenomic replicon generated from the
Con1 strain. In this replicon, the HCV IRES within the 5’ non-translated region (NTR)
drives translation of the first 32 amino acids of the core protein fused to humanized
Renilla luciferase (hRluc). The encephalomycarditis virus (EMCV) IRES lies 3’ of the
hRluc open reading frame (ORF) and drives translation of non-structural (NS) proteins
NS3 through NS5B, which is flanked at its 3’ end with the HCV 3’ NTR (for schematic,
see Figure 1a).
To prepare CA32 replicon cells, the replicon-encoding plasmid APP660 was
linearized with the unique restriction endonuclease ScaI (NEB, Ipswitch, MA) at 37°C for
4 h. The reaction was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1)
(Invitrogen) and once with chloroform:isoamyl alcohol (24:1). The linearized plasmid
was precipitated with 1/10 volume 3M sodium acetate (pH 5.2) (Sigma-Aldrich, St.
Louis, MO) and 2 volumes ethanol, and the pellet was washed twice with 70% ethanol.
Linearized DNA (1 μg) was in vitro transcribed (IVT) using T7 polymerase from the

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MEGAscript® T7 Kit (Invitrogen). RNA size and integrity were verified by resolving 1 μg
IVT RNA on a standard formaldehyde-agarose gel and staining with ethidium bromide
(Sigma-Aldrich). Approximately 24 h prior to electroporation, Huh-7.5 cells were seeded
at a density of 5.5 x 106 cells per 150 cm2 T-flask. On the day of electroporation, cells
were washed once with PBS prior to incubation with trypsin-EDTA for up to 5 min at
37°C. After the cells detached, complete medium was added, and cells were pelleted
via centrifugation at 200 x g for 5 min at 4°C. Cells were washed by resuspending in
ice-cold PBS and pelleting at 200 x g for 5 min at 4°C, resuspended once more in ice-
cold PBS, passed through a 70 μm cell strainer (VWR, Radnor, PA) and counted prior
to a final centrifugation at 200 x g for 5 min at 4°C. After resuspension in ice-cold PBS
at a density of 1.5 x 107 cells per ml, 6 x 106 cells were combined with 1 μg IVT RNA
and electroporated in a 2 mm-gap electroporation cuvette (Bio-Rad, Hercules, CA)
using the BTX Electrosquare Porator with the following settings: 820V, 99 μsec pulse
length, 5 pulses, 1.1 sec intervals. Following electroporation, cells recovered at RT for
10 min prior to plating in complete medium. Replicon-containing cells were passaged
as they reached 70-80% confluence and were seeded at a density no lower than 13,000
cells/cm2.
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Compound screening with CA32 replicon cells
Replicon cells were seeded into 96-well plates at 20,000 cells/well in screening medium,
consisting of DMEM with 10% FBS, 1X penicillin/streptomycin, 1X non-essential amino
acids, and 100 ng/ml Fungizone (Invitrogen). After 4 h attachment, cells were treated
with compound in 1% DMSO (Sigma-Aldrich) in a final volume of 200 μl for 24 to 48 h.

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Pilot studies indicated that 1% DMSO caused no adverse effects on cell viability for this
assay duration. For primary screening, compounds were added at a single dose (10
μM) in single replicate without toxicity evaluation. Compounds that caused a 50%
reduction in Renilla luciferase levels were progressed to secondary screening for EC50
and CC50 determination. Identical culture conditions were used for EC50 and CC50
determination. For secondary screening, compound was added to wells using a five
point 3-fold serial dilution series with four replicate treatments per dose. Renilla
luciferase levels were assayed using the Renilla Luciferase Assay Kit (Promega,
Madison, WI), and toxicity/cell viability was assayed using CellTiter-Glo Luminescent
Cell Viability Assay (Promega). Both EC50 (the concentration at which a 50% reduction
in Renilla luciferase levels was observed relative to an untreated control) and CC50 (the
concentration resulting in a 50% decrease in cell viability) values were determined from
the raw data using a proprietary software program based on a Hill Plot calculated from a
four-parameter logistic model.
Compound synthesis and enantiomer separation
AP89652 [3-chloro-5-(furan-2-yl)-N-(thiophene-2-yl-methyl)—7-(trifluoromethyl)-4,5,6,7-
tetrahydropyrazolo[1,5,a]pyrimidine-2-carboxamide] (Figure 1b) was synthesized in two
steps using a procedure described by Dalinger et al. (15). The pyrazolo[1,5-a]pyrimidine
ring was assembled by condensation of 5-amino-4-chloro-1H-pyrazole-3-carboxylic acid
with 4,4,4-trifluoro-1-(furan-2-yl)-butane-1,3-dione and the pyrimidine ring was reduced
stereoselectively with sodium borohydride in ethanol, yielding only one pair of
diastereoisomers that corresponds to 2,4-syn-isomers. Separation of two enantiomers

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(AP80977 and AP80978) was achieved by an HPLC chromatography method using a
chiral column Chiralcel OD-H and heptane/2-propanol as a mobile phase. Absolute
configuration of AP80978 was determined by x-ray diffraction (oXray Ltd.) and the
active enantiomer was found to be (5S,7R) 3-chloro-5-(furan-2-yl)-N-(thiophene-2-yl-
methyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo [1,5,a]pyrimidine-2-carboxamide.
The synthetic procedure for AP89652, its separation into two enantiomers, and
determination of the absolute configuration for the active AP80978 enantiomer were
described previously (16, 17).
Control inhibitors
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Control inhibitors ribavirin, a viral RNA synthesis inhibitor (18), and CsA, an HCV
replication inhibitor (19), were obtained from Sigma Aldrich (St. Louis, MO). VX950, an
NS3-4A protease inhibitor (10), was synthesized by Stereochem Research Centre
(Hyderabad, India). 2’C-meA, an NS5B polymerase inhibitor (20), was obtained from
Carbosynth Limited (Berkshire, UK).
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Replicon clearance assay
The genotype 1b replicon cells used in this assay harbor the replicon Con1/SG-
Neo(I)hRLuc2aUb, which was created at Apath, LLC and is encoded by plasmid
APP76. In this replicon, the HCV IRES within the 5’ NTR drives translation of the first
15 amino acids of the core protein fused to Neo. The EMCV IRES is 3’ of Neo and
drives translation of an hRluc-ubiquitin (Ub)-foot-and-mouth disease virus 2a peptide
(FMDV2a)-NS3-NS5B fusion protein (Figure 2A). The presence of FMDV2a and Ub

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increases the likelihood of cleavage of the hRluc reporter from the C-terminal HCV non-
structural proteins. Preparation of stable replicon cells was carried out as described for
the genotype 2a replicon J6/JFHEMCVIRES2aRlucNeo.
Con1/SG-Neo((I)hRLuc2aUb replicon cells were seeded into 12-well cluster
plates at a density of 5 x 104 cells/well in complete Huh-7.5 media lacking G418
(Invitrogen). After 4 h attachment, triplicate wells were harvested for RNA extraction as
a 0 h control. Additional wells were treated in triplicate with either 1% DMSO (untreated
control), 7.5 μM VX950, 2.5 μM CsA, or 3.75 μM or 15 μM AP80978 (replicon clearance
phase). Cell density was monitored daily, and when the cells reached 80% confluence,
a sample was collected for RNA extraction and a subset of the remaining cells were
split at a 1:3 ratio into the appropriate compound-containing media. At the fourth
passage, cells were collected and passaged into media lacking compound in the
presence of 250 μg/ml G418 to initiate the rebound phase. Untreated cells surviving the
treatment were collected as they reached 80% confluence. RNA from all time points
and treatments was extracted using a 96-well RNeasy RNA Extraction Kit (Qiagen,
Valencia, CA). RNA levels of HCV 3’ NTR and the housekeeping gene GAPDH were
quantified via RT-qPCR. GAPDH RT-qPCR was carried out using the following primers:
forward,
5'-CCTGCACCACCAACTGCTTA
probe,
-3';
reverse,
5’-
5'-
GCAGTGATGGCATGGACTGT-3';
Cy5-
CTGGCCAAGGTCATCCATGACAACT-BHQ-2-3’. HCV RNA levels were normalized to
those of GAPDH and were expressed as HCV log10 change relative to the HCV RNA
level in untreated cells on day 0 using the comparative Ct method (Applied Biosystems).
At each time point, normalization of HCV to GAPDH was determined as follows:

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dCt=(CtHCV)-(CtGAPDH). Calculation of ddCt value relative to t = 0 was determined as
follows: ddCt=(dCt time x)-(dCt time 0). HCV RNA log change was calculated as log(2^((-
1)*ddCt)).
Evaluation of AP80978 against genotype 1a and 2a replicons
The genotype 2a replicon, J6/JFHEMCVIRES2aRlucNeo, was created at Apath, LLC
and is encoded by the plasmid APP40. It is a stable subgenomic replicon with elements
from both the JFH and J6 strains. The JFH IRES within the 5’ NTR drives translation of
the first 19 amino acids of the JFH core protein linked to an hRluc-neomycin
phosphotransferase (Neo) fusion protein. The EMCV IRES is 3’ of the hRluc-Neo ORF
and drives translation of JFH NS3-NS5B, which is flanked at its 3’ end with the J6 3’
NTR. The differences between the JFH and J6 3’NTRs are within the variable region.
Preparation of stable genotype 2a replicon cells was performed as outlined
above for CA32 cells except that 24 h after plating, medium was removed and replaced
with selection media (i.e. complete media containing 1 g/L G418 (Invitrogen). Cells
were monitored daily and passaged as needed to maintain a subconfluent culture.
Selection was considered complete when cells electroporated in parallel with a
polymerase-deficient replicon lacked any viable cells. After this observation, the
concentration of G418 in the media was decreased to 250 μg/ml for culture
maintenance. Screening with the resulting genotype 2a replicon cells (APC140) was as
described for CA32 replicon cells except that seeding density was 12,000 cells/well and
cells were assayed 48 h post-treatment in the absence of selection agent.
The genotype 1a replicon cells (Huh-7.5 containing the H/SG-Neo(L+I) replicon,
APC89) was previously reported (21). Replicon cells were seeded into 12-well plates at

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a density of 40,000 cells/well. After 4 h attachment, cells were treated in triplicate using
a five point 3-fold serial dilution series in 1% DMSO, which caused no adverse effects
on cell viability for this assay duration in pilot studies. After 72 h, RNA was extracted
using the RNeasy RNA Extraction Kit (Qiagen). Total RNA concentration was
determined using Ribogreen RNA Quantitation Reagent (Invitrogen), and were
normalized between samples. HCV RNA levels were quantified via quantitative RT-PCR
(RT-qPCR) using MultiCode-RTx HCV Viral Load Primer Mix and RNA Reagent Set
(EraGen Biosciences, Madison, WI) on an Applied Biosystems 7300 Real-Time PCR
System. EC50 values were determined as described above.
Evaluation of AP80978 against other viruses
BHK-S cells harboring a subgenomic, puromycin-selectable Renilla luciferase reporter
yellow fever virus (YFV) replicon (YF-hRUPac) (22) were seeded at 10,000 cells/well
into 96-well plates. The serotype 2 dengue virus replicon was constructed by removing
a structural protein-coding region, between capsid gene codon 28 and the last 26
codons at the 3’ end of the envelope gene from an infectious clone DEN2 16681 (23).
This deletion was replaced with the humanized Renilla luciferase-ubiquitin-puromycin
acetyl transferase (hRUPac) cassette from the yellow fever replicon (22) to generate
pD2-hRUPac. BHK-S cells harboring the dengue virus replicon were seeded at 8,000
cells/well in 96-well plates. The respiratory syncytial virus (RSV) replicon is similar to
that reported by Malykhina et al. (24) except that it also contains a Renilla luciferase
reporter located between the GFP and NS1 coding sequence. Screening was carried
out by seeding 1,500 RSV BHK-SR19-T7 replicon cells and 8,500 BHK-S parental

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cells/well in 96-well plates. For West Nile Virus (WNV), BHK-WNV-Rep replicon cells
(22) were seeded at 10,000 cells/well into 96-well plates. The ebola virus (EBOV) mini-
genome expresses a Renilla luciferase reporter-encoding plasmid that carries cis-acting
elements for ebola virus replication. The EBOV minigenome was transfected into BHK-
SINRep T7 cells, along with four expression plasmids that encode the replicase proteins
NP, VP35, L, and VP30 (25). EBOV mini-genome cells were seeded at 20,000
cells/well into 96-well plates.
BHK-S cells harboring a subgenomic, puromycin-selectable firefly luciferase
reporter Sindbis virus replicon (SINrep19.FLuc.Pac) (26) were seeded at 10,000
cells/well into 96-well plates. The influenza A virus (FluA) mini-genome expressing
firefly luciferase reporter carries cis-acting elements for FluA replication. The FluA
minigenome was transfected into 293T cells, along with four expression plasmids that
encode the replicase proteins NP, PA, PB1, and PB2. FluA mini-genome cells (27)
were seeded at 20,000 cells/well into 96-well plates.
For determination of anti-replicon activity, all cells were treated with compounds
using a five point 3-fold serial dilution series with four replicate treatments per dose. A
24h incubation period was chosen to maximize screening throughput, following which,
Renilla (dengue, YFV, RSV, WNV, EBOV) or firefly (Sindbis or FluA) luciferase levels
were evaluated to determine an EC50 value. In parallel, cell toxicity/cell viability was
assayed on parental cells using CellTiter-Glo Luminescent Cell Viability Assay.
Cytotoxicity evaluation in other cell types

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HepG2 cells, Caco-2, MRC-5, and Jurkat cells were obtained from the ATCC
(Manassas, VA). HepG2 and MRC-5 cells were maintained in Eagle’s Minimal
Essential Medium (Invitrogen) supplemented with 10% FBS, 100 units/ml penicillin, and
100 μg/ml streptomycin. Caco-2 cells were grown in the same media except with 20%
FBS. Jurkat cells were maintained in RPMI-1640 medium supplemented with 10%
FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cells were incubated at
37°C in a humidified 5% CO2 incubator. Cytotoxicity screening was carried out by
incubating cells (20,000 cells/well for HepG2, Caco-2, and MRC-5 cell lines, and 40,000
cells/well for Jurkat cells) in 96-well plates in the presence of serial dilutions of
compound for 24 hours, a time point chosen as a realistic interval to observe control
inhibitor activity, and which also maximized assay throughput. At the end of the
incubation period, cell viability was assessed by the CellTiter-Glo Luminescent Cell
Viability Assay.
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Generation and evaluation of AP80978-resistant replicons
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Clone A cells (28) contain a genotype 1b (Con1) stable subgenomic replicon, and were
used for resistant mutant selection. Cells were seeded at a density of 1 x 106 cells per
75 cm2 T-flask. After 4 h attachment, cells were treated with complete medium
supplemented with either 1% DMSO (untreated control) or 10 μM AP80978. Medium
was changed every 3-4 d to replenish compound and G418, and cells were split as
needed to maintain subconfluent cultures. After 12 d of culture in the presence of 10
μM AP80978, the concentration was increased to 20 μM, and cells were cultured for an
additional 19 d. After this period, a sample of cells from both AP80978-treated and
control cells were collected for evaluation of response to AP80978 as follows: cells

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were seeded into 12-well plates at a density of 40,000 cells/well in the absence of
G418. After 4 h attachment, cells were treated in triplicate with AP80978 using a five
point, 3-fold serial dilution series in 1% DMSO. After 72 hours, total RNA was extracted
using the RNeasy RNA Extraction Kit (Qiagen). Total RNA was quantified using the
Ribogreen RNA Quantitation Reagent (Invitrogen), and concentrations were normalized
between samples. HCV RNA levels were quantified via quantitative RT-PCR using
MultiCode-RTx HCV Viral Load Primer Mix and RNA Reagent Set on an Applied
Biosystems 7300 Real-Time PCR System. EC50 values were determined as described
above.
Evaluation of cell-associated versus replicon-associated resistance
Total RNA (10 μg) extracted from control and AP80978-treated cells was electroporated
into the Clone A parental cell line Huh7. Cells were selected with 1 mg/ml G418, and
after selection was complete (determined by complete cell death in cells electroporated
without RNA), cells were pooled and evaluated for response to AP80978 as described
above.
Replicon sequence analysis
Total RNA was extracted from control and AP80978-resistant replicon cell lines using
the RNeasy RNA Extraction Kit (Qiagen). RT-PCR was carried out with replicon-specific
primers using SuperScript III reverse transcriptase (Invitrogen) and iProof high fidelity
polymerase (BioRad). PCR amplicons were sequenced and compared against the wild-
type reference sequence using Clone Manager software. The four mutations identified

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within NS4B in the AP80978-resistant cells (G60 no change, F98V, F98L, S238Y) were
introduced individually into the genotype 1b replicon-encoding plasmid APP76 using
standard PCR-based techniques. The mutated plasmids were sequenced to verify that
only the desired mutations had been introduced.
Colony formation assay
IVT RNA corresponding to wild-type replicons or replicons with F98V or F98L resistance
mutations were electroporated into Huh-7.5 cells as described for the genotype 2a
replicon. Electroporated cells (225, 450, 900, or 1800) were seeded in triplicate into 6-
well cluster plates along with cells electroporated with a polymerase-defective construct
to achieve a total cell number of 90,000 cells/well. After 24 h attachment, cells were
treated with either 1 mg/mL G418 alone or G418 in the presence of 20 μM AP80978.
Media was changed every 3-4 days to replenish compound and remove dead cells.
After 2-3 wks, media were removed, cells were fixed with 7% formaldehyde and stained
with 1% crystal violet (Sigma-Aldrich) in 50% ethanol, and colonies were counted.
Generation and testing of intergenotypic 2a/1b replicons
Standard PCR-based techniques were used to produce a series of genotype 2a
intergenotypic replicons (encoded by plasmid APP40, description above) in which the
genotype 2a NS4B-encoded amino acids were replaced with the corresponding
sequence from genotype 1b: NS4B(Con1 7-254), NS4B(Con1 7-52), NS4B(Con1 53-254),
NS4B(Con1 219-254), NS4B(Con1 7-52, 219-254), NS4B(Con1 53-219). Polymerase-defective
versions of each construct were prepared in parallel to evaluate replication of each

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intergenotypic replicon. The resulting plasmids were sequenced to verify that the
desired constructs had been produced. Preparation of intergenotypic replicon cells and
compound screening was performed as outlined above for genotype 2a replicon cells.
The genotype 2a replicon containing the NS4B configuration NS4B(Con1 7-254) did not
replicate by comparison with the polymerase-defective counterpart.
Generation and testing of AP80978-sensitive virus
To construct an AP80978-sensitive hepatitis C virus, a 3355 bp SpeI-RsrII (NEB) DNA
fragment from the genotype 2a replicon, encoding genotype 1b NS4B amino acids 53
through 219 (i.e. NS4B(Con1 53-219)) was excised and inserted in the same restriction
sites in the genotype 2a/2a J6/JFH Jc1 (29) HCV plasmid construct (see Figure 6A).
This HCVcc construct is referred to as J6/JFH Jc1/Con 1 NS4B 53-218
.
The resulting plasmid was linearized with XbaI (NEB), and DNA was prepared for
in vitro transcription and electroporation as described above for the replicons. Following
electroporation, cell culture supernatants were collected and concentrated using Amicon
Centrifugal Filter Units with a 100,000 molecular weight cut-off (EMD Millipore
Corporation, Billerica, MA). The virus titer was determined by infecting replicate wells
containing Huh-7.5 cells with serially-diluted virus, staining 4 d later with NS5A
monoclonal antibody, and calculating the TCID50/ml from the number of positively-
stained replicates for each dilution.
To evaluate the effect of AP80978 on J6/JFH Jc1/Con1 NS4B
virus, Huh-
53-218
7.5 cells were seeded into 96-well plates at a density of 20,000 cells/well. After 24 h
attachment, cells were treated in quadruplicate with serially diluted compound in 1%

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DMSO in the presence of 2000 TCID50 units/well virus (MOI=0.1). Following 48 h
incubation, cells were washed and RNA was extracted using RNeasy RNA Extraction kit
(Qiagen). RNA levels of HCV 3’ NTR were quantified via RT-qPCR and were
expressed as HCV log change relative to untreated control cells using the comparative
Ct method.
Results
Identification of AP89652 in a genotype 1b replicon assay
In order to identify new classes of antiviral compounds that inhibit HCV replication, a
high throughput cell-based screen was carried out using a transient genotype 1b
subgenomic Renilla luciferase replicon (Figure 1A). A primary screen of 93,000
commercially available compounds against these replicon cells identified 209
compounds that decreased Renilla luciferase levels by 50%. As the primary screen did
not employ a method to identify cytotoxic compounds, these 209 compounds were
further evaluated in a secondary assay to calculate efficacy (EC50) and toxicity (CC50).
Evaluation of these compounds in the secondary screen identified 38 compounds with
EC50 values less than 10 μM and CC50 greater than 100 μM. The 38 compounds
belonged to 13 distinct chemical classes, with 15 compounds categorized as orphan
compounds.
One compound, AP89652 (Figure 1B), that belongs to
a
tetrahydropyrazole-[1,5,a]-pyrimidine chemical series was selected for further study
based on its favorable physico-chemical properties, novelty, and amenability of this
chemical series to SAR (structure activity relation) studies.

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AP89652 contains two chiral centers (Figure 1B), yielding four potential isomers.
However, based on the literature describing the synthesis of tetrahydropyrazolo-[1,5a]-
pyrimidine chemical series (15) and our results, it was found that AP89652 is a racemic
mixture of two syn-enantiomers, as shown in Figure 1C.
To determine which
enantiomer of AP89652 was responsible for HCV replication inhibitory activity, the
optically pure enantiomers were isolated via chiral chromatography and evaluated
against the replicon cell line. Screening against CA32 genotype 1b subgenomic
replicon cells revealed that only one of the two enantiomers, AP80978, had replicon-
inhibitory activity, with an EC50 of 0.63 μM, while the other, AP80977, lacked activity
(Table 1). Similar results were obtained when replicon levels were quantified using RT-
qPCR (data not shown).
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HCV Replicon Clearance by AP80978
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The HCV replicon clearance assay served as a cell culture model for virus clearance.
At the onset of the assay, cells harboring a stable HCV replicon were cultured under
non-selection conditions in the presence or absence of compound. During this “replicon
clearance phase”, cells were able to proliferate independently of HCV replicon
replication. The “rebound phase” was initiated by inhibitor removal and resumption of
G418 selection, allowing growth of only HCV replicon-harboring survivor cells.
In this assay, the cells harboring the stable genotype 1b subgenomic replicon
Con1/SG-Neo(I)hRluc2aUb (Figure 2A) were treated with either 15 μM or 3.75 μM
AP80978 in the absence of neomycin for 17 d. To compare efficacy of AP80978 with
other antiviral compounds, parallel cultures were treated with 2.5 μM CsA or 7.5 μM
VX950. Compound-treated cells were passaged as needed to maintain a subconfluent

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culture, replenishing compound and collecting an aliquot of cells at each passage. On
d17, cells were seeded in G418-containing selection media in the absence of
compound, and cells were cultured for an additional 7 d, during which untreated cells
approached confluence. Massive cell death was observed in cultures that had been
treated with compounds. At each passage, HCV RNA levels were quantified and
normalized relative to GAPDH via RT-qPCR for treated and control samples. HCV RNA
levels remained similar over the course of the assay in control cells grown in the
absence of inhibitors, while cells treated with both doses of AP80978 exhibited a
progressive decrease in HCV RNA levels during the replicon clearance phase, with a
reduction of 1.5-1.75 log10 RNA copies by d17 of the assay (Figure 2B). Similar to
results with AP80978, treatment with CsA and VX950 was associated with a maximal
1.5 to 2 log10 decrease in HCV RNA levels. During the rebound phase, HCV RNA levels
in all inhibitor-treated cells, including AP80978 and control CsA and VX950, were
undetectable in the presence of G418, indicating the absence of functional replicon
RNA within these cells (Figure 2B).
In control untreated cells, replicon RNA was
slightly lower than baseline, but within the variation typically observed in RT-qPCR in
the clearance assay.
Genotype- and Virus-specificity of AP80978
To evaluate the genotype specificity of AP80978, the compound was screened against
cells harboring the genotype 1a replicon H/SG-Neo(L+I) (21) and the genotype 2a
replicon J6/JFHEMCVIRES2aRlucNeo. AP80978 inhibited replication of the genotype
1a replicon, with an EC50 value (0.62 μM) similar to that for the genotype 1b replicon

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(Table 2). However, the compound was completely inactive against the genotype 2a
replicon (EC50 >25 μM). Screening against genotype 1a, 1b, and 2a replicons by two
external laboratories confirmed similar EC50 values for genotypes 1a and 1b, and
inactivity against genotype 2a (data not shown).
To evaluate whether AP80978 was active against other viruses, the compound
was tested against virus-free mini-genome and replicon systems that have been
developed for high-throughput screening to identify replication inhibitors of negative-
strand and other positive-strand viruses. AP80978 was inactive against infection-free
negative-strand RNA virus systems for ebola virus, influenza A virus, and respiratory
syncytial virus, although the controls (ribavirin, CsA, and 2’C-meA) for each of these
systems behaved as expected (Table 3). Similarly, the compound was inactive against
the alphavirus Sindbis replicon, as well as other positive-strand flavivirus replicons for
dengue virus, yellow fever virus, and West Nile virus, indicating that AP80978 is an
HCV-specific antiviral compound. To confirm that the compound that was used in these
studies was active, AP80978 was tested in parallel against HCV replicon cells, and
showed efficacy.
Cytotoxicity of AP80978 in Different Cell Types
To determine the effect of AP80978 on other cell types, cytotoxicity was evaluated
against a panel of cell lines from an array of different tissues. Similar to observations
using the Huh-7.5 cell line that supports HCV replication in cell culture, AP80978 was
non-toxic against another human liver cell line, HepG2, with a CC50 value >100 μM

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(Table 4). It was also non-toxic in human cell lines derived from intestinal epithelium
(Caco-2), lung fibroblasts (MRC-5), and T-lymphocytes (Jurkat).
Mechanism of Action of AP80978
Selection and characterization of AP80978 resistance mutations
Preliminary biochemical studies showed that AP80978 had no activity against the HCV
NS3-4A serine protease and NS5B polymerase (not shown), suggesting a novel viral or
host target. Due to the error-prone nature of the HCV RNA polymerase, culture of HCV
replicon cells in the presence of replication inhibitors can result in the selection of
inhibitor-resistant replicons. In an effort to gain insight into the molecular target of
AP80978, Clone A cells resistant to AP80978 were selected by sequential passaging in
the presence of compound. Clone A cells are a human hepatoma cell line that contains
a stable genotype 1b (Con1 strain) subgenomic replicon (28). Cells were cultured
under neomycin-containing conditions for 12 d in medium supplemented with 10 μM
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AP80978, followed by culture for an additional 19 d in 20 μM AP80978. Control Clone A
cells grown in the presence of diluent only were cultured in parallel. After this culture
period, cells were evaluated for response to AP80978 by culturing treated and control
cells in the presence of serial dilutions of AP80978 for 72 h and subsequently
quantifying HCV RNA levels via RT-qPCR to determine the EC50. Clone A cells that
had undergone selection exhibited greater than 10-fold decreased sensitivity to
AP80978 (EC50 >20 μM) relative to the control cells (EC50=1.8 μM).
To determine whether resistance was encoded by the replicon, total RNA was
extracted from both AP80978-resistant and control cells, and RNA was re-introduced

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via electroporation into naïve Huh7 cells, the parental cell line of Clone A cells.
Electroporated cells that harbored actively replicating replicons were selected with
G418, pooled, and assayed for sensitivity to AP80978 as described above. Cells
electroporated with RNA from control cells were sensitive to AP80978 (EC50=0.91 μM),
while those electroporated with RNA from AP80978-resistant cells maintained
resistance to the compound (EC50 >20 μ ), indicating that the resistance was
associated with the replicon.
To identify nucleotide changes that conferred resistance to AP80978, RNA
extracted from AP80978-resistant and control Clone A cells was reverse transcribed
and amplified using replicon-specific primers. Sequence analysis of the amplified
replicons revealed four point mutations present within the sequence encoding NS4B
from the AP80978-resistant cells that were absent from the control or wild-type replicon
sequence. Three of the four mutations resulted in amino acid changes corresponding to
F98L, F98V, and S238Y within NS4B. One silent mutation at codon 60 was also
observed.
Each mutation was introduced independently into a stable genotype 1b
subgenomic reporter replicon plasmid (Figure 3A) via site-directed mutagenesis.
Sequencing of the resulting plasmids revealed that they were void of additional
mutations. IVT RNA generated from each mutant and parental replicon plasmid was
introduced into Huh-7.5 cells, a highly permissive human hepatoma cell line. Resulting
replicon cell lines were evaluated for response to AP80978 and the positive control
compounds CsA and VX950. A differential response was observed between cells
electroporated with different replicon RNAs. Cells containing replicons with F98V and

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F98L amino acid substitutions were resistant to AP80978 (EC50 >20 μM), while those
containing parental wild-type replicon (EC50 = 0.29 μM), the S238Y substitution (EC50
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0.41 μM) or the silent mutation (G60nc, EC50 =0.24 μM) remained sensitive to AP80978
(Table 5). These data suggest that NS4B is the target of AP80978 and that changing
the amino acid at residue 98 of NS4B to either valine or leucine was sufficient to confer
resistance to the compound. Cells electroporated with IVT RNA from each construct
were responsive to CsA (EC50 values ranged from 0.1 to 0.16 μM) and VX950
(replicons cleared from all cell lines, data not shown), indicating that resistance to
AP80978 did not confer cross-resistance to either of these compounds.
Alignment of the NS4B amino acid sequences from genotypes 1b and 2a revealed
a leucine at codon 98 of NS4B in genotype 2a (Figure 3B). Since mutation that
changed phenylalanine to leucine at this position in the genotype 1b replicon conferred
resistance to AP80978, mutagenesis was carried out to determine whether a
phenylalanine at this position in the genotype 2a replicon was sufficient to confer
sensitivity to the compound. The resulting genotype 2a replicon with the L98F
substitution remained resistant to the compound (EC50 > 20 μM), indicating that a
mutation resulting in amino acid substitution in this residue alone was insufficient to
confer AP80978 sensitivity to an HCV genotype 2a subgenomic replicon.
The effect of AP80978 resistance mutations was evaluated in a long-term colony
formation assay. IVT RNA from either wild-type or resistant replicon constructs was
electroporated into naïve Huh-7.5 cells and plated at varying densities in the presence
of cells that had been electroporated with polymerase-defective constructs in order to
allow transient growth of cells in the presence of G418. Cells that had been

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electroporated with all constructs grew in the presence of G418, with similar numbers of
colonies. However, only cells that had been electroporated with IVT RNA encoding
resistance mutations grew in the presence of AP80978 (Figure 4).
In cells
electroporated with RNA encoding the F98L resistance-associated amino acid change,
there were fewer and smaller colonies in the presence of AP80978, suggesting that
maintaining resistance to this compound may be associated with a fitness cost.
However, colony number and size were similar in the absence and presence of
AP80978 for cells electroporated with the construct encoding the F98V substitution.
Evaluation of AP80978 against intergenotypic genotype 2a/1b replicons
To confirm NS4B as the molecular target of AP80978, an alternate approach was taken
that took advantage of the differential response of genotypes 1b and 2a to AP80978.
Using the plasmid encoding J6/JFHEMCVIRES2aRlucNeo (a genotype 2a replicon that
is insensitive to AP80978) as a template, six intergenotypic constructs were made, in
which regions encoding the following amino acids of NS4B were replaced with the
corresponding amino acids from genotype 1b NS4B (sensitive to AP80978): (i) 7-254,
(ii) 7-52, (iii) 53-254, (iv) 219-254, (v) 7-52 and 219-254, and (vi) 53 to 218 (Figure 5).
Testing the resulting intergenotypic constructs would show which portions of genotype
1b NS4B protein confer AP80978 response. IVT RNA generated from each of the six
intergenotypic replicon cDNAs as well as the genotype 2a parental replicon was
electroporated into Huh-7.5 cells and evaluated for response to AP80978 and the
control compound CsA (Figure 5). The intergenotypic replicon that contained the nearly
full-length NS4B from genotype 1b (amino acids 7-254) did not replicate, similar to the

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replication-defective control replicon, while the remaining intergenotypic replicons
replicated to varying degrees based on reporter levels. Compound screening was
initially carried out in unselected cells 96 h post-electroporation, and again following
selection with G418, with similar results. Two intergenotypic replicons that encoded
amino acid sequences from genotype 1b within the central region of NS4B (amino acids
53-254 and amino acids 53 to 218) were sensitive to AP80978, with EC50 values less
than 1 μM, while all other intergenotypic replicons, as well as the genotype 2a parental
replicon, did not respond to AP80978 treatment. Thus, the molecular target of AP80978
was located between amino acids 53 and 218 within NS4B of genotype 1b. All of the
intergenotypic and wild-type control constructs were responsive to CsA (not shown).
Evaluation of AP80978 activity against cell culture-produced HCV
To evaluate the effect of AP80978 on virus replication in the context of the fully-
infectious replication cycle, an AP80978-sensitive virus construct was produced by
cloning residues 53-218 from genotype 1b into a J6/JFH1 Jc1 construct, to produce
J6/JFH Jc1 (Con 1 NS4B 53-218) (Figure 6A). IVT RNA produced from this construct was
electroporated into Huh-7.5 cells, and intergenotypic virus was collected from the cell
culture medium. Titers were low, suggesting that the virus replicated poorly. Huh-7.5
cells were infected with either the intergenotypic or wild-type virus in the presence of
either AP80978, AP80977 (the second syn enantiomer that was inactive in the replicon
assay), AP89652 (the racemic mixture), or AP80935 (an analog of AP80978 with an
aromatic six-membered ring). Parallel cultures were treated with the HCV inhibitors
2’C-meA, VX950, and CsA. After 48 h, RNA was extracted and HCV RNA levels were

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quantified. As expected, in cells infected with the J6/JFH Jc1 virus, HCV RNA levels did
not change appreciably in cells treated with 0.06-5 μM AP80978, AP80977, AP89652,
or AP80935 (Figure 6B), although high concentrations of the control inhibitors 2’C-meA
and VX950 were associated with a 2-3 log10 reduction (Figure 6C). Lower inhibitory
potency was observed for CsA for J6/JFH Jc1 HCV RNA (Figure 6C). In cells infected
with intergenotypic J6/JFH Jc1 (NS4B 53-218), HCV RNA levels decreased >1 log10 at 70
nM AP80978, and ~0.7 log10 at 20 nM (Figure 6D). Unexpectedly, AP80977, the
enantiomer that was inactive in the genotype 1b replicon assay, had inhibitory activity
against the intergenotypic virus, with a reduction of ~0.7 log10 HCV RNA at 0.67 μM.
Treatment with AP89652 and AP80935 also resulted in decreases in HCV RNA levels,
but potency was less than AP80978. However, AP80935 had similar potency as
AP80978 in a 24h genotype 1b replicon assay (not shown). One key finding is that
AP80978 maximally decreased HCV RNA by 1.5 log10 at 220 nM, which was lower than
the replicon assay EC50 and suggestive of enhanced activity in the context of replicating
virus. At a comparable concentration (190 nM), inhibitory efficacy was decreasing or
lost for the HCV inhibitors 2’C-meA, VX950, and CsA (Figure 6E).
Discussion
The purpose of this study was to identify new classes of anti-HCV compounds,
particularly ones with novel viral targets. Such compounds may be critical in patients
failing existing therapies, and/or who develop drug-resistant viruses. We identified a
novel small-molecule inhibitor, AP80978, with activity against genotype 1a and 1b, but
not 2a HCV, which was not toxic in multiple cell lines. AP80978 was similar to VX950,

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an approved therapeutic for chronic HCV infection, in potency and clearance rate in the
replicon clearance assay and against replicating virus. Preliminary studies indicated that
AP80978 activity was not directed against HCV protease and polymerase, suggesting a
mechanism of action distinct from many compounds currently being evaluated in clinical
studies. Through two complementary approaches, resistant mutant generation and
evaluating the differential response of intergenotypic genomes to the compound, we
demonstrated that AP80978 acts against a novel target, NS4B.
Experimental data from numerous studies suggest several molecular functions for
the NS4B protein, including (i) formation of a membranous web, the modified membrane
structure located in the endoplasmic reticulum that is the proposed site for HCV
replication (30), (ii) modulation of NS5B RNA-dependent RNA polymerase activity (31),
(iii) modulation of HCV and host cellular translation (32-34), (iv) nucleotide binding and
GTPase activity (35), (v) modulation of NS5A hyperphosphorylation (36, 37) (vi) HCV
RNA binding (38). In theory, antiviral compounds could target any of these functions of
NS4B.
Other labs have recently identified compounds that inhibit HCV replication by
targeting NS4B, attesting to its potential as an effective drug target against HCV
(reviewed in (39)). Clemizole was identified after screening a compound library for
inhibitors of NS4B-HCV RNA binding, by using in vitro protein expression coupled with a
microfluidic affinity analysis (38). This compound inhibited HCV replication in cell
culture, and mutations conferring resistance to this compound displayed increased
affinity for the viral RNA. Viropharma discovered several classes of compounds that
interacted with NS4B using a biochemical binding assay, and demonstrated anti-

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replicon activity, presumably by apoptosis induction of NS4B-expressing cells (40). A
new class of NS4B inhibitors was identified at Stanford University, by screening for
inhibitors of vesicle aggregation, which is mediated by the NS4B AH2 domain, an
amphipathic helix between amino acids 42-66 that is involved in membrane
rearrangement (41). Of particular interest is anguizole, an NS4B antagonist that targets
the AH2 domain and changes its subcellular distribution (42). Anguizole and AP80978
share two-dimensional structural similarity in their pyrazolopyrimidine core and
substitution nature and pattern, and it would be interesting to determine whether they
have similar mechanistic activity. A novel NS4B inhibitor with activity against genotype
1a and 1b replicons is PTC725, a small molecule whose inhibitory activity is additive to
synergistic with α-interferon and HCV protease and polymerase inhibitors, although
activity against replicating HCV was not assessed (43). Similar to our data, F98 was
implicated in the response to PTC725, with mutants encoding F98L/C amino acid
substitutions reduced in both inhibitor susceptibility and replicon fitness compared to
wild-type (43). In that study, immunofluorescence experiments of replicon cells showed
no changes in intracellular distribution of NS4B in the presence of the inhibitor or with
replicons expressing F98C-substituted NS4B (43). As AP80978 and PTC725 are
dissimilar in chemical structure, it would be informative to compare their three
dimensional models to determine if they share similar electrostatic, van der Waals
and/or hydrophobic interactions with the target, since both compounds select for
mutations encoding resistance at the same amino acid position. In this context, it will be
worthwhile to further understand the mechanistic properties of AP80978, particularly
whether it antagonizes NS4B by affecting interactions with HCV RNA and/or membrane

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rearrangements. Finally, the fact that AP80978-resistant replicons remained sensitive
to CsA and VX950 suggests that drug combinations may be very effective. In this
regard, it would also be important to know whether AP80978 is potentially additive or
synergistic when combined with other inhibitors.
One unexpected finding from our study was the increased potency of both
AP80978 and AP80977 in the virus assay relative to the replicon assay. In the replicon
assay, AP80977 was inactive, while in the infection assay using cell culture-produced
intergenotypic J6/JFH Jc1 (Con 1 NS4B 53-218) virus, its activity was similar to that of the
control HCV inhibitors. Similarly, AP80978, which was already active in the replicon
assay, had enhanced potency in the infection assay. The greater inhibitory effect on
infectious virus vs. replicon is unsurprising, because in addition to creating RNA
genome replication foci, NS4B also interacts with other replicase proteins as well as
itself during infectious virion production (44-46). Therefore, an inhibitor targeting NS4B
may block numerous steps in the context of fully infectious virus replication. Whether
this assay or replicon-based assays more closely reflect inhibitor performance in
patients with chronic HCV infection is unknown. It is also not known whether the lower
potency of the racemate in the replicon assay was due to dilution of the activity of
AP80978 by AP80977, or because of an inhibitory effect of AP80977 on the activity of
AP80978. Overall, however, the potency of AP80978 against fully-replicating HCV does
provide promise for in vivo efficacy and future mechanistic studies.
Additional studies to determine how this compound affects NS4B function, as well
as structure-activity relationship studies to further improve potency and genotype
coverage are warranted. Since genotype 1 is the most prominent HCV in North America