Identification of Disubstituted Sulfonamide Compounds as Specific
Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA
Dawei Cai,aCourtney Mills,bWenquan Yu,bRan Yan,aCarol E. Aldrich,cJeffry R. Saputelli,cWilliam S. Mason,cXiaodong Xu,b
Ju-Tao Guo,aTimothy M. Block,a,bAndrea Cuconati,band Haitao Guoa
Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania, USAa;
Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown, Pennsylvania, USAb; and Institute for Cancer Research, Fox Chase Cancer Center,
Philadelphia, Pennsylvania, USAc
tions are transient, approximately 5 to 10% of infected adults and
over 90% of infected neonates fail to mount a sufficient immune
response to clear the virus and develop a life-long chronic infec-
tion (23, 27). Chronic hepatitis B is currently a substantial public
health burden, affecting approximately 350 million individuals
worldwide. These patients have an elevated risk of liver cirrhosis,
hepatocellular carcinoma, and other severe clinical sequelae (1,
23). It is therefore a global health priority to cure chronic HBV
infection and prevent its dire consequences.
HBV is a noncytopathic, liver-tropic DNA virus belonging to
the Hepadnaviridae family. Upon infection, the viral genomic re-
laxed circular DNA (rcDNA) is transported into the cell nucleus
and converted into episomal covalently closed circular DNA
pregenomic RNA (pgRNA) is assembled with HBV polymerase
and capsid proteins to form the nucleocapsid, inside which poly-
which is subsequently copied into plus-strand DNA to form the
progeny rcDNA genome. The mature nucleocapsids are then
either packaged with viral envelope proteins to egress as virion
particles or shuttled to the nucleus to amplify the cccDNA
way (reviewed in references 1, 29, and 37).
cccDNA is an essential component of the HBV replication cy-
cle and is responsible for the establishment of infection and viral
with hepatitis B virus (HBV). Although most adulthood infec-
persistence. The details of the molecular mechanism by which
rcDNA is converted into cccDNA remain poorly understood.
Considering the subcellular location and unique structures of
these two viral DNA molecules, virus trafficking and a number of
specific biochemical reactions can be predicted to occur during
cccDNA formation. To begin with, the cytoplasmic rcDNA pres-
ent in nucleocapsids needs to be transported into the nucleus via
karyopherin-dependent recognition of nuclear localization sig-
nals (NLS) on the capsid protein (19, 34). On the other hand,
minal features of rcDNA, including (i) completion of viral plus-
strand DNA synthesis, (ii) removal of the 5=-capped RNA primer
at the 5= terminus of plus-strand DNA, (iii) removal of the viral
polymerase covalently attached to the 5= end of minus-strand
DNA, (iv) removal of one copy of the terminal redundancies on
minus-strand DNA (40), and (v) ligation of both strands to gen-
erate cccDNA. Recently, a protein-free rcDNA form without co-
valently bound viral polymerase has been identified, which was
Received 2 March 2012 Returned for modification 11 April 2012
Accepted 17 May 2012
Published ahead of print 29 May 2012
Address correspondence to Haitao Guo, Haitao.Guo@drexelmed.edu, or Andrea
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 56 Number 8Antimicrobial Agents and Chemotherapyp. 4277–4288aac.asm.org
designated deproteinized rcDNA (DP-rcDNA). This viral DNA
demonstrated as one, if not the only, functional precursor inter-
mediate for cccDNA formation (6, 11, 12). DP-rcDNA thus pro-
vides a potential antiviral target for cccDNA intervention.
To date, there is no definitive cure for chronic hepatitis B.
feron (IFN-?) and five nucleos(t)ide analogues (lamivudine
[3TC], adefovir, entecavir, telbivudine, and tenofovir). IFN-?
therapy yields sustained virological responses in less than 40% of
side effects. The five nucleos(t)ide analogues all act as potent in-
emergence of drug-resistant virus dramatically limits their long-
term efficacy (31). The major limitation of current treatment is
the failure to eliminate the preexisting cccDNA pool and/or pre-
vent cccDNA formation from trace levels of wild-type or drug-
resistant virus. Thus, there is an urgent need for the development
However, screenings for anti-cccDNA agents have not been
conducted because of the lack of efficient in vitro HBV infection
models, and a practical approach for measuring cccDNA in a
high- to mid-throughput format was unavailable. Although there
are primary human hepatocytes and the recently established
HepaRG cell culture system available to support cccDNA-depen-
dent HBV replication, the efficiency of HBV infection in both
ity in primary hepatocytes from donor to donor, with some
batches proving to be uninfectable. The HepaRG system requires
much manipulation for experimental setup (8). These character-
screening of compound libraries for cccDNA inhibitors. Alterna-
lar amplification pathway in stably transfected HBV cell cultures
that constitutively or conditionally replicate the HBV genome (2,
11, 22, 39), as represented by the HepG2.2.15 line (35, 38). How-
ever, direct cccDNA detection from HBV cell lines by either
cccDNA in HepG2.2.15 cells since most of the viral products are
derived from an integrated viral transgene and are indistinguish-
able from cccDNA contributions. To surmount this problem, we
have previously reported that the production of secreted HBV e
antigen (HBeAg) was predominantly cccDNA dependent in
(22, 45). In the present study, we used an upgraded version of a
solely cccDNA-dependent HBeAg-producing cell line, termed
HepDE19 (11), in a 96-well format assay for the screening of
agents that inhibit cccDNA formation and/or maintenance. As
developed, the assay is adaptable to high-throughput screening
formats and full automation.
This report presents the results of that screening campaign.
Our screening effort led to the identification of two disubstituted
sulfonamides (DSS) as inhibitors of cccDNA production. Both
(EC50s) in cell culture. We further demonstrated that the DSS
compounds synchronously reduced the levels of viral cccDNA
suggested that the DSS compounds primarily interfered with
rcDNA conversion into cccDNA. This is the first attempt, to the
best of our knowledge, to identify small molecules that directly
target cccDNA formation and has resulted in two novel leads for
the development of new hepatitis B therapeutics.
MATERIALS AND METHODS
College, New York, NY) were maintained in Dulbecco’s modified Eagle’s
medium-F12 (Mediatech, Manassas, VA) supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 400
?g/ml G418. Tetracycline-inducible HBV producer cells, specifically,
HepDE19 and HepDES19 cells, were maintained in the same way as
HepG2.2.15 cells but with the addition of 1 ?g/ml tetracycline (11). To
initiate HBV replication and cccDNA formation in HepDE19 and Hep-
cells were cultured for the indicated time period.
Compound sources and handling. The in-house compound library
(Institute for Hepatitis and Virus Research [IHVR] small-molecule col-
lection) consists of 85,000 compounds from the complete libraries of
ChemDiv, Inc. (San Diego, CA), Asinex Inc. (Winston-Salem, NC),
Chembridge Inc. (San Diego, CA), Maybridge Inc. (Cornwall, United
Kingdom), LOPAC1280 (Sigma-Aldrich, St. Louis, MO), the Micro-
Source SPECTRUM Collection (Gaylordsville, CT), and the Johns Hop-
selected from the large libraries of these companies on the basis of their
“drug-like” properties and diversity. The library was “cherry-picked” by
the vendors as requested using their own cheminformatic tools to gener-
ate smaller sets. Web-based tools (especially ADME-Tox) available at
sembled library. While the ?47,000-compound ChemDiv and Asinex
collections are combinatorial, the remainder of the IHVR library
(?38,000) is highly diverse. Overall, the IHVR library has an average
molecular mass of ?350 Da and a maximum cLogP value (the logarithm
of a compound’s partition coefficient between n-octanol and water) of
5.0. The LOPAC, MicroSource, and John Hopkins collections are anno-
tated compounds that are drugs in clinical use, drug candidates that have
known targets. Compounds were purchased as dry powder in 96-well
format “mother” plates, resuspended in ultrapure dimethyl sulfoxide
(DMSO) to a final concentration of 10 mM, and diluted in DMSO into
working stock (“daughter”) plates at 1 mM. Compounds were stored in
covered polypropylene plates at ?20°C. Resynthesis of CCC-0975 and
CCC-0346 was carried out by ChemDiv, Inc. (San Diego, CA), and
Pharmabridge Inc. (Doylestown, PA), respectively.
Compound screening with a 96-well format assay. HepDE19 cells
cells were then trypsinized and seeded into 96-well plates at a density of
5.0 ? 104cells/well with tetracycline-free medium to induce HBV repli-
cation. Immediately following cell seeding, compounds were added to
screening plates by means of automated liquid handling (Beckman
screening plate consisted of 80 compound test wells, 4 wells of cells with
1.0% DMSO only, 4 wells with DMSO and without cells, and 4 wells of
HBeAg (precore) accumulation was assayed with an in-house-developed
indirect enzyme-linked immunosorbent assay (ELISA) with a Z factor of
fixed with 100% ice-cold methanol for 20 min, followed by two washes
(150 ?l each) with phosphate-buffered saline (PBS) containing 0.5%
Tween 20 (PBST) with 1 min of incubation at room temperature. Wells
were blocked for 12 h at 4°C with 100 ?l PBST containing 2% bovine
Cai et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
BSA containing a mouse anti-HBeAg monoclonal antibody (clone
followed by the addition of 25 ?l PBST-BSA containing a horseradish
peroxidase-conjugated anti-mouse antibody (diluted 1:5,000) and incu-
absorbance at a wavelength of 650 nm was determined with a reference
reading at 490 nm. Wells where the signal was reduced by 50% were
defined as containing a hit compound. The primary screening took ap-
proximately 5 months at a pace of 4,300 compounds per week.
To confirm the activity of hit compounds, EC50s were determined by
incubating cells with compounds in duplicate wells at concentrations of
and performing best-fit curve analysis of the results with XLfit 4.0 (IDBS;
Bridgewater, NJ). Degrees of inhibition were calculated against multiple
In addition, each plate had multiple wells containing 50 ?M 3TC as a
ing the manufacturer’s directions (International Immuno-Diagnostics,
Foster City, CA).
centrations (CC50s) were determined by plating cells at 1.0 ? 104/well
(20% confluence) to detect inhibition of cell growth compared with that
which occurred in the absence of compounds. Cell plates were then incu-
for the compound screen assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
a final concentration of 0.5 mg/ml (28). Plates were again incubated at
37°C for 4 h, after which 10% SDS–0.01 N HCl was added to each well in
to solubilize the reaction product. Colorimetric absorbance was read in a
Rainbow spectrophotometer (Tecan US Inc., Durham, NC) at 570 nm
The selectivity index (SI) of each compound was determined as fol-
lows: SI ? CC50/EC50. Compound hits with SIs of ?4 were counter-
screened through HepG2.2.15 cells, in which the majority of HBeAg ex-
pression is from integrated HBV DNA, not cccDNA. Those compounds
forwarded to the secondary assay.
Viral nucleic acid analysis. Total cellular RNA was extracted with
1.5% agarose gel containing 2.2 M formaldehyde and transferred onto
fer (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Intracellular
hepadnaviral core DNA and cccDNA (Hirt DNA) were extracted as de-
scribed previously (10–13, 15). Half of the DNA sample recovered from
one well of a six-well plate was resolved by electrophoresis into a 1.2%
agarose gel and blotted onto Hybond-XL membrane. Membranes were
hepadnaviral minus-strand-specific (for detection of DNA) or plus-
strand-specific (for detection of RNA) full-length riboprobe and exposed
nals were visualized by Typhoon FLA-7000 (GE Healthcare) and quanti-
fied with ImageQuant TL software (GE Healthcare).
was performed as previously described but with modifications (12).
Briefly, an EPR mixture was assembled with 10 ?l of HBV virion stock
(?5 ? 107genome equivalents) concentrated from the supernatant of
HepDE19 cells cultured in tetracycline-free medium (11), or 10 ?l of
DHBV virion stock (?2 ? 107genome equivalents) partially purified
from DHBV-positive duck serum, plus 15 ?l of 2? EPR buffer, which
consisted of 0.3 M NaCl; 0.1 M Tris-HCl (pH 8.0); 20 mM MgCl2; 2 mM
and dTTP; and 10 ?M [?-32P]dCTP. Compounds were added as indi-
cated, and the mixture was supplemented with water to bring the volume
to 30 ?l. As inhibitory controls, 0.2 mM dGTP in 2? EPR buffer was
net sodium (PFA) was added to the DHBV EPR mixture, respectively.
After incubation at 37°C for 1 h, the reaction solution was dotted onto a
trichloride acid for 30 min at room temperature and then washed three
minute) of acid-insoluble32P was measured with a liquid scintillation
counter (Perkin-Elmer, Waltham, MA).
HBV precore/core immunoblotting. Cells in one well of a six-well
plate were washed once with PBS buffer and lysed in 300 ?l of 1? Laem-
mli buffer. Thirty microliters of the cell lysate was resolved by 12% SDS-
PAGE, and proteins were transferred onto an Immobilon-FL polyvi-
nylidene difluoride membrane (Millipore). The membrane was blocked
with Western Breeze blocking buffer (Invitrogen) and probed with anti-
bodies against the C-terminal 14 amino acids of HBV precore/core pro-
tein (12) and ?-actin (Millipore). Bound antibodies were revealed by
IRDye secondary antibodies and visualized using the Li-COR Odyssey
system (Lincoln, NE).
were purchased from Metzer Farms (Gonzales, CA). Primary duck hepa-
tocyte (PDH) cultures were prepared from 1-week-old DHBV-negative
ducklings (33). PDHs were plated into 60-mm-diameter tissue culture
dishes in L15 medium supplemented with 5% fetal bovine serum and
maintained at 37°C. The next day, the cultures were shifted to serum-free
L15 medium. The cells were infected on day 2 after seeding with congen-
itally DHBV-infected duckling serum containing approximately 1.5 ?
107enveloped virus particles. The medium was replaced after 16 h and
changed every other day thereafter. Cultures were harvested at the time
points indicated. The plates were washed once with chilled PBS, and viral
as previously described (10). All PDH experiments were reviewed and
Chase Cancer Center.
Production of HBeAg is cccDNA dependent in HepDE19 cells.
The cccDNA-dependent assay mechanism of the HepDE19 cell
line is illustrated in Fig. 1. Briefly, a more-than-one-genome
length of the HBV genome spanning the entire viral pgRNA re-
gion under the control of the tetracycline-regulated cytomegalo-
virus immediate-early (tet-CMV) promoter is integrated into the
chromosomal DNA of HepG2 cells. A point mutation of the pre-
core start codon at the 5= end of the transgene is introduced to
prevent the expression of precore/HBeAg from the transgene.
viral core protein and polymerase and initiate reverse transcrip-
tion to generate rcDNA, resulting in cccDNA formation via the
intracellular amplification pathway. Meanwhile, the start codon
of the C-terminally truncated precore open reading frame (ORF)
at the 3= end of the pgRNA is copied into the viral DNA sequence
and the precore ORF is restored during rcDNA conversion into
cccDNA. Thus, the authentic precore mRNA will be transcribed
only from cccDNA, with the translated precore protein product
being further processed into HBeAg (42), which is secreted into
HBV cccDNA Inhibitors
August 2012 Volume 56 Number 8aac.asm.org 4279
the culture fluid and serves as a marker for cccDNA in HepDE19
To validate cccDNA-dependent HBeAg production in
HepDE19 cells, we compared HBV replication, cccDNA forma-
tion, and precore/HBeAg production in parallel in HepG2.2.15
cells displayed constitutive HBV RNA transcription, DNA repli-
cation, and precore/core protein expression when the cells
reached confluence (Fig. 2A, left panel); ELISAs showed that the
HBeAg level rapidly increased from day 0 to day 4 and continued
time point in this experiment (Fig. 2A, lanes 1 to 5), which dem-
onstrated that the HBeAg is expressed predominantly from the
HBV transgene and not from cccDNA, if there is any, in
pression, DNA replication, and cccDNA synthesis gradually in-
creased in a time-dependent manner and precore protein expres-
sion was detected only after cccDNA reached detectable levels
proportional to the intracellular cccDNA level (Fig. 2B). There-
fore, the results confirmed a quantitative relationship between
cccDNA and HBeAg levels and support the use of HepDE19 cells
for screening to identify compounds that affect HBV cccDNA
formation and/or maintenance.
Identification of DSS compounds as novel cccDNA inhibi-
tors. Using the HepDE19 cell-based assay described above, we
screened an in-house chemical compound library consisting of
pC, C, pol, pS1, pS2, S, and X represent ORFs for the precore protein; the core protein; the polymerase; the preS1, preS2, and S domains of the HBV surface
antigen; and the X protein, respectively. DR represents identical direct repeat sequences 1 and 2. pA is a polyadenylation site. Upon the removal of tetracycline
(tet), pgRNA is transcribed and the viral core protein and polymerase are produced (B), resulting in pgRNA packaging, reverse transcription of pgRNA to
minus-strand DNA (C), and sequential plus-strand DNA synthesis and circulation into rcDNA (D). rcDNA is converted to the cccDNA template, in which the
precore ORF is restored, giving rise to authentic precore mRNA (E) and pgRNA (F). (G) cccDNA-derived precore mRNA serves as the template to translate the
precore, which is further processed into secreted HBeAg through proteolytic cleavage of the N-terminal signal peptide and the C-terminal domain (CTD) (42).
Cai et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
Compounds were added simultaneously with tetracycline with-
drawal in order to identify inhibitors of cccDNA establishment
and/or maintenance. HBeAg was detected by ELISA as described
in Materials and Methods. Nontoxic compounds causing a 50%
reduction of the HBeAg level were declared primary hits. Hits
were counterscreened in HepG2.2.15 cells, in which HBeAg is
produced predominantly in a cccDNA-independent manner. A
total of 329 compounds that selectively reduced HBeAg levels in
HepDE19 cells, but not HepG2.2.15 cells, were chosen for fol-
low-up assays. Following EC50and CC50determinations, eight
incubated with each compound at 10 ?M after tetracycline with-
drawal for a total incubation time of 14 days to ensure sufficient
DSS, termed 2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-
yl)-benzamide (CCC-0346), which reduced HBeAg with accept-
able SIs of ?11 and ?7 in the screening assays, respectively (Fig.
3), emerged as final confirmed cccDNA inhibitor hits.
DSS compounds were deemed interesting in that they had not
been identified as hits in other antiviral screenings we had con-
ducted in-house, which suggested that their activity is specific
tural relationship with DSS compounds and other sulfonamide-
containing antiviral molecules have been described in the litera-
ture as inhibitors of viral polymerases, proteases, integrases,
transcription, and entry (41); this chemical distinction under-
scores the novelty of the anti-HBV DSS compounds. Both DSS
compounds possess very attractive drug-like properties that meet
Lipinski’s rule of five (24), including cLogP values of ?5.0, mo-
lecular weights of ?500, ?5 H bond donors, ?10 H bond accep-
tors, and acidic pKa values that suggest good oral bioavailability.
In addition, the structures are highly chemically tractable for fu-
ture lead optimization and structure-activity relationship (SAR)
dor or prepared by resynthesis and retested with HepDES19 cells,
which have a higher level of cccDNA production than HepDE19
DSS compounds inhibit accumulation of HBV cccDNA and
DP-rcDNA in HepDES19 cells. Treatment with CCC-0975 re-
sulted in a dose-dependent reduction of cccDNA in HepDES19
cells with an EC50of 10 ?M, along with a significant reduction of
els). The reduction of DP-rcDNA and cccDNA was proportional,
compared with that in the untreated control. CCC-0346 has a
higher level of toxicity than CCC-0975 in cell growth assays (Fig.
3); however, at concentrations that proved nontoxic to confluent
FIG 2 Evaluation of cccDNA-dependent HBeAg production in HepDE19
cells. (A) Kinetics of intracellular virus production in HepG2.2.15 and
HepDE19 cells. HepG2.2.15 cells were seeded at a density of 1.2 ? 106cells/
then the cells were continuously cultured for 8 days. HepDE19 cells were
cultured in tetracycline-containing medium until confluent (day 0), and then
tetracycline was removed from the medium and the cells were maintained for
another 8 days. HepG2.2.15 and HepDE19 cells and culture fluid were har-
vested every other day from day 0 to day 8. Total cellular RNA was extracted,
and HBV RNA was detected by Northern blotting as described in Materials
are indicated. HBV core DNA and cccDNA (Hirt DNA) were extracted and
analyzed by Southern hybridization. The positions of rcDNA (RC), single-
stranded DNA (SS), DP-rcDNA (DP-rc), and cccDNA are indicated. The ex-
pression of the HBV core and precore proteins was detected by Western blot-
ting, with the levels of ?-actin serving as a loading control. Since the core
antibody recognizes the 14-amino-acid epitope at the C terminus of the core
protein (12), the detected precore band represents an HBeAg precursor (p22)
with an intact C-terminal domain (18, 42). (B) Correlation between the levels
of HBV cccDNA and HBeAg in HepDE19 cells. HBeAg levels in the culture
medium harvested from panel A were determined by commercial HBeAg
ELISA (International Immuno-Diagnostics, Foster City, CA) and plotted as a
are representative of two separate trials.
HBV cccDNA Inhibitors
August 2012 Volume 56 Number 8aac.asm.org 4281
cells, CCC-0346 displayed an antiviral effect against DP-rcDNA
and cccDNA accumulation similar to that of CCC-0975 but had
an EC50of 3 ?M (Fig. 4B, bottom panels).
Although cccDNA formation in HepDES19 cells is driven by
mediates under DSS compound treatment at high concentrations
(Fig. 4, top and middle panels) did not quantitatively account for
the reduction of cccDNA, and their decrease ought to be a conse-
tributes approximately 10% of the total viral RNA and DNA pro-
duction in stably HBV-transfected cell lines (2, 14, 45). We thus
speculated that DSS-mediated cccDNA reduction was not, or at
transcription or DNA replication. This notion was further sup-
ported by the observations that DSS compounds did not inhibit
HBV polymerase activity in the in vitro endogenous polymerase
dition, DSS compounds did not reduce the steady-state levels of
(day 4) of treatment (Fig. 6, top and middle panels), when the
cccDNA was undetectable by Southern blotting (Fig. 6, bottom
panel). Strikingly, DSS compounds led to a significant reduction
deproteinization and/or the stability of DP-rcDNA is influenced
by DSS compounds. Nevertheless, the possibility that DSS-medi-
ated cccDNA reduction, seen at later time points, is due to DP-
rcDNA independent mechanisms is not ruled out.
DSS compounds do not alter the decay kinetics of HBV DP-
rcDNA and cccDNA. To further determine whether DSS com-
pounds reduce HBV DP-rcDNA and cccDNA through direct in-
hibition of the biosynthesis of these two DNA molecules or by
promoting their degradation in cell culture, a HepDES19 cell-
based experimental system was used to study the stability of
cccDNA upon compound treatment. As shown in Fig. 7, removal
of tetracycline from HepDES19 cells led to the accumulation of
HBV RNA, core DNA, DP-rcDNA, and cccDNA at day 12 (Fig.
7B, lane 1), at which point the culture fluid was supplemented
pgRNA transcription and viral DNA replication, respectively,
thereby preventing the de novo replenishment of cccDNA forma-
tion. After another 4-day period, the levels of viral RNA and core
els); however, HBV DP-rcDNA and cccDNA remained at high
levels (Fig. 7B, lane 2, bottom panel), which was consistent with
previous reports demonstrating that hepadnaviral cccDNA per se
dominantly from cccDNA-based transcription and maintained
thereafter (Fig. 7B, top panel). The observed slight increase in
cccDNA from day 12 to day 16 might have been due to the con-
tinued conversion of DP-rcDNA to cccDNA (Fig. 7B [compare
lanes 2 and 1], C, and D), which, for unknown reasons, is ex-
The decay kinetics of the preexisting DP-rcDNA and cccDNA
were then determined without or with DSS compound treatment
in the presence of tetracycline and 3TC. As shown in Fig. 7B by
in the cells following continuous culture with similar half-lives of
not alter the decay kinetics of DP-rcDNA and cccDNA. Those
observations, along with the fact that DSS compounds inhibited
the accumulation of DP-rcDNA and cccDNA in the context of
HBV DNA replication (Fig. 4 and 6), thus suggested that the an-
tiviral mechanism(s) of DSS compounds is to block the biosyn-
thesis of cccDNA, perhaps through reducing the amount of DP-
rcDNA, which is a functional precursor of cccDNA formation
above-described evaluations of DSS compound potency against
cccDNA biosynthesis were done with stably HBV-transfected cell
lines, in which cccDNA formation relies largely on transgene-de-
rived viral replication. Therefore, a critical issue was whether or
and subsequent viral replication during an authentic hepadnavi-
infection and cccDNA formation with high efficiency (26). In
lication and cccDNA formation permit shorter incubation times.
As shown in Fig. 8, pretreatment of PDHs for 2 h, DHBV in-
oculation, and continued treatment with CCC-0975 for 5 days
cccDNA biosynthesis, with an EC50of 3 ?M (Fig. 8A, top panel).
Because viral replication totally relies on cccDNA in DHBV-in-
fected PDHs, viral core DNA was reduced proportionally with
FIG3 DSS compounds as inhibitors of HBeAg production in HepDE19 cells.
The chemical structures and properties of CCC-0975 (A) and CCC-0346 (B)
are presented. Graphs represent inhibition of HBeAg production (top) and
loss of HepDE19 cell viability (bottom), and each point is the average of du-
plicate samples. Incubation was for 7 days. EC50and CC50values (?M) were
calculated with XLfit 4.0 (IDBS, Surrey, United Kingdom). MW, molecular
Cai et al.
aac.asm.org Antimicrobial Agents and Chemotherapy
cccDNA inhibition (Fig. 8A, bottom panel). Interestingly, CCC-
0346 did not exhibit significant toxicity in PDHs compared to
(Fig. 8B). Such a discrepancy might be due to cell type- or virus-
specific conditions. In a parallel experiment, treatment of PDH
with DSS compounds at a single dose of 10 ?M postinoculation
with DHBV resulted in similar effects against cccDNA formation
and viral DNA replication (Fig. 8C). The primary hepatocytes
were monitored daily by microscopy following compound treat-
throughout the experiments (data not shown). In addition, nei-
ther DSS compound inhibited DHBV polymerase activity in an
especially CCC-0975, exhibit antiviral activity against cccDNA
formation in multiple hepadnavirus systems.
Current treatments of chronic hepatitis B are limited to IFN ther-
apy and nucleos(t)ide analogue reverse transcriptase inhibitors,
all of which are used for prolonged periods but cure the infection
FIG 5 DSS compounds do not inhibit HBV polymerase activity. HBV virion
particles purified from HepDE19 culture fluid were subjected to endogenous
polymerase reaction with [32P]dCTP plus CCC-0975 or CCC-0346 at the in-
dicated concentrations. DMSO at 0.1% was used as a solvent control, and
ddGTP was a reference chain terminator in the reaction. Incorporated
relative polymerase activity was plotted as a percentage of the readout of
counts per minute in the control reaction (y axis). Values are averages of
triplicate samples, and error bars represent standard deviations.
FIG 4 DSS compounds reduce the levels of cccDNA and DP-rcDNA in HepDES19 cells. Upon withdrawal of tetracycline, HepDES19 cells were left untreated
(UNT) or treated with CCC-0975 (A) or CCC-0346 (B) at the indicated concentrations; tetracycline-free medium was changed every other day with fresh
(top), core DNA (middle), and Hirt DNAs (DP-rcDNA and cccDNA) (bottom) were extracted and analyzed by Northern blotting and Southern blotting,
DNA species in each sample is expressed as a percentage of that from untreated cells and is indicated below each blot.
HBV cccDNA Inhibitors
August 2012 Volume 56 Number 8aac.asm.org 4283
virus genome, cccDNA, which is inherently stable in the nuclei of
infected hepatocytes and is only indirectly affected by current
therapies (25). In order to clear the infection, a durable, curative
antiviral therapy that directly reduces the level of HBV cccDNA
without killing infected hepatocytes is needed.
It is generally acknowledged that cccDNA formation and per-
to be many molecular opportunities for intervention, ranging
from biosynthesis to maintenance of cccDNA. The putative ap-
following. The first is inhibition of cccDNA establishment. Such
lead from mature cytoplasmic rcDNA to the presence of cccDNA
in the nucleus (12). The second is inhibition of cccDNA main-
tenance factors. Although it is still unclear whether a cccDNA-
specific maintenance factor(s) exists, a small molecule may
specifically recognize and alter the stability of the cccDNA
minichromosome. The third is chemical alteration of cccDNA.
Compared to the host chromatin, cccDNA should exhibit in-
creased sensitivity to DNA damage due to the very dense cod-
ing of the HBV genome (30). The fourth is epigenetic silencing
of cccDNA transcriptional activity (32). The fifth is induction
clearance of cccDNA during acute infection can occur through a
noncytolytic mechanism that is largely independent of adaptive
immune function (9, 43), indicating that cccDNA can be reduced
by innate cellular defenses that may be activated by a biologically
active stimulator or a small-molecule compound.
of HBeAg, we discovered two structurally related sulfonamide
compounds that significantly reduced the levels of HBV cccDNA
in cell cultures. Further mechanistic studies revealed that the DSS
compounds directly block the conversion of HBV rcDNA into
cccDNA, rather than inhibiting the production of rcDNA as ex-
of-concept evidence that it is feasible to develop small-molecule
precursor, an essential but unexploited step in the HBV replica-
The exact target(s) of DSS compounds is still unclear, consid-
ering that there are many molecular details yet to be elucidated in
the understanding of cccDNA formation and metabolism.
cccDNA is formed from both the incoming viral genome during
initiation of infection and newly synthesized mature viral DNA,
through conversion of the viral rcDNA genome, most possibly by
employing the host DNA repair machinery in the nucleus (1). In
one attempt to identify the potential intermediate(s) bridging
rcDNA-to-cccDNA conversion, a linear woodchuck hepatitis vi-
rus genome that contains a terminal duplication of the cohesive
region (between DR1 and DR2) from rcDNA by displacement
synthesis through the cohesive overlap was proposed to be a
cccDNA precursor by serving as a substrate for DNA repair
through legitimate recombination. The authors found that some
integrated viral DNA appeared to have this linear DNA as a pre-
cursor; however, the extrachromosomal form of such hypothetic
terious (44). Our recent studies focusing on a DP-rcDNA mole-
cule which loses the covalently bonded viral polymerase revealed
that DP-rcDNA is a bona fide precursor of cccDNA formation,
ular pathway of cccDNA formation has been proposed in which
the completion of viral plus-strand DNA inside the nucleocapsid
The deproteinization reaction is tightly associated with a nucleo-
capsid structural shift, resulting in exposure of the NLS at the C
terminus of capsid protein, which in turn initiates the
plexes. Finally, DP-rcDNA is released from the capsid in the nu-
cleus and is converted into cccDNA presumably through DNA
as well as cccDNA, without directly affecting viral DNA replica-
tion, thus providing another line of evidence that DP-rcDNA is a
precursor of cccDNA formation. Nevertheless, it is still possible
that DSS compounds may inhibit the production of another un-
discovered cccDNA precursor(s) or DNA repair mechanism(s)
involved in cccDNA formation.
The mechanisms underlying the deproteinization of hepadna-
FIG 6 DSS compounds reduce the level of HBV DP-rcDNA, but not viral
RNA and core DNA, in HepDES19 cells with short-term treatment. Upon the
removal of tetracycline, HepDES19 cells were left untreated or treated with
CCC-0975 or CCC-0346 at the indicated concentration; tetracycline-free me-
dium was changed every other day with fresh compound supplementation.
The DMSO concentration in the whole experiment was normalized at 0.1%.
Northern blotting and Southern blotting, respectively.
Cai et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
virus rcDNA remain elusive. The viral polymerase is covalently
linked to the 5= phosphate group of the rcDNA minus strand
through a tyrosine residue (Y63 for HBV, Y96 for DHBV) in the
terminal protein (TP) domain, as a consequence of TP-mediated
protein priming during the initial reverse transcription of viral
rcDNA ought to be an essential step in cccDNA formation. Our
removed by endonuclease cleavage of the sequences proximal to
the linkage between the rcDNA terminus and polymerase, based
of rcDNA deproteinization are narrowed down to the following.
(i) The phosphodiester bond is hydrolyzed by a phosphodies-
terase, one candidate being a recently discovered human 5=-ty-
rosyl DNA phosphodiesterase (TDP2) that removes topoisomer-
ase covalently conjugated at the 5= end of a chromosomal DNA
break (3). (ii) Polymerase is removed by proteolytic digestion,
which results in a small peptide, or at least the tyrosine residue,
being left on the DP-rcDNA. Therefore, it will be important to
tivity of DSS compounds in the future.
The pool size of cccDNA ranges from 1 to 50 copies per cell,
and once established, nascent cccDNA converted from progeny
rcDNA, coupled with its stability within the infected hepatocyte,
is poorly understood. The precise half-life of HBV cccDNA has
been reported to range from days to months in different animal
days later, one set of cells (lane 3) was cultured further with medium containing tetracycline and 3TC and another two sets of cells were treated with CCC-0975
(10 ?M, lanes 4, 7, 10, and 13) or CCC-0346 (3 ?M, lanes 5, 8, 11, and 14) in the presence of tetracycline and 3TC for the indicated period of time. The DMSO
concentration in all experimental groups was 0.1%. (B) Cells were harvested at the indicated time points, and the levels of viral RNA, core DNA, and Hirt DNA
were determined as described in Materials and Methods. The relative intensities of viral DP-rcDNA and cccDNA signals in each sample are expressed as
HBV cccDNA Inhibitors
August 2012 Volume 56 Number 8aac.asm.org 4285
cells under 3TC treatment (Fig. 7). Because cccDNA plays a cen-
tral role in HBV persistence, elimination of cccDNA is the ulti-
mate goal of antiviral therapy. Unfortunately, current antiviral
treatment with nucleos(t)ide analogues fails to eliminate the pre-
existing cccDNA pool and/or prevent cccDNA formation from
trace level wild-type or drug-resistant virus (48). Although DSS
compounds are unable to promote the degradation of the preex-
isting cccDNA in cell cultures, their mechanism of action is cer-
tainly distinct from that of the nucleos(t)ide analogues, and thus,
they will likely retain activity in preventing cccDNA formation
even when the virus has become resistant to polymerase inhibi-
tors. In addition, DSS compounds may also exhibit synergistic
effects in combination therapy with replication inhibitors. It is
envisioned that the combination of inhibition of viral replication
by nucleos(t)ide analogue with its amelioration in liver function
(25) and the inhibition of de novo cccDNA formation by a DSS
compound may enhance the eventual clearance of cccDNA. The
antiviral effect of DSS compounds in combination with a nucle-
os(t)ide analogue and the potential inhibitory effect of DSS com-
pounds against drug-resistant virus cccDNA formation will be
tested in future studies.
0975, also inhibited DHBV cccDNA formation in virus-infected
PDHs. This observation indicates a wide spectrum for these com-
pounds in hepadnavirus cccDNA intervention and also provides
an opportunity to evaluate their antiviral activity (alone or in
combination with nucleos[t]ide analogues) in the duck model.
Other hepadnavirus animal models, including humanized uPA/
SCID mice and woodchucks (4, 21), are also available for in vivo
testing of DSS compounds. First, however, the potency of the hit
compounds needs to be improved. This work is under way
through a SAR study. The structural features of these DSS com-
pounds offer multiple modification opportunities for lead opti-
ally explored for SAR (e.g., electronic effect, steric effect, etc.)
investigation and to improve antiviral profiles.
In summary, we have, for the first time, discovered two struc-
turally related novel inhibitors of HBV cccDNA biosynthesis
current antiviral agents, the unique antiviral mechanism of DSS
compounds is inhibition of the formation of cccDNA from its
rcDNA precursor and may involve the inhibition of rcDNA de-
proteinization, a possible intermediate step during cccDNA for-
FIG8 Antiviral effects of DSS compounds in DHBV-infected PDHs. PDHs were plated in 60-mm dishes and pretreated with control solvent (0.1% DMSO) or
with CCC-0975 (A) or CCC-0346 (B) at the indicated concentrations. Two hours later, PDHs were infected in the presence of the test compounds with
DHBV-positive duck serum containing approximately 1.5 ? 107virions. After overnight incubation, the PDHs were rinsed twice with regular medium and the
treatments were continued with medium and compound replenishment every other day. In a parallel experiment (C), PDHs were infected overnight without
DNA species in each sample are expressed as percentages of those in the untreated (UNT) cells and are presented below each blot.
Cai et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
mation. Thus, these two inhibitors also provide a promising re-
search tool to identify a viral/host protein(s) involved in cccDNA
formation. Clearance of cccDNA is the ultimate goal for the cure
available therapeutics alone; the further development of DSS
compounds may ultimately lead to drugs that change the land-
scape of hepatitis B management.
1A2 (to Andrea Cuconati and Haitao Guo), and R01AI094474 (to Haitao
appropriation of the Commonwealth of Pennsylvania. Work in William
Haitao Guo is the Bruce Witte fellow of the Hepatitis B Foundation.
1. Block TM, Guo H, Guo JT. 2007. Molecular virology of hepatitis B virus
for clinicians. Clin. Liver Dis. 11:685–706, vii.
2. Chou YC, et al. 2005. Evaluation of transcriptional efficiency of hepatitis
bined with the restriction enzyme digestion method. J. Virol. 79:1813–
3. Cortes Ledesma F, El Khamisy SF, Zuma MC, Osborn K, Caldecott
KW. 2009. A human 5=-tyrosyl DNA phosphodiesterase that repairs to-
poisomerase-mediated DNA damage. Nature 461:674–678.
4. Dandri M, Petersen J. 2012. Chimeric mouse model of hepatitis B virus
infection. J. Hepatol. 56:493–495.
5. Galibert F, Mandart E, Fitoussi F, Charnay P. 1979. Nucleotide se-
quence of the hepatitis B virus genome (subtype ayw) cloned in E. coli.
6. Gao W, Hu J. 2007. Formation of hepatitis B virus covalently closed
HBeAg-positive chronic hepatitis B. Gastroenterology 133:1437–1444.
B virus. Proc. Natl. Acad. Sci. U S A. 99:15655–15660.
9. Guidotti LG, et al. 1999. Viral clearance without destruction of infected
cells during acute HBV infection. Science 284:825–829.
10. Guo H, Aldrich CE, Saputelli J, Xu C, Mason WS. 2006. The insertion
domain of the duck hepatitis B virus core protein plays a role in nucleo-
capsid assembly. Virology 353:443–450.
11. Guo H, et al. 2007. Characterization of the intracellular deproteinized
relaxed circular DNA of hepatitis B virus: an intermediate of covalently
closed circular DNA formation. J. Virol. 81:12472–12484.
12. Guo H, Mao R, Block TM, Guo JT. 2010. Production and function of the
13. Guo H, et al. 2005. Identification and characterization of avihepadnavi-
ruses isolated from exotic anseriformes maintained in captivity. J. Virol.
14. Guo H, et al. 2007. Regulation of hepatitis B virus replication by the
phosphatidylinositol 3-kinase-akt signal transduction pathway. J. Virol.
15. Hirt B. 1967. Selective extraction of polyoma DNA from infected mouse
cell cultures. J. Mol. Biol. 26:365–369.
16. Hoofnagle JH, di Bisceglie AM. 1997. The treatment of chronic viral
hepatitis. N. Engl. J. Med. 336:347–356.
17. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. 2007. Management
of hepatitis B: summary of a clinical research workshop. Hepatology 45:
18. Ito K, Kim KH, Lok AS, Tong S. 2009. Characterization of genotype-
specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen pre-
cursor and identification of furin as the candidate enzyme. J. Virol. 83:
19. Kann M, Schmitz A, Rabe B. 2007. Intracellular transport of hepatitis B
virus. World J. Gastroenterol. 13:39–47.
20. Köck J, et al. 2010. Generation of covalently closed circular DNA of
manner. PLoS Pathog. 6:e1001082. doi:10.1371/journal.ppat.1001082.
21. Kulkarni K, Jacobson IM, Tennant BC. 2007. The role of the woodchuck
model in the treatment of hepatitis B virus infection. Clin. Liver Dis.
22. Ladner SK, et al. 1997. Inducible expression of human hepatitis B virus
(HBV) in stably transfected hepatoblastoma cells: a novel system for
screening potential inhibitors of HBV replication. Antimicrob. Agents
24. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 2001. Experimental
and computational approaches to estimate solubility and permeability in
drug discovery and development settings. Adv. Drug Deliv. Rev. 46:3–26.
ideal drug here yet? J. Hepatol. 51:416–418.
26. Mason WS, Taylor JM. 1989. Experimental systems for the study of
hepadnavirus and hepatitis delta virus infections. Hepatology 9:635–645.
27. McMahon BJ. 2005. Epidemiology and natural history of hepatitis B.
Semin. Liver Dis. 25(Suppl 1):3–8.
28. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and sur-
vival: application to proliferation and cytotoxicity assays. J. Immunol.
29. Nassal M. 2008. Hepatitis B viruses: reverse transcription a different way.
Virus Res. 134:235–249.
30. Newbold JE, et al. 1995. The covalently closed duplex form of the hep-
adnavirus genome exists in situ as a heterogeneous population of viral
minichromosomes. J. Virol. 69:3350–3357.
31. Pawlotsky JM, et al. 2008. Virologic monitoring of hepatitis B virus
approach. Gastroenterology 134:405–415.
32. Pollicino T, et al. 2006. Hepatitis B virus replication is regulated by the
33. Pugh JC, Summers JW. 1989. Infection and uptake of duck hepatitis B
virus by duck hepatocytes maintained in the presence of dimethyl sulfox-
ide. Virology 172:564–572.
34. Rabe B, Vlachou A, Pante N, Helenius A, Kann M. 2003. Nuclear import
of hepatitis B virus capsids and release of the viral genome. Proc. Natl.
Acad. Sci. U S A. 100:9849–9854.
35. Schmidt K, Korba B. 2000. Hepatitis B virus cell culture assays for anti-
viral activity. Methods Mol. Med. 24:51–67.
36. Schulze-Bergkamen H, et al. 2003. Primary human hepatocytes—a valu-
FIG 9 DSS compounds do not inhibit DHBV polymerase activity. DHBV
virion particles derived from the sera of congenitally DHBV-infected ducks
were subjected to an endogenous polymerase reaction with [32P]dCTP plus
CCC-0975 or CCC-0346 at 10 ?M. DMSO at 0.1% was used as a solvent
control. PFA served as a positive control for polymerase inhibitor. Incorpo-
rated [32P]dCTP radioactivity was measured with a liquid scintillation coun-
ter. Relative polymerase activity was plotted as a percentage of the counts per
minute read in the control reaction mixture (y axis). Values are averages of
triplicate samples, and error bars represent standard deviations.
HBV cccDNA Inhibitors
August 2012 Volume 56 Number 8aac.asm.org 4287
able tool for investigation of apoptosis and hepatitis B virus infection. J. Download full-text
37. Seeger C, Mason WS. 2000. Hepatitis B virus biology. Microbiol. Mol.
Biol. Rev. 64:51–68.
38. Sells MA, Chen M, Acs G. 1987. Production of hepatitis B virus particles
in hepG2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl.
Acad. Sci. USA. 84:1005–1009.
39. Sells MA, Zelent AZ, Shvartsman M, Acs G. 1988. Replicative interme-
40. Sohn JA, Litwin S, Seeger C. 2009. Mechanism for CCC DNA synthesis in
41. Supuran CT, Innocenti A, Mastrolorenzo A, Scozzafava A. 2004. Anti-
viral sulfonamide derivatives. Mini Rev. Med. Chem. 4:189–200.
42. Wang J, Lee AS, Ou JH. 1991. Proteolytic conversion of hepatitis B virus
compartment. J. Virol. 65:5080–5083.
43. Wieland SF, Spangenberg HC, Thimme R, Purcell RH, Chisari FV.
2004. Expansion and contraction of the hepatitis B virus transcriptional
44. Yang W, Mason WS, Summers J. 1996. Covalently closed circular viral
infected liver. J. Virol. 70:4567–4575.
45. Zhou T, et al. 2006. Hepatitis B virus e antigen production is dependent
upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and
may serve as a cccDNA surrogate in antiviral screening assays. Antiviral
46. Zhu Y, et al. 2001. Kinetics of hepadnavirus loss from the liver during
inhibition of viral DNA synthesis. J. Virol. 75:311–322.
47. Zoulim F. 2005. New insight on hepatitis B virus persistence from the
study of intrahepatic viral cccDNA. J. Hepatol. 42:302–308.
48. Zoulim F, Locarnini S. 2009. Hepatitis B virus resistance to nucleos(t)ide
analogues. Gastroenterology 137:1593–1608.e1-2.
Cai et al.
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