Novel hepatitis B virus capsid assembly modulator induces potent antiviral responses in vitro and in humanized mice

Article abstract of DOI:10.1128/AAC.01701-19

Hepatitis B virus (HBV) affects an estimated 250 million chronic carriers worldwide. Though several vaccines exist, they are ineffective for those already infected. HBV persists due to the formation of covalently closed circular DNA (cccDNA) the viral minichromosome in the nucleus of hepatocytes. Current nucleoside analogs and interferon therapies rarely clear cccDNA, requiring lifelong treatment. Our group identified GLP-26, a novel glyoxamide derivative that alters HBV nucleocapsid assembly and prevents viral DNA replication. GLP-26 exhibited single-digit nanomolar anti-HBV activity, inhibition of HBV e antigen (HBeAg) secretion, and reduced cccDNA amplification, in addition to showing a promising preclinical profile. Strikingly, long term combination treatment with entecavir in a humanized mouse model induced a decrease in viral loads and viral antigens that was sustained for up to 12 weeks after treatment cessation.

Full text of DOI:10.1128/AAC.01701-19

AAC Accepted Manuscript Posted Online 11 November 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.01701-19
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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Novel Hepatitis B Virus Capsid Assembly Modulator Induces Potent
Antiviral Responses in Vitro and in Humanized Mice
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Franck Amblard1#, Sebastien Boucle1#, Leda Bassit1, Bryan Cox1, Ozkan Sari1, Sijia Tao1, Zhe Chen1, Tugba
Ozturk1, Kiran Verma1, Olivia Russell1, Virgile Rat2, Hugues de Rocquigny2, Oriane Fiquet3,4, Maud
Boussand3,4, James Di Santo3,4, Helene Strick-Marchand3,4, and Raymond F. Schinazi1*
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Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory
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University School of Medicine, 1760 Haygood Drive, Atlanta, GA 30322, USA;
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Morphogenèse et Antigénicité du VIH et des Virus des Hépatites, Inserm – U1259 MAVIVH, Hôpital
Bretonneau, 10 boulevard Tonnellé – BP 3223 37032 Tours Cedex 1, France;
3 Innate Immunity Unit, Institut Pasteur, 75724 Paris, France;
4 Inserm U1223, 75724 Paris, France;
Running title: Efficacy of HBV Capsid Assembly Modulator
Dr. Raymond F. Schinazi, Center for AIDS Research, Laboratory of Biochemical Pharmacology, 1760
Haygood Drive, Atlanta, GA 30322, USA. Telephone: +1-404-727-1414. Email: rschina@emory.edu
#Contributed equally
Keywords: Capsid, hepatitis B virus, antiviral, cccDNA
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23 Abstract
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Hepatitis B virus (HBV) affects an estimated 250 million chronic carriers worldwide. Though several
vaccines exist, they are ineffective for those already infected. HBV persists due to the formation of
covalently-closed circular DNA (cccDNA) – the viral minichromosome – in the nucleus of hepatocytes.
Current nucleoside analogs and interferon therapies rarely clear cccDNA, requiring lifelong treatment. Our
group identified GLP-26, a novel glyoxamide derivative that alters HBV nucleocapsid assembly and prevents
viral DNA replication. GLP-26 exhibited single-digit nanomolar anti-HBV activity and inhibition of HBeAg
secretion, and reduced cccDNA amplification in addition to a promising pre-clinical profile. Strikingly, long
term combination treatment with entecavir in a humanized mouse model induced decrease in viral loads and
viral antigens that was sustained for up to 12 weeks after treatment cessation.
34 Introduction
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Despite the availability of effective vaccines, epidemiologic data estimate that approximately 250 million
people are chronically infected with hepatitis B virus (HBV) (more than the HIV and HCV carriers combined)
and are at high risk for development of hepatitis, cirrhosis and hepatocellular carcinoma (1). Current anti-
HBV treatment options include pegylated interferon alpha2a (pegIFN) and/or nucleoside analogs that require
lifetime use to suppress the virus. Two key events in the HBV replication cycle involve first the generation of
cccDNA transcriptional template, either from input viral DNA or newly replicated capsid-associated DNA,
and second, reverse transcription of the viral pre-genomic (pg) RNA to form HBV DNA genomes that are
encapsidated into de novo viral particles (2). HBV persists in long-lived hepatocytes due to the establishment
and maintenance of cccDNA in the nucleus of host cells (3) where it is not targeted by current therapies and
serves as a viral reservoir (4). Since hepatocytes have a long half-life, elimination of cccDNA by hepatocyte
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turnover can be considered as a means of viral clearance only if the cccDNA is disrupted or silenced while
replication of new HBV is stopped. HBV rebounds after cessation of treatment with currently approved
nucleoside analog inhibitors. To address this issue, novel antiviral agents are now being investigated including
entry inhibitors, hepatitis B surface antigen (HBsAg) inhibitors and capsid assembly modulators (CAM) (5).
HBV capsid assembly plays an essential role in many steps of the viral replication cycle (6). Notably, HBV
capsid is responsible for trafficking relaxed circular DNA (rcDNA) to the nucleus thereby establishing and
maintaining cccDNA levels as a “refill” mechanism. Further, the HBV capsid protein is found in the nucleus
of hepatocytes and interacts with host factors responsible for transcriptional regulation (7). Therefore, it is
hypothesized that targeting disruption of the nucleocapsid could impact cccDNA stability and potentially lead
to eradication of HBV (8). Based on the promise of sustained antiviral activity, several CAM have been
studied such as GLS-4 (1) (phase II) (9), RG-7907 (Roche, phase I), AT-130 (2) (10), DVR-23 (11), NVR 3-
778 (3) (12) (Novira/JnJ, phase IIa), AB-423 (13) and AB-506 (14) (Arbutus), JNJ-379 (Phase IIa) (15) and
ABI H0731 (16) (Assembly Bioscience, Phase 1a) (SI Appendix, Fig. S1). Structurally these compounds are
heteroaryldihydro-pyrimidines (HAPs), phenylpropenamides (PP) or sulfamoylbenzamides (SBA). Here we
report the discovery and the preclinical characterization of GLP-26, a novel CAM with a unique
glyoxamidopyrrolo backbone, obtained through chemical optimization of early SBA derivatives identified by
our team (17).
GLP-26 (Fig. 1) is an HBV capsid assembly modulator displaying substantial effects at low nanomolar ranges
on both HBV DNA replication and HBV e antigen (HBeAg) secretion, with greater than 1 log reduction of
cccDNA amplification along with promising pre-clinical profile. Most interestingly, sustained decreases in
HBeAg and HBV surface antigen (HBsAg) levels were observed in an HBV-infected humanized mouse
model treated with GLP-26 in combination with entecavir up to 3 months after drug cessation.
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70 Results
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GLP-26 is a non-toxic inhibitor of HBV DNA, HBeAg and cccDNA production in vitro.
The in vitro anti-HBV activity of GLP-26 was determined by measuring secreted HBV DNA from HepAD38
cells and from infected primary human hepatocytes (PHH). GLP-26 displayed potent antiviral activity, with
EC50 of 0.003 M and 0.04 M in HepAD38 cells (Table 1) and PHH (Table 2), respectively. GLP-26 did
not show toxicity up to 100 µM in human hepatoma cell lines (HepG2) nor in a panel of other relevant cell
types (SI Appendix, Table S1) yielding a wide selectivity index (SI, HepG2 cells > 33,333). It is noteworthy
that GLP-26 was 25-120x more potent in these assays than GLS4, a CAM currently evaluated in the clinic. In
addition, GLP-26 did not show signs of mitochondrial toxicity at concentrations up to 50 µM and no increase
in lactic acid production (% of lactic acid/ % of nuclear DNA) was observed at concentrations up to 25 µM,
which is well above the EC50/90 antiviral values (SI Appendix, Table S2). As a correlate to cccDNA levels,
GLP-26 was evaluated for inhibition of HBeAg production in HepAD38 cells (18). GLP-26 effectively
inhibited HBeAg secretion with an EC50 = 0.003 M, which was over 50 times more potent than GLS4 (EC50
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= 0.16 M, data not shown). As expected, the nucleoside analog 3TC had minimal effect on inhibition of
HBeAg secretion (Fig. 2A). The same system was used to investigate the effects of agents on cccDNA using
RT-qPCR. Both GLP-26 and GLS-4 equally exhibited potent inhibition of HBV cccDNA amplification, with
> 1 log reduction relative to untreated mock control (Fig. 2B).
GLP-26 stabilizes HBV capsid particles and induces their accumulation in the cytoplasm.
Direct binding of GLP-26 to HBV capsid protein was evaluated using a fluorescent thermal shift assay by
measuring changes in the thermal stability of capsids upon complexation with GLP-26 (19). The HBV capsid
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protein fragment 1-149aa (Cp149) was expressed and isolated as dimers as previously described, and capsid
particles were formed from Cp149 dimers by decreasing pH and increasing salt concentration. GLP-26
reproducibly increased the melting temperature of HBV Cp149 capsids (Tm = 87 ± 0.3oC) to a greater extent
than GLS4 (Tm = 85 ± 0.3oC) (Fig. 3A). Fitting the titration of GLP-26 to HBV Cp149 capsids provides a Kd
= 0.7 ± 1.5 M which was ~60x fold lower than GLS4 (Kd = 41 ± 13 µM) indicating that GLP-26 binds to and
stabilizes HBV capsids.
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The effect of GLP-26 on cellular localization of capsids was determined by confocal microscopy in HepAD38
cells (SI Appendix, Fig. S3). In the absence of drug (Fig. 3B), 50% of the cells contained HBV core proteins
corresponding to HBV capsids in both the nucleus and the cytoplasm. In contrast, treatment with GLP-26 for
24 h emptied the nucleus and led to an accumulation of capsid particles exclusively in the cytoplasm (Fig. 3C)
while with GLS4, capsids formed large aggregates spread in the cell (Fig. 3D) as previously reported for this
type of HAP derivatives. (20, 21, 22, 23)
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GLP-26 induces the formation of firm HBV capsid particles.
The effects of GLP-26 on capsid assembly were observed using negative-stain electron microscopy (TEM).
To determine the effects on the assembly process, Cp149 dimers were first incubated with drug followed by
addition of salt to initiate assembly. In the absence of drug, HBV capsids formed regular icosahedrons with a
diameter of approximately 40 nm (Fig. 4A-B). Addition of GLP-26 to Cp149 dimers followed by assembly
generated clusters of small and misshapen particles in contrast to the large, aberrant capsid morphologies
observed induced by GLS4 (SI Appendix, Fig. S4). To determine the effects post-assembly, images of pre-
formed HBV capsid particles treated with compounds were collected. Addition of GLS4 to pre-formed
capsids resulted in larger, broken assemblies, similar in appearance to cracked egg shells (SI Appendix, Fig.
S4). Unlike GLS4, fewer particles were observed upon addition of GLP-26, and those that remained exhibited
smaller and firmer morphologies like “hard boiled eggs” (Fig. 4E-F).
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GLP-26 demonstrated synergistic antiviral activity with nucleoside analogs in vitro.
Anticipating that HBV CAMs such as GLP-26 will be administered in combination with existing direct acting
agents, we evaluated its interaction with entecavir (ETV), a potent nucleoside analog inhibitor of HBV
replication. For the median-effect analysis, the drugs were combined at a 5:1 ratio (GLP-26 + ETV) based on
their EC50 values. These two agents resulted in a combination index (CI) of 0.6 (SI Appendix, Table S3)
indicating that GLP-26 interacted synergistically with ETV in the HepAD38 system.
GLP-26 has a favorable in vitro and in vivo pharmacokinetic profile.
In preparation for in vivo applications, the stability of GLP-26 was evaluated. GLP-26 demonstrated favorable
stability profiles in plasma with half-lives > 24 h in human, mouse, and dog and a t of ~ 8.5 h in rat plasmas
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(SI Appendix, Table S4). Liver microsome stability was also satisfactory with t of 71 min and 7.6 h in
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mouse and human respectively (SI Appendix, Table S6).
The pharmacokinetic characteristics of GLP-26 were evaluated in CD-1 mice where it displayed favorable
oral bioavailability (SI Appendix, Figure S5) with AUC0-7hobs of 1,587 and 1,306 hr.ng/mL by oral (PO) and
intravenous (IV) routes respectively. The t½ was much longer from PO administration (> 6 h) compared to
that from IV (1.5h), providing prolonged concentrations high above the in vitro EC50. Due to the favorable
oral absorption profile and long half-life, we decided to deliver GLP-26 in vivo using oral dosing.
GLP-26 decreases HBV DNA, HBsAg and HBeAg levels in HBV-infected humanized mice.
To evaluate GLP-26 activity in vivo, HBV-infected BRGS-uPA mice with chimeric humanized livers
(HUHEP mice) (24, 25) were treated with either GLP-26 alone (60 mg/kg/day) or in combination with ETV
(0.3 mg/kg/day) by oral administration (Fig. 5A-D). At the start of treatment all mice had serum hAlb levels
above 100 g/ml and serum HBV DNA levels above 106 copies/ml. Over a period of 10 weeks, the untreated
cohort increased HBV DNA (>1 log10) and HBsAg (0.5-2 log10) with no significant change in HBeAg
expression. Treatment with GLP-26 alone led to decreases in viral loads (1-3 log10), HBsAg (0.3-2 log10)
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and HBeAg (0.3-1 log10). In comparison, mice treated with ETV alone had a 2.7-3.3 log10 decrease in HBV
DNA (one mouse had levels below limits of detection); however, the ETV-treated group showed minimal
decreases in HBsAg and HBeAg (0.7 log10 and 0.2 log10, respectively).
GLP-26 and ETV act synergistically to reduce viral DNA and antigens with long-term sustained activity
post-treatment in HBV-infected humanized mice.
Based on our in vitro results showing a synergistic effect between capsid assembly modulator GLP-26 and
nucleoside analog ETV, a similar combination was evaluated in vivo. HBV-infected mice were treated
concomitantly with both agents for 10 weeks. In the six mice treated with combination ETV (0.3 mg/kg/day)
+ GLP-26 (60 mg/kg/day), viral loads strongly decreased with a mean -4 log10 HBV DNA and half the mice
had undetectable viremia at the end of treatment (Fig. 5A). Furthermore, all the mice had significantly
decreased viral antigen loads – mean -1.8 log10 HBsAg and -1 log10 HBeAg (Fig. 5B-C).
As half the mice in the ETV + GLP-26 group had viral loads below the limit of detection after 10 weeks, viral
kinetics were monitored for 11-12 weeks post-treatment cessation. During the rebound phase, viral loads
returned rapidly in HBV-infected HUHEP mice that had received ETV alone, consistent with previous results
(26). However, in the ETV+GLP-26 combination treatment group, of the three mice that had undetectable
viremia, two remained aviremic for several weeks. HBV DNA was undetectable in one mouse for 5 weeks
and the other for 11 weeks off treatment (Fig. 5A). Interestingly, even if HBeAg levels remained stable or
slightly increased in most mice during the rebound phase, reduction of HBeAg up to -2 log10 was observed in
two mice (Fig. 5C). HBsAg levels decreased substantially even after treatment cessation with the exception of
two mice. One mouse showed undetectable levels of HBsAg (lower limit of detection = 0.1 IU/ml) despite
being weakly viremic (Fig. 5B).
158 Discussion
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Current treatments for chronic HBV are limited to nucleoside polymerase inhibitors and/or PEG-interferon.
These strategies rarely achieve functional cure and development of novel therapeutic agents interfering with
other essential steps of the viral replication cycle are needed. GLP-26 is a novel nontoxic CAM that displays
low nanomolar activity against HBV in both HepAD38 and PHH cells. GLP-26 is highly specific for HBV
and did not show activity against a panel of viruses including Dengue virus, West Nile virus, Chikungunya
virus, Zika virus and HIV-1 up to 30 µM (SI Appendix, Table S6). Unlike heteroaryldihydropyrimidine
derivatives (HAP) such as GLS4, GLP-26 binds to the capsid and induces formation of tight, intact particles.
These biochemical outcomes are similar to those observed for the alternate class of CAMs that includes the
propenamides (AT-130) and sulfamoylbenzamides (SBA, such as AB-423 or NVR-3-778) derivatives.
GLP-26 decreases cccDNA in vitro and decreases HBeAg (a biomarker of cccDNA) levels in vitro and in
vivo. HBV maintains cccDNA levels by recycling mature relaxed circular (rc) DNA to the nucleus (27). This
process relies on the proper biophysical properties of HBV capsid protein for rcDNA formation and nuclear
transport. Since GLP-26 affects capsid assembly and possibly transport into the nucleus, the agent likely
disrupts cccDNA maintenance from this cycle leading to an overall reduction in cccDNA levels.
GLP-26 in combination with ETV potently decreased HBsAg and HBeAg levels in a humanized mouse model
of infection both during and more importantly after treatment. It is worth noting that previous studies have
shown decreased HBV viral antigens during combination treatments (28), yet continued antiviral effects on
these markers after treatment have not been yet reported for other CAMs from the same class (AB-423 and
JNJ-632 respectively) (13, 29). The mechanisms resulting in sustained response could arise from the very
potent antiviral activity of GLP-26 (10-100 times more potent than AB-423 and JNJ-632) combined with a
prolonged exposure from oral administration leading to sustained efficacious levels of GLP-26. Since HBsAg
seroconversion is more likely with low levels of HBsAg (30), these observations suggest there is potential for
seroconversion when these biomarkers are decreased with GLP-26 treatment. In addition, the model used in
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this study did not reconstitute the mice with humanized immune systems, and we anticipate improved activity
and seroconversion in immunocompetent animal models or humans upon treatment with GLP-26.
Overall, GLP-26 displayed favorable metabolic stability with high oral bioavailability and no adverse effects
were observed after oral administration for up to 10 weeks, highlighting the relative safety of this compound.
Optimization of the treatment period, oral dosing and drug combinations (ETV, pegIFN, etc.) will be essential
to deliver a more pronounced and lasting antiviral effect in animal models and eventually in humans.
Conclusion: We identified GLP-26 as a highly potent and promising HBV CAM. Direct effects of GLP-26 on
HBV capsid assembly was established using electron microscopy, confocal microscopy and thermal shift
assays. GLP-26 inhibited HBV DNA, HBeAg and cccDNA amplification and did not display any toxicity in
vitro. Oral bioavailability in mice and stability in both plasma and liver microsomes strengthen an already
excellent preclinical profile. Combination treatment of GLP-26 with ETV in a humanized mouse model of
HBV infection delivered sustained antiviral response up to 12 weeks after treatment cessation offering the
hope that similar effects can be reached in humans with this novel CAM.
196 Materials and Methods
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Synthesis of compounds. Synthesis and characterization of GLP-26 is detailed in Supplementary Information
(SI) Section. GLS-4 was prepared according to the chemistry and methods previously described (31). Both
compounds had a purity > 95% as determined by proton 13NMR and HPLC analysis. Entecavir (ETV),
lamivudine (3TC), tenofovir disoproxil fumarate (TDF) were purchased from commercial vendors and
confirmed > 95% purity using standard analytical methods such as mass spectrometry and NMR.
Cytotoxicity assays. In vitro cytotoxicity was determined using the CellTiter 96 non-radioactive cell
proliferation colorimetric assay (MTT assay, Promega) in primary human peripheral blood mononuclear cells
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(PBMC), human T lymphoblast (CEM) and human hepatocellular carcinoma (HepG2) cell lines. Toxicity
levels were measured as the concentration of test compound that inhibited cell proliferation by 50% (CC50).
HBV Assay in HepAD38. The HBV assay was performed in HepAD38 cells as previously described (32).
Briefly, HepAD38 cells were seeded onto 96-well plates and incubated for two days at 37oC in a humidified
5% CO2 atmosphere. On day two, medium with tetracycline (Tet) was removed and cells were washed with
1X PBS. Antiviral drugs were prepared in medium without Tet and added in duplicate at various
concentrations. After a total of seven days incubation, total DNA was extracted using DNeasy 96 Tissue kit
(Qiagen), and HBV DNA was amplified by real-time PCR (18). Antiviral activity was measured by
determining the average threshold cycle for the HBV DNA amplification with the compounds (alone or in
combination), which was subtracted from the average cycle of the untreated-tetracycline control (∆CT). Drugs
were first tested individually for effective concentration, which inhibited 50% and 90% of HBV DNA
replication (EC50 and EC90) using CalcuSyn software program (Biosoft, Ferguson, MO, USA).
Evaluation of HBeAg secretion. The effect of GLP-26 on the levels of cccDNA amplification was assessed
using the HepAD38 cells to measure HBeAg as a cccDNA-dependent marker (33). In this system, HBV
replication is controlled with tetracycline: its presence in the medium blocks pre-genomic (pg) RNA
synthesis, and in its absence, synthesis of pgRNA and HBV DNA replication occur. In addition, when cells
are re-treated with Tet, new cccDNA formation is restored and HBe Ag production can be measured as a
reporter for levels of intracellular cccDNA. HepAD38 cells were incubated with or without test compounds
for seven days in medium without Tet, and another seven days in medium with Tet when cccDNA formation
and virus production relies exclusively on restored cccDNA and not on the transgene. Supernatants were
harvested at day-14, clarified by centrifugation at 2,550 x g for 5 min, and stored at -70oC until use. Levels of
HBeAg secreted in the culture medium were measured by using HBeAg ELISA kit (BioChain Institute Inc.
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Hayward, CA) according to the manufacturer’s protocol. The concentration of compound that reduced levels
of secreted HBeAg by 50% (EC50) was determined by linear regression.
Evaluation of Intracellular HBV DNA and cccDNA Levels. DNA was extracted from HepAD38 cells for
cccDNA detection. On day 14 of the experiments, DNA was purified from cells using a commercially
available kit (Plasmid Miniprep Kit, Qiagen) or a modified Hirt extraction (34). All samples were treated with
plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease (PSAD) (Epicentre, Lucigen
Corporation, Middleton, WI) at 37oC for 18 h, followed by incubation at 70oC for 30 min to inactivate PSAD.
For HBV cccDNA amplification, we used TaqMan primers as previously shown (35) to specifically amplify
cccDNA
(forward
primer:
5’
ACTCTTGGACTCBCAGCAATG3’,
and probe:
reverse
primer:
5’-
CTTTATACGGGTCAATGTCCA-3’,
5’-FAM-
CTTTTTCACCTCTGCCTAATCATCTCWTGTTCA-TAMRA-3’) using the LightCycler 480 instrument
(Roche).
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Anti-HBV Evaluation in Primary Human Hepatocytes (PHH). PHH were seeded on collagen-coated 48-
well plates at 1.4x105 cells/well in InVitroGro CP (BioIVT) medium for three hours, and then replenished
with maintenance HI medium - InVitroGro HI plus Torpedo antibiotic mix (BioIVT). After 24 h, cells were
incubated with HBV inoculum at multiplicity of infection (MOI) of 1,000 genome equivalent per cell in
maintenance HI medium containing 4% PEG-8000. The HBV inoculum was removed 24 h post infection, and
the cultures were maintained in HI medium for four days. Infected PHH cells were then incubated with the
indicated concentrations of test compounds for seven days, when the medium with or without compound was
replenished. After a total of 10 days, supernatants were harvested and HBV DNA production was quantified
by qRT-PCR using the following HBV specific primers: HBV-AD38-qF1 (5’-CCGTCT GTG CCT TCT CAT
CTG-3’), HBV-AD38-qR1 (5’- AGT CCA AGA GTY CTC TTATRY AAG ACC TT-3’), and HBV-AD38-
qP1 (5’-FAM-CCG TGT GCA /ZEN/CTT CGCTTC ACC TCT GC-3’BHQ1).
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Electron Microscopy. Samples of HBV Cp149 dimers and capsids were prepared as previously described
(17). Samples were fixed onto a charged carbon grid and stained by uranyl acetate contrast agent for 15 min.
EM images were collected using a JEOL JEM-1400 electron microscope operating at 120 kV at 25,000 -
35,000 x magnification (Emory University Robert P. Apkarian electron microscopy core facility).
Thermal Shift Fluorescence Assay. Samples were prepared containing 2 µM HBV Cp149 capsids and
varying concentrations of compounds (1-80 µM) with < 1% DMSO to a final volume of 40 µL. Sypro orange
was added to each well at 2 µL of 1:50 dilution. Each measurement was made in triplicate across two samples.
Sypro orange fluorescence was monitored continuously as temperature scanned from 45-95oC at a rate of
1oC/min on a Light Cycler 480 (Roche).
Confocal microscopy. Experiments were performed on HepAD38 cells. Cells were maintained in DMEM
supplemented with 10% fetal bovine serum (Gibco, France) and 1% antibiotics (penicillin/streptomycin:
Gibco, France) at 37oC in 5% CO2. HepAD38 cells were seeded for 6 hours, washed and incubated in DMEM.
Next, 1% v/v DMSO or 100 µM of GLP-26 (in 1% v/v DMSO) were added to this medium. After 24 h of
treatment, the medium was refreshed and cells were incubated for additional 24 h. HepAD38 cells were fixed
with 4% paraformaldehyde/PBS, permeabilized with 0.2% triton/PBS and blocked for 45 min with 0.4% of
BSA. Cells were then incubated with a human anti-HBc antibody (36) and after successive washing in PBS,
with Alexa fluor 488 goat anti-human (ThermoFisher scientific). The last wash contained DAPI and cells
were kept in PBS at 4oC until observation. Fluorescence confocal images were taken using a confocal
microscope LEICA SP8 gSTED equipped with 63x PL APO 1.40 CS2 Oil, a laser diode at 405 nm for DAPI
and an argon laser at 488 nm for Alexa 488.
Drug Combination Study. Drug interactions were analyzed using CalcuSyn (Biosoft, Ferguson, MO, USA)
computer software For the median-effect analysis, the drugs were combined at a 5:1 ratio (GLP-26 + entecavir
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- ETV) based on their EC50 values. Five to six concentrations of each single drug, or in combination, were
performed in at least two independent experiments (37).
Stability in Mouse, Rat, Dog and Human Plasma. One mL of mouse, rat, dog or human plasma containing
5 mM MgCl2 were used for the stability assay. Propantheline bromide at 10 M was used as a positive
control. The reaction was started by adding 10 µL of 1 mM stock solution of GLP-26 to give a final
concentration of 10 µM and incubated at 37oC. At selected times (0, 0.25, 0.5, 1, 2, 4, 6 and 24 h), 100 µL
aliquots were taken and the reaction was stopped by mixing with 400 µL of ice-cold acetonitrile. The samples
were centrifuged and 100 µL of the supernatant was mixed with 100 µL of LC-MS mobile phase then
subjected to LC-MS/MS analysis.
Stability in Mouse and Human Liver Microsomes. The reaction mixture was prepared in a total volume of
1.5 mL containing 5 mM of MgCl2, 100 mM of potassium phosphate buffer (pH 7.4), 1 mg/mL mouse or
human liver microsome and 1 µM compound. The reaction was initiated by adding 1 mM NADPH to the
mixture and incubated at 37oC. At selected times (0, 5, 15, 30, 45, 60 and 90 min), 200 µL aliquots were taken
and the reaction stopped by mixing with 200 µL of 70% ice-cold methanol. The samples were centrifuged and
supernatant were subjected to LC-MS/MS analysis. Propranolol at 10 M was used as a positive control.
Pharmacokinetic Studies: GLP-26 (3 mg/mL) in PBS containing 20% DMSO and 20% PEG-400, was given
by intravenous (IV) injection (15 mg/kg) and orally (PO) 30 mg/kg to female CD-1 mice. At the given time
points (0.5 h, 2 h, 4 h, and 7 h), blood samples were collected using heparinized capillaries. Samples were
centrifuged at 15,000 g for 10 min. Subsequently, blood plasma was collected and frozen at -80oC until
analysis using LC-MS/MS (ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 3 mm X 30 mm and Turbo-
IonsprayTM Interface in the negative ion-mode on AB Sciex 5500Qtrap). IACUC approval was obtained
prior to initiation of these mouse studies.
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Generation of HUHEP Mice, HBV Infection and Treatment. BALB/c Rag2-/-IL-2Rc-/-NOD.sirpa uPAtg/tg
(BRGS-uPA) mice were intrasplenically injected with 7 x 105 freshly thawed human hepatocytes (BD
Biosciences, Corning) to generate HUHEP mice as previously described (25). Liver chimerism of HUHEP
mice was evaluated with a species-specific human albumin (hAlb) ELISA (Bethyl Laboratories) on plasma
samples as previously described (25). HUHEP mice with ≥100 μg/ml hAlb were intraperitoneally infected
with 1x107 HBV genome equivalents as previously described (24). HBV-infected mice with >106 HBV DNA
copies/ml were treated per os with either ETV 0.3 mg/kg/day (Baraclude, BMS), or GLP-26 60 mg/kg/day
(dissolved in PEG400, Sigma), or the combination of ETV+GLP-26 at the same doses, delivered in MediDrop
Sucralose (Clear H2O) continuously for 10 weeks. For the rebound phase, mice were returned to regular
drinking water. Animals were housed in isolators under pathogen-free conditions with humane care.
Experiments were approved by an institutional ethical committee at the Institut Pasteur (Paris, France) and
validated by the French Ministry of Education and Research (MENESR # 02162.02).
Virological Measurements in HUHEP Mice. HBV DNA was extracted from plasma and quantified by
qPCR as previously described (24). HBeAg was quantified with an ELISA chemiluminescent immunoassay
kit (Autobio, China), and HBsAg was quantified with the MONOLISA HBsAg Ultra kit (Bio-Rad) following
manufacturer’s protocols.
ACKNOWLEDGMENT: This work was supported in part by NIH Grant 1-R01-AI-132833 (RFS), and
5P30-AI-50409 (CFAR) (RFS), by Agence Nationale de Recherches sur le Sida et les Hépatites Virales
(ANRS) Grants #2016-16180 and #2016-16365, European Commission Seventh Framework Programme
PATHCo (HEALTH-F3-2012-305578), Institut Pasteur, and Institut national de la santé et de la recherche
médicale (HSM); by ANRS and Institut national de la santé et de la recherche médicale (INSERM) (HDR).
We gratefully acknowledge the Center for Translational Science and the Animalerie Centrale of the Institut
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Pasteur for productive collaboration. We also thank Pierre-Ivan Raynal and Julien Burlaud-Gaillard from
Electron Microscopy (EM) Facility (IBiSA) of the Tours University (http://microscopies.med.univ-tours.fr)
for technical support. We gratefully acknowledge Elizabeth Wright, Ph.D. (University of Wisconsin,
Madison) and Hong Yi (Emory University Robert P. Apkarian electronmicroscopy core) for their advice and
assistance with the EM studies.
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Figure 1. Structure of GLP-26.
447
448
A.
20

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450
B.
Tet ON
3TC, µM
10
GLS4, µM
GLP-26, µM
1 10
1
10
0.00
-0.50
-1.00
-1.50
-2.00
-2.50
-3.00
-3.50
HBV cccDNA
HBV DNA
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Figure 2. Decrease of cccDNA markers in HepAD38 cells by GLP-26. A) Percent inhibition of HBeAg
secretion by ELISA; Cells with or without drugs were incubated in the absence of tetracycline for 7 days,
followed by addition of Tet to culture of both untreated and drug-treated cells for another 7 days (7-14 days).
Tet ON, cells cultured in the presence of tetracycline for 14 days. Inhibition (%) of HBeAg secretion was
determined relative to untreated Tet off/on control. and B) The levels of HBV DNA and cccDNA were
quantified by qPCR and log viral reduction was determined relative to untreated mock Tet off control. All
values represent the average of at least two independent experiments and samples were performed in duplicate
± SD.
453
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459
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Figure 3. Effects of GLP-26 binding to HBV capsids. A) Thermal shift fluorescence assay thermograms for
vehicle (dotted line), GLS4 (dashed line) and GLP-26 (solid line) treated HBV Cp149 capsids. Confocal
immunofluorescence microscopy images showing HBV Core protein (in green) distribution in HepAD38
hepatocytes after 24 h for B) vehicle (DMSO) , C) GLP-26 (1 M). D) GLS-4 (1 M). Nuclei are DAPI
stained (blue).
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Figure 4. Effects of GLP-26 on the HBV Cp149 capsid morphology determined by negative-stain
electron microscopy. A and B) HBV Cp149 capsid particles treated with vehicle and. C and D) HBV Cp149
treated with GLP-26 (25 M) prior to assembly initiation. E and F) Pre-formed HBV-Cp149 capsid particles
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471
472
473
474
475
476
477
treated with GLP-26 (25 M). Black bars represent 50 nm scale.
23

478
103
102
A.
C.
101
100
101
100
10-1
10-2
10-3
10-4
10-1
10-2
10-3
0
2
4
6
8
10 12 14 16 18 20 22
0
2
4
6
8
10 12 14 16 18 20 22
week post treatment
week post treatment
HUHEP HBV
HUHEP HBV ETV
HUHEP HBV GLP-26
HUHEP HBV ETV+GLP-26
101
100
103
102
D.
B.
101
100
10-1
10-2
10-3
10-4
10-1
10-2
0
2
4
6
8
10 12 14 16 18 20 22
0
2
4
6
8
10 12 14 16 18 20 22
week post treatment
week post treatment
479
480
481
482
483
484
485
486
Figure 5. Sustained antiviral activity of GLP-26 in HBV-infected humanized mice by oral
administration. Effect of entecavir (ETV) 0.3 mg/kg/day, GLP-26 60 mg/kg/day or the combination of ETV
+ GLP-26 at the same doses on A) HBV DNA, B) HBsAg, C) HBeAg, and D) human Albumin in HBV-
infected HUHEP mice. Treatment period is indicated in the grey shaded area followed by a rebound period,
the lower limit of detection is shown as a thin dotted horizontal line in A) and B). The thick dotted horizontal
line shows the reference point of one to evaluate fold changes. Each full line represents the longitudinal
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results from an individual HBV-infected HUHEP mouse either untreated (black line), ETV (green line), GLP-
26 (blue line), or ETV+GLP-26 (red line) treated.
Table 1. Anti-HBV activity of GLP-26 in HepAD38 cells.
Anti-HBV Activity (µM)
Druga
EC50
EC90
GLP-26
GLS4
3TC
0.003 ± 0.002
0.08 ± 0.02
0.41 ± 0.36
0.014 ± 0.002
0.28 ± 0.06
1.65 ± 0.92
ETV
0.0006 ± 0.0003 0.011 ± 0.002
0.005 ± 0.0004 0.070 ± 0.010
TDF
491
492
493
494
a3TC: Lamivudine; ETV: Entecavir. TDF: Tenofovir disoproxil fumarate
All values represent the average of at least two independent experiments and samples were performed in
duplicate ± SD.
495
496
Table 2. Anti-HBV activity of GLP-26 in primary human hepatocytes (PHH).
Anti-HBV Activity (µM)
PHH
CC50
(µM)
>10
Drug
EC50
GLP-26
GLS4
TDFa
0.04 ± 0.01
4.34 ± 1.62
0.27 ± 0.23
>10
>10
25

aTDF: Tenofovir disoproxil fumarate. All values represent the
average of at least two independent experiments and
samples were performed in duplicate.
497
26