Drug Discovery Towards
HBV Global Eradication
Raymond F. Schinazi, PhD, DSc
Frances Winship Walters Professor
Emory University/VA Medical Center
Atlanta, Georgia, USA
[email protected]
Paris
June 17, 2014
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My talk is dedicated to my friend Baruch Blumberg
Hepatitis B Virus
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450 million chronic infections. More than HIV and HCV
Causes cirrhosis. Major cause of hepatocellular carcinoma
1 million deaths / year
Discovered 1965
Double stranded DNA
Complete Dane particle 42 nm, 28 nm electron dense core,
containing HBcAg and HBeAg. The coat and the 22 nm free
particles contain HBsAg
At least 4 phenotypes of HBsAg are recognized;
adw, adr, ayw and ayr. The HBcAg is of a single serotype
• HBV classified into 8 genotypes (A-H). in Asia, genotypes
B and C predominate.
Surface protein
Lipid envelope
Capsid/core protein (Cp)
Nucleic acid and RT
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Clinically Important Antiviral Agents for HBV 2014
Drugs
FDA Status, yr
Company
Approved 1991/2005
Merck/Roche
Lamivudine (Epivir, Zeffix, 3TC)
Approved 1998
GSK/Shire
Hepsera (Adefovir dipivoxil, Preveon)
Approved 2002
Gilead
Entecavir (Baraclude, BMS-200475)
Approved 2005
BMS
L-Thymidine (LdT, Telbivudine)
Approved 2006
Novartis/IDIX
Tenofovir DF (PMPA-DF, TDF)
Approved 2008 (2001)
Gilead
Clevudine (CLV)
Approved in S. Korea
Bukwang
Tenofovir alafenamide (TAF, GS-7340)
Pending approval
Gilead
Emtriva (FTC, emtricitabine)
Approved for HIV
Gilead
Truvada (FTC + TDF)
Approved for HIV
Gilead
Interferons
(Intron A, Pegasys, various)
Off-label use:
NUCLEOSIDES CONTINUE TO DOMINATE THE ANTIVIRAL FIELD
HBV Eradication: cure or remission?
Cure
Remission
Infectious diseases model
Cancer model
(Sleeping beauty)
Long term health in absence
of anti-HBV drugs
Elimination of all
HBV infected cells
HBV DNA “undetectable”
DSL DNA & HBsAg negative
Sterilising cure
Throwing in the kitchen sink
HBV DNA < 1,000 IU/ml
Functional cure
(immune tolerance)
Elite controllers
(Inactive carriers - occult)
HBV Replication Cycle
rcDNA
rcDNA in capsid
entry
Transportation
and uncoating
cccDNA
synthesis
rcDNA = relaxed circular
DNA in nucleocapsid
cccDNA = covalently
closed circular DNA in
nucleus
Host
chromosome
cccDNA
regulation
transcription
mRNA
pgRNA
release
translation
RT
Assembly
and RT
HBV Persistence
cccDNA persistence is thought to be the cause of chronic
HBV disease
– cccDNA exists as a minichromosome in the nucleus
– cccDNA persists in the absence of active viral
replication
– cccDNA levels reduced, but not eliminated with
treatment/ liver regeneration
HBV Cure:
Elimination, suppression or control of cccDNA
What are the Possible Mechanisms of Targeting cccDNA?
Goal of cccDNA Inhibition is to either:
1. Prevent cccDNA formation
2. Eliminate existing cccDNA
3. Silence cccDNA transcription
Targeting any of the following steps should
lead to cccDNA control:
1. Capsid disassembly
2. Inhibition of rcDNA entry into nucleus
3. Inhibition of conversion of rcDNA to
cccDNA
4. Physical elimination of cccDNA
5. Inhibition of cccDNA transcription
(epigenetic control)
6. Inhibition of viral or cellular factors
contributing to cccDNA
stability/formation
7. Other??
rcDNA in capsid
2
1
DP-rcDNA
cccDNA
synthesis
cccDNA
transcription
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4
5
mRNA
HBV Capsid Protein (HBc) and Inhibition
Multiple roles of HBc during replication
• Capsid assembly
• Encapsidation of RNA
• DNA synthesis
• Transport of rcDNA into the nucleus
• Virus maturation, budding and release.
• Epigenetic role?
• Other?
= HBc (aka core antigen, HBcAg,
p21.5)
mRNA
Capsid effector molecules (HAPs) thought to
promote mis-assembly
Effect of HAP on cccDNA control mostly
unknown
translation
Formation of cccDNA and Interaction with Capsid
= Sites of HBc interaction with HBV nucleic acid
nucleus
Guo et al. 2007
Interaction of capsid with cccDNA
precursors
Role of Capsid effectors in the context of cccDNA
remains unknown
Direct Interaction with
cccDNA
• Capsid was found to bind
to CpG islands.
• Negative correlation with
methylation.
• Epigenetic role as
transcription activator?
Y-H Guo et al. 2011
Technical Limitations for cccDNA Studies
• Lack of efficient in vitro model for cccDNA
studies.
– Primary hepatocytes hard to obtain/maintain
– Hepatoma cell lines not always permissive to
infection/do not produce large amount of cccDNA
– Constitutively induced stable cell lines not a good
model for cccDNA studies
• Commonly used system: inducible HepAD38
cell line or 2.215 cells
• Feedback loop of virus replication cycle makes
mechanistic studies difficult
HBV Replication Cycle:
Opportunity for design of novel antiviral targets
infection by
endocytosis
envelopment of surface
proteins and virus secretion
RNA
containing
capsid
There is a human
analog to every
viral enzyme
genome maturation
DNA containing
capsid
There is no
analog
protein
kinase
Capsid
subunits
assembly
to viral
pol
capsid proteins
binding of the capsid
in the NPC and
transport through the
nuclear pore
disassembly
pregenomic
mRNA
Adapted from Schroder et al,
Science 2003;299:893-896.
Viral
cccDNA
transcription
Formation
of
cccDNA
Reassembly to
empty capsids (?)
pol-DNA complex
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Capsid inhibitors (CpAMs):
Multifaceted mechanism of action
Capsid inhibitors have two distinct mechanisms:
Capsid stabilization
Induce formation of non capsid polymers
Promotion of excess assembly
Misdirected assembly, decreasing
Stability of normal capsids
Inhibition of all downstream events including trafficking to nucleus and cccDNA formation
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Capsid Inhibitors (CpAMs)
Existing heteroaryldihydropyrimidines and phase of development
Abandoned?
BAY-41-4109 (Aicuris)
Preclinical
Preclinical
HAP-1 (racemic mixture)
Preclinical
AT-61
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Zlotnick et al, J. Mol. Recognit. 2006; 19: 542–548, Deres et al, Science Vol 299 7, February 2003, King et al, Antiv. Agents Chem., Dec. 1998, p. 3179–3186
Quinazolin analogs
F
F
Cl
O
Cl
O
F
N
F
N
N
H
N
H
N
F
F
F
IC50 = 1.05µM
NA
F
F
Cl
O
Cl
O
N
N
N
H
N
H
N
IC50 = 0.44µM
N
IC50 = 25.10µM
C chain - has a hydrophobic groove that
HAP fills
Leu 30
Trp 102
Ile 105
Tyr 118
Thr 33
Ser 106
Ile 139
Leu 140
Phe 110
Confidential - RFS
C chain
HAP
intercalates
in between
D chain
Confidential - RFS
HAP1 causes gross changes in capsid structure
A
B
D
C
Overlay
BLUE -HAP1
RED +HAP1
Co-crystal of HAP1 bound to crosslinked capsid
Bourne, Finn, Zlotnick (2006) J. Virol, 80:11055-61.
Anti-HBV activity and cytotoxicity of capsid
inhibitors in human hepatoma cells
Code
HepAD38
EC50, µM
HepAD38
EC90, µM
HepG2
CC50, µM (TI)
AZ-02 (B-70)
2.3
5.9
8.4 (3.7)
AZ-03 (B-73)
7.6
> 10
3.7 (0.5)
AZ-05 (B-79)
1.3
2.9
61 (47)
AZ-06 (B-80)
0.4
2.8
5.8 (15)
AZ-07 (B-81)
4.3
8.9
4.5 (1.0)
AZ-08 (B-83)
7.7
> 10
18 (2.3)
AZ-09 (B-89)
1.2
5.2
> 100 (> 83)
AZ-10 (B-108)
1.3
7.9
> 100 (> 77)
EC50 and EC90, effective concentrations required for reducing HBV replication by
50% and 90%, respectively. CC50, cytotoxicity concentration of test compound
that inhibits cell growth by 50%. TI, therapeutic index (CC50/EC50).
Anti-HBV activity and cytotoxicity of capsid
inhibitors in human hepatoma cells
Code
HepAD38
EC50, µM
HepAD38
EC90, µM
HepG2
CC50, µM (TI)
AZ-12 (B-120)
0.3
0.9
32 (99)
AZ-13 (B-121)
0.4
1.0
19 (53)
AZ-14 (B-122)
6.9
> 10
47 (6.9)
AZ-16 (B-124)
7.6
> 10
20 (2.7)
AZ-17 (B-125)
8.8
> 10
> 100 (> 11)
AZ-18 (B-142)
5.9
9.2
5.6 (1.0)
AZ-01 (B-61)
0.49
0.52
1.0
1.0
> 100 (> 200)
> 100 (> 200)
3TC (+ control)
0.06
0.2
> 100 (> 1000)
EC50 and EC90, effective concentrations required for reducing HBV replication by
50% and 90%, respectively. CC50, cytotoxicity concentration of test compound
that inhibits cell growth by 50%. TI, therapeutic index (CC50/EC50).
Inducible System Used for cccDNA Inhibitor Screening
tetCMV
HBV
Screened for 85,000 compounds
X
cccDNA
synthesis
transcription
mRNA
Modified from Cai et al. 2012
pgRNA
translation
RT
X
3TC
Nucleoside analog
treatment blocks
viral replication cycle
HBeAg
Cai et al. 2012, HepDE19/HepDES19 system
Discovery of Disubstituted Sulfonamide Compounds as
Inhibitors of cccDNA
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Micromolar activity in cell culture
Block conversion of rcDNA to cccDNA
Mechanism still unclear
More SAR possible
In development by OnCore Biopharma, Inc.
Cai et al. 2012
Capsid Inhibitors + existing therapy + novel anti-HBV targets:
Tandem approach to cure HBV
Inhibition of ongoing viral replication
Inhibition of establishment of latency
Inhibition of pro-HBV cellular environment
=
CURE FOR HBV
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New Tools towards HBV Lead
Discovery
• Sodium Taurocholate Cotransporting Polypeptide NTCP - Yang et
al., eLife 2012
– New functional receptor interacts with L proteins of HBV and HDV
– New insights on mechanism of entry and interaction with host cells
• Hepatoma Cell Line (HLCZ01) Supports Complete Replication of HBV
and HCV (Yang et al., PNAS 2014)
– Mechanism of entry and replication
– Interaction between host and virus
• Large scale Production, Structure and Function of Human HBV
Polymerase (Voros et al., J Virol 2014)
– Metal-dependent and -biding modulator of HBV pol
– Calcomine Orange 2RS inhibits priming activity of recombinant hHBV
pol
Benzimidazole (BM601)
 First inhibitor of HBV secretion and budding in this series
 Interferes with protein aggregation
CC50 = 24.5 µM
TI = > 40
HBVDNA
EC50 = 0.6 µM
HBsAg
EC50 = 1.5 µM
Xu et al Antiviral Research 2014
HBV Cure Strategy: Lymphotoxin-β receptor
(LTβR) Agonists to clear cccDNA
Inducing nuclear deaminases—for example, by lymphotoxin-β receptor
activation—allows the development of new therapeutics that, in combination
with existing antivirals, may cure hepatitis B infection.
J. Lucifora et al.: Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343, 1221, 2014.
HBV Cure Strategy: RNAi therapeutics versus
nucleoside treatment of chronic hepatitis B
siRNA
ARC-520 (Arowhead Research) : Inclusion of two siRNAs is predicted to provide
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activity against 99.6% of all known HBV genomes
HBV Cure Strategy: Elimination of cccDNA Using
Engineered DNA Cleavage Enzymes
Critical factors of successful DNA cleavage enzyme therapy:
• Vector choice for gene delivery (immune clearance, number of genes delivered/
vector, etc)
• Specific gene delivery to hepatocytes (minimizing toxicity and maximizing
efficiency)
• Enzyme-DNA target binding affinity and cleavage efficiency
• Number of doses needed for cure
• Development of resistance and “cleavage enzyme combination therapy”
Author Conclusions
• Importance of first dose in gene therapy:
– Should be very high to maximize cccDNA elimination
– Should have multiple enzymes to minimize resistance development
• Sequential doses with different vectors harboring different
enzymes should be considered
• Gene therapy should be combined with antiviral therapy
(e.g., entecavir) in order to maximize viral suppression
• Key challenge will be measuring therapeutic success:
– Difficult to detect low residual cccDNA
– Exceedingly small number of cccDNA-containing reservoir cells may
be sufficient for reactivation upon cessation of antiviral therapy
Unknowns and Priorities in
Eliminating Latent HBV
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Specificity: how to only kill cells that contain latent HBV and eliminate cccDNA.
Target new formation of cccDNA, silencing, degradation, and/or dilution.
Delivery: how to deliver the “deadly punch”, or drug(s) to initiate cell apoptosis. Role
of siRNA? Mircludex-B? TLR7 agonist? HAP?
Better in vitro and animal models to evaluate new strategies, alone and in
combination. Ideal model?
Drug development to increase specificity for latently infected cells and/or enhanced
tissue delivery. Nanoparticles?
Improve understanding of relationship between capsid inhibition and cccDNA
decline.
Better understanding of the immune system in controlling latency and activation.
Role of IFN?
How do we best measure latent and active viral replication in vivo in different
compartment? What other cells have the HBV receptor (NTCP) other than liver
cells? How do we identify extra-hepatic HBV reservoirs? Why liver tropism? Cellular
DNA ?
Defining “cure” and degree of viral load reduction to achieve ‘cure’ not yet clear.
NIH/ANRS HBV Collaboratory Network
NIH/ANRS
HBV
IS HBV ERADICATION POSSIBLE?
Impossible n‘est pas
français
Everything is theoretically
impossible until done
Robert Anson Heinlein,
American Science Fiction writer
The best is yet to come!
Supported by NIH, CFAR, and the Department of Veterans Affairs
COI: I am a founder & shareholder of Idenix & RFS Pharma LLC