Professor Stephen Locarnini

1
Anti-HBV Drugs Molecular Structure and Antiviral
Activity

• Professor Stephen Locarnini
• Victorian Infectious Diseases Reference
  Laboratory,
• North Melbourne, Victoria 3051,
• AUSTRALIA
• www.vidrl.org.au/publications/hep_updates.htm

2
Hepatitis B Molecular pathogenesis

• HBV replicates its DNA genome via reverse
  transcription of pregenomic RNA
• HBV is not generally cytopathic to hepatocytes
• Precore protein / HBeAg essential for
  establishing persistent infection
• Host immune responses (generally inadequate
  and/or in appropriate) are responsible for the
  liver disease of chronic hepatitis B
• Two therapeutic approaches
• (i) direct antiviral agents lamivudine,
  adefovir and entecavir.
• (ii) immune modulation interferon alpha and
  thymosin-alpha

3
Key Step 1 Conversion of RC DNA into cccDNA
4
Key Step 2 Reverse Transcription
Key Step 1 Conversion of RC DNA into cccDNA
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Early Events cccDNA Generation
Nucleus
ER/Golgi
Cytosol
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Genomic Replication Reverse Transcription
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Late Events Assembly Release Versus Recycling

• Precore protein inhibits core dimerization.
• Precore protein essential for persistent
  infection.

8
IFN-? (i) 2?5?-OA Synthetase RNaseL(ii) PKR
(iii) MxA
LA PROTEIN
Gordien et al (2001) J Virol 752684
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Inhibition of Reverse Transcription
Nucleos(t)ide Analogues
transport to cell
nucleus
CCC DNA
DNA
uncoating
Attachment and
repair
LA PROTEIN
Penetration
MINICHROMOSOME
Nucleus
Re-entry
pregenomic RNA
HBV RNA
Golgi
transcripts
complex
HBV polymerase
protein

envelope proteins S, M, L
Release
core proteins
Nucleos(t)ide Analogues eg.
LMV, ADV, ETV
HBV DNA SYNTHESIS
Delaney et al (2001) Antiviral Chem Chemother.
121-35
Chain Termination
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What Causes Antiviral Drug Resistance?

• Antiviral drug resistance reflects reduced
  susceptibility of a virus to the inhibitory
  effect of a drug
• Antiviral drug resistance results from a process
  of adaptive mutations under therapy
• High replication rates
• Low fidelity of the viral polymerase
• Selective pressure of the drug
• Role of replication space (liver turnover)
• Fitness of mutant

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Escape Mutants and Interferon-Alpha Responses

• No specific mutation conclusively associated with
  IFN-a treatment failure
• In vivo
• Correlation between BCP mutations and poor IFN- a
  response for gentypes B and C HBV
• (Kao et al (2000) J.Hepatol 33998)
• HBV genotype A is more sensitive to IFN- a than
  genotype D
• (Erhadt et al (2005) GUT 541009)
• this was independent of HBeAg status
• IFN-a treatment associated with emergence of
  novel pre-C sequences, some of which prevent
  HBeAg expression
• (Gunther et al (1992) Virology 187271)
• In vitro
• HBV genotype C with G1896A precore stop codon
  mutation resistant to IFN- a
• (Wang et al (2005) World J Gastroenterol 11649)

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Resistance Rates in Naïve Patients Treated with
LMV, ADV or LdT
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Clinical Consequences of Drug Resistant HBV

• Flares in serum ALT
• Reduced HBeAg seroconversion
• Histological progression of liver disease
• Increased recurrence post-OLT
• Changes in HBsAg envelope antigenicity
• Transmission of drug-resistant HBV

14
Time to Disease Progression by YMDD StatusFrom
Liaw et al, 2004 NEJM 3511521
25
Placebo (n215)
21
YMDDm (n209) (49)
20
Wild Type (n221)
Placebo
with disease progression
15
13
YMDDm
10
5
5
WT
0
0
6
12
18
24
30
36
Time after randomisation (months)
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Three Groups of Nucleoside/Nucleotide Analogues
used in Chronic Hepatitis B

• L-Nucleoside Group
• lamivudine (3TC)
• emtricitabine (FTC)
• telbivudine (L-dT)
• clevudine (L-FMAU)
• Acyclic Phosphonate Group
• adefovir (PMEA)
• tenofovir (PMPA)
• Cyclopentane/pentene Ring Group
• entecavir
• abacavir/carbovir

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L-Nucleoside Group
LAMIVUDINE
EMTRICITABINE
TELBIVUDINE
CLEVUDINE
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Acyclic Phosphonate Group
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Cyclopentane Group
ABACAVIR / CARBOVIR
ENTECAVIR
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HBV Antiviral Drug Resistance Patterns (Approved
Therapies)

• Monotherapy Lamivudine
• Group 1 rtL180M rtM204V/I
• 2 rtM204I
• 3 rtL80V/I rtM204I
• 4 rtV173L rtL180M rtM204V
• 5rtI169 T rtV173L rtL180M rtM204V
• 6 rtA181T
• 7 rtL180M rtM204S
• 8 rtT184S
• 9 rtQ215S

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HBV Antiviral Drug Resistance Patterns (Approved
Therapies)

• Monotherapy Adefovir Dipivoxil
• Group 1 rtN236T
• 2 rtA181V/T
• 3 rtV84M / rtS85A
• 4 rtV214A / rtQ215S
• Monotherapy Entecavir LMV Backbone
• Group 1 rtI169T rtV173L rtL180M
  rtT184G rtS202I rtM204V
• Group 2 rtI169T rtV173L rtL180M
  rtM204V rtM250V
• Group 3 Various Combinations of Mutations at
  codons 184, 202 and 250

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HBV Antiviral Drug Resistance Patterns (Under
Investigation)

• Monotherapy Telbivudine
• Group 1 rtM204I
• 2 Other Lamivudine Associated
  Mutations ?
• Monotherapy Tenofovir
• Group 1 rtL180M rtA194T rtM204V
• 2 rtV214A, rtQ215S

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Key Resistance Mutations
LMV Resistance rtL80V/I rtV173L/rtL180M
rtM204V/I/S rtQ215S L-dT Resistance
rtM204I ADV Resistance rtV84M rtA181T/V rtV214A
rtQ215S rtN236T TDF Resistance
rtA194T/rtV214A/rtQ215S ETV Resistance
rtS184G rtS202I rtM250V
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Resistance Mutations Associated with the
Nucleos(t)ide Analogue Class
Selection of LAM-Resistant Mutants Affects Future
Treatment Options
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Molecular Mechanisms of Antiviral Resistance in
Hepatitis B

• Mutations Cause
• Impaired Incorporation of NA into viral DNA
  resulting in reduced binding affinity.
• (i) Steric Hindrance
• Catalytic domain mutations alter the ability of
  the POL to bind NA relative to the natural
  substrate (dNTP)
• (ii) Catalytic Efficiency
• Suboptimal nucleophilic attack geometry for
  incorporation of NA into viral DNA

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HBV Polymerase
Orange Domain A Yellow Domain B Green Domain
C Red Domain F White Domain D Pink Domain E
Bartholomeusz et al (2004) Antiviral Therapy
9149-60.
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HBV Polymerase LMV Resistance
Bartholomeusz et al (2004) Antiviral Therapy
9149.
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3 Clusters of ADV Mutations
Orange Domain A Yellow Domain B Green Domain
C Red Domain F White Domain D Pink Domain E
Group 2
Group 1
Group 3
Bartholomeusz et al (2004) Hepatology 40(No. 4
Suppl.1)246A Abstract 185
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Wildtype rtN236
rtN236T
Indirect perturbation of the triphosphate binding
site and alteration of the Mg binding site
Wild type HBV polymerase with ADV
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Group 2 ADV Mutations
Wildtype
rtA181V
Indirect steric hinderance via the C domain
rtM204
Wild type HBV polymerase with ADV
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Group 3 ADV Mutations rtQ215S
Wild Type
rtQ215S

• Mutations in the C-D hinge region can potentially
  alter both the C and the D Domain relative to the
  active site
• rtQ215S mutation located on the external region
  of RT.
• Potential to alter the interaction with the
  Polymerase complex (TP, Spacer or RNAse H).

31
Role of rtM250V in ETV Resistance
Wild type Pol with Entecavir
rtM250V with Entecavir

• M250V alters the binding interaction between the
  primer stand and the incoming dNTP (Entecavir)

• M250 interacts with the primer strand

Warner et al (2004) Hepatology 40(No. 4
Suppl.1)245A Abstract 183
32
ETV Resistance Group 2
Wild type polymerase
rtT184G rtS202I

• Altered geometry of the Nucleotide Binding pocket

• rtT184 and rtS202 stabilize the interaction
  between the B and C domains

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Indications of Emergence of Drug-Resistant Virus

• 1. Increasing viral load (?1.0 log IU/ml)
• 2. Increasing serum ALT level
• 3. Clinical deterioration
• 4. Identification of known genotypic markers of
  drug resistance within viral polymerase

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Antiviral Treatment Failure
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Management of Patients With Drug-Resistant
Hepatitis B
typically Lamivudine
36
Management of Patients With Drug-Resistant
Hepatitis B
typically Lamivudine
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Ways to Prevent Resistance (D. Richman)

• Maximize antiviral activity
• increase maximum tolerated dose
• select most effective regimen
• nucleoside analogue potentiation
• Maximize genetic barriers to resistance
• avoid sequential therapy
• choose drugs requiring multiple resistance
  mutations
• choose drugs where patient is naïve
• Increase pharmacologic barriers
• patient compliance
• raising trough levels
• tissue distribution (no sanctuaries)

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New Targets/New Directions for Hepatitis B Therapy
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Immune Based ApproachThe Innate Immune System

• Evolutionarily ancient
• Universal - all multicellular organisms
• Constitutive - germ-line
• Immediate pathogen response (hours)
• Components Pattern Recognition Central
• Pattern recognition receptors pathogen-assoc.
  molecular patterns
• Cell-surface eg Toll-like receptors
• Secreted
• Intracellular
• Phagocytes eg. dendritic cells
• Antimicrobial peptides eg. cathelicidins,
  defensins
• Alternate complement pathway

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TLRs What Are They?

• Type I integral membrane glycoproteins
• Members of larger superfamily that includes IL-1
  receptors (considerable homology in cytoplasmic
  regions)
• Highly conserved

Akira S, Takeda K (2004) Toll-like receptor
signalling Nat Rev Immunol 4499-511
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TLR Signaling
IFN?/?
TNF?
Akira S, Takeda K. (2004) Toll-like receptor
signalling. Nat Rev Immunol 4499-511.
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IMPAIRED TOLL-LIKE RECEPTOR EXPRESSION IN CHRONIC
HEPATITIS B IMPLICATIONS FOR PATHOGENESIS AND
THERAPY

• Visvanathan K1, Skinner N1, Riordan S2, Sozzi V3
  , Edwards R3, Chang J4, Lewis S4 and Locarnini S3.

1Murdoch Childrens Research Institute,
Melbourne, Australia 2Gastrointestinal and Liver
Unit, The Prince of Wales Hospital and University
of New South Wales, Sydney, Australia, 3Victorian
Infectious Diseases Reference Laboratory,
Melbourne, Australia and 4Alfred Hospital,
Melbourne Australia.
Hepatology 40 (No. 4, Suppl 1) 2004 598A
Abstract 1005)
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In vivo Correlation of the Precore TLR
Relationship in HBeAg-positive and negative CH-B
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HBeAg and TLR Expression Hep G-2 Transduced
HBeAg-Pos or HBeAg-Neg / Recombinant
HBV-Baculovirus
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TOLL 2 Precore Interaction
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TOLL 2 Precore Interaction
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Summary and Conclusion

• TLRs expressed on hepatocytes in vivo and in
  vitro
• TLRs expressed on Kupffer cells in vivo
• Precore protein/HBeAg downregulates TLR-2
  expression and functional signalling
• Absence of Precore protein/HBeAg upregulates
  TLR-2 expression and functional signalling
• Identification of an important interaction of the
  precore protein/HBeAg and the innate immune
  response to HBV
• Substantial pathogenic and therapeutic
  implications in the management of HBeAg-Pos and
  HBeAg-Neg CH-B

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Future Directions Drug Resistance

• resistance emerges when replication occurs in the
  presence of the drug selection pressure
• (No Replication No Resistance NRNR)
• the best cost-effective strategy is to prevent or
  avoid the emergence of antiviral drug-resistance
• combination chemotherapy should increase the
  efficiency of antiviral therapy
• select patients for therapy who are most likely
  to HBeAg-seroconvert and/or sustain the
  virological suppression achieved with antiviral
  therapy
• develop better treatment strategies based on
  viral replication and molecular pathogenesis
• HAART-type approach PLUS Innate Immune Based
  Therapies