Professor Stephen Locarnini

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Antiviral Resistance Novel Mechanisms for
Different Blood Borne Viruses HBV / HCV / HIV

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

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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 (cell turnover
  lymphoid vs hepatic compartments)
• Fitness of mutant

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Viral Replication and Mutational Frequency

• High virion production 1012-13 virions per day
• Wild-type HBV HCV/HIV Pol lacks proof-reading
  function
• High mutational rate10-5 substitution/base/cycle
• 1010-11 point mutations produced per day
• All possible single base changes can be produced
  per day
• Single / double mutations pre-exist in HBV from
  patients prior to therapy WHY MOST MONOTHERAPIES
  FAIL
• Triple / quadruple mutations require replication
  in the presence of selection pressure and rarely
  pre-exist WHY COMBINATION TREATMENT WORKS
  (Colgrone Japour. 1999. AVR. 4145)

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Viral Mutations Occur Spontaneously as a Natural
Result of Viral Replication
Wild Type Virus
Mutant
Pawlotsky JM. Therapy of Hepatitis C From
Empiricism to Eradication. Hepatology.
200643(Suppl 1)S207-S220.
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Inhibition of Wild-Type Virus May Lead to
Selection of Naturally-Occurring Resistant Mutants
Wild Type Virus
Wild Type Virus
Drug
Mutant
Mutant
Pawlotsky JM. Therapy of Hepatitis C From
Empiricism to Eradication. Hepatology.
200643(Suppl 1)S207-S220.
6
Combination Therapy May Optimize the Chance for
SVR Before Resistance Can Occur
Wild Type Virus
Drug 1
SVR
Drug 2
Mutant
Pawlotsky JM. Therapy of Hepatitis C From
Empiricism to Eradication. Hepatology.
200643(Suppl 1)S207-S220.
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Antiviral Selection Pressure
Richman 2000 Hepatology32866-867
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Replication Space (Modified from Dr. Jake Liang)

• Uninfected Cells are Created by
• Normal Growth of the Target Organ
• Cell Proliferation and Turnover
• c. Loss of Viral Transcriptional template

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Replication Fitness/Competence

• the ability to produce offspring in the setting
  of natural selection
• is NOT a yield measurement
• can be measured virologically using in vitro
  co-infection competition assays
• Enzyme processivity the ability of the Pol to
  catalyze the incorporation of nucleotides during
  synthesis of cDNA products before enzyme
  disassociates from its template
• Enzyme Fidelity the ability of the Pol to
  correctly base-pair the template during
  polymerization

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Genomic Replication Reverse Transcription
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Inhibition of Reverse Transcription
Nucleos(t)ide Analogues
transport to cell
nucleus
CCC DNA
DNA
uncoating
Attachment and
repair
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
Chain Termination
Delaney et al (2001) Antiviral Chem Chemother.
121-35
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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
• Identification of known genotypic markers of drug
  resistance within viral polymerase
• primary resistance mutations
  (rtM204V/I)
• secondary/compensatory mutations
  (rtV173L)

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LMV MonoTherapy Major Issues

• In HIV
• Compensatory Mutations Fix The Primary Drug
  Resistance Mutations

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LMV MonoTherapy Major Issues
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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

Doo and Liang. 2001. Gastroenterology1201000-100
8. Bartholomeusz et al. 2004. AVT949
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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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ETV Resistance Group 2
Wild type polymerase
rtT184G rtS202I
rtT184 and rtS202 stabilize the interaction
between the B and C domains
Altered geometry of the Nucleotide Binding pocket
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HCV Replication in Infected Host Cell
Y
Y
NUCLEUS
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HCV Enzymes are the Targets for New HCV Therapies
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HCV NS3/4A Protease

• generates NS proteins from HCV polyprotein
• part of replication complex
• essential for HCV replication
• blocks hosts IRF-3 mediated immune response by
  proteolysis of essential signalling proteins
  (Foy et al. Science 2003. 3001145)
• Proteolytic activity inhibited by BILN 2061,
  VX-950, SCH6, SCH503034 and ITMN-191
• Macrocyclic tri-peptide/peptidomimetic which
  block different parts of active site
• Active against predominantly genotype 1

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Antiviral Drug Resistance 1

• RdRP is an error-prone enzyme (no proof reading
  capacity)
• A) Resistance in vitro NS3/4A
• in vitro selection identified in NS-3 R155Q,
  A156T, D168A/V
• 20-100 fold increase in IC50
• BILN 2061 A156 V/T
• R155 Q
• D168 V/A
• VX 950 (Telaprevir) A156 S/V/T
• V36M
• SCH 6 A156 V/T
• R109 K
• SCH 503034 A156 S/T
• T54A
• V170A
• ITMN-191 D168 A/V/E
• (InterImmune)
• 3D modelling shows

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Mechanisms of Resistance NS3/4A

• Steric hindrance
• Direct effect BILN 2061 A156T
• Indirect effect BILN 2061 D168V
• D168 forms salt bridge with R155
• Decreased Interaction with Inhibitor
• Direct effect VX-950 R155T
• threonine is smaller (less interaction with P2)
• Indirect effect VX-950 V36M
• methionine is smaller (does not directly touch
  inhibitor)

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Macrocyclic Inhibitor is Bound in the NS-3 Active
Site

BILN-2061 co-complex with HCV NS3-NS4A genotype
1b
Kindly provided by J. Courcambeck and P Halfon
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Drug-Resistance Mechanism
Direct contact between BILN-206I and mutated
residues at R155Q and A156T
BILN-2061 co-complex with HCV NS3-NS4A genotype
1b, strain R155Q
Kindly provided by J. Courcambeck and P. Halfon
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Indirect Resistance Mechanism
D168A/V removes two salt bridges to R123 and R155
? affecting conformational stablilization
resulting in unfavourable van der Waals
interactions
BILN-2061 co-complex with HCV NS3-NS4A genotype
1b, strain D168A
Kindly provided by J. Courcambeck and P. Halfon
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HCV Enzymes are the Targets for New HCV Therapies
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HCV NS5B

• HCV polymerase, essential for replication
• 3D structure has been solved by X-ray
  crystallography
• characteristic polymerase configuration
  resembling right hand
• Small molecule inhibitors of NS5B act at
  different sites
• Active site
• nucleoside analogues
• chain terminators
• pyrophosphate mimetics complex with catalytic
  cations (Mg)
• carboxylic acid derivatives
• diketoacid derivatives
• Allosteric sites
• non-nucleoside inhibitors (NNI) include
  heterocyclics
• benzimidazole derivatives
• benzothiodiazine derivatives

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Antiviral Drug Resistance 2

• 1. HCV NS5-B Non Nucleoside Inhibitors
• in vitro selection identified single amino-acid
  substitutions in NS5-B
• M414T, C451R. G558R and H95R causing a 50-fold
  increase in IC50
• 3D modelling reveals
• Site A (Thumb Finger Tips) Indole N-Acetamide
  (Merck) P494
• Benzimidazoles P496
• Site B (Thumb) Thiophene-COOH (Shire) M423
• Site C (Palm) Benzothiadiazine
  (GSK/Abbott) M414T A837093 (Abbott) Y448
• Site D (R200 Hinge)
• Benzofurans (Viropharma/ Wyeth) C316
• HCV-796 V201
• indicating multiple mechanisms for resistance
  (Tomei et al. 2004 JV
  78938)
• Active against Genotype 1 ONLY
• C316N 40 of Genotype 1b

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Antiviral Drug Resistance 3

• HCV NS5-B and Ribonucleoside Inhibitors
• Single mutation S282T within active site of NS-5B
  (Migliaccio et al. 2003 JBC 27849164)
• B) Resistance in vitro NS-5B
• 1) Nucleoside analogues (Active site)
• i) 2-C-Methyl-Pyrimidine/ Purine
• NM283 (cytidine) S282T
• 1.0 log drop
• ii) 4-Azido Cytiduo
• R1626 (prodrug R1479) S282T
• 1.2 log drop S96T
• iii) Tricyclic analogues (Biota)
• iv) PSI-6130 (Pharmasset/ Roche)
• Cytidine analogue
• F at 2-C
• v) MK-0608
• 7-deaza-2-C-methyl adenine S282T
• 0.2mg/Kg gt1.0 log drop- chimps
• 1mg/Kg gt 4.5 log drop-chimps

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Mechanisms of Resistance NS5B

• Steric Hindrance
• Direct effect S282T
• (palm domain)
• Indirect effects
• NNI TOO MANY C316
• allosteric interactions (non-competitive
  inhibition)

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HCV RNA-Dependant RNA Polymerase
Thumb
Fingers
Flap
NNI
Palm
Kindy provided by Dr John McHutchison
(Butcher et al., Nature 410, 2001)
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Combination of Inhibitors
AASLD 2006 Poster 417 A848837 NS-5B NNI
Log Reduction
0.0 -1 -2 -3 -4 -5 -6 -7
Placebo
IFN low dose
A848837
A848837 IFN
SCH ALONE
IFN high dose
A848837 SCH
P.0 P.1 P.2 P.3 P.4 P.5

• CONCLUSIONS
• A848837 IFN at least additive
• A848837 SCH (PI) Synergistic

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Summary

• NS3/4A and NS-5B excellent drug targets but drug
  resistant mutants of HCV emerge rapidly
• A number of drugs very active against HCV NS3/4A
  and NS-5B
• as monotherapy, resistance appear very rapidly
• All isolates sensitive to IFN-a
• Risk of resistance is reduced if use combination
  therapy
• NS5B S282T severely replication impaired
  (fitness)
• C316N found 40 of genotype 1b isolates
  naturally
• NS3/4A complex profiles
• A156T/V less fit

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Future Directions Drug Resistance

• Resistance emerges when replication occurs in the
  presence of the drug selection pressure
• The best cost-effective strategy is to prevent or
  avoid the emergence of antiviral drug-resistance
• (No Replication No Resistance NRNR)
• combination chemotherapy should increase the
  efficiency of antiviral therapy
• Single/double mutants pre-exist why most
  monotherapies fail
• Triple/quadruple mutants selected out why
  combination therapy works

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Antiretroviral Drugs Available Clinically
Nucleoside RT Nonnucleoside Protease
Inhibitors RT Inhibitors Inhibitors (NRTIs)
(NNRTIs) (PIs) Abacavir Delavirdine fosA
mprenavir Didanosine Nevirapine Indinavir Lamiv
udine Efavirenz Nelfinavir Stavudine
Ritonavir Zalcitabine
Fusion Saquinavir/r Zidovudine
Inhibitor Lopinavir/r Tenofovir
Enfuvirtide Atazanavir Tipranavir/r
Darunavir/r
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Mechanisms of NRTI Resistance (1)
1. Discrimination (between drug and natural
substrate) (i) M184V Causes drug (lamivudine)
to bind to active site with reduced affinity (kd)
compared to natural substrate (dCTP) and also
reduced catalysis to DNA (kpol). (ii) K65R
Causes drug (TFV, ddI) to bind to active site
with normal affinity but reduced catalysis (kpol)
into DNA. (iii) Q151M Causes decreased
electrostatic interaction between drugs (NRTIs)
and enzyme, resulting in reduced catalysis to
DNA.
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Mechanisms of NRTI Resistance (2)

• 2. Excision Mutations
• NRTI binds to RT and is incorporated into new
  DNA chain.
• Inhibitor is excised allowing elongation to
  continue.
• Mutations that facilitate this excision are
• TAMs (41, 67, 70, 210, 215)
• T69S insertion mutations.

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Thymidine-Associated Mutations (TAMs)

• Are ZDV-associated mutations
• (M41L, D67N, K70R, T215F/Y, L210W, K219Q)
• Also known as excision mutations
• They increase the ability of the RT to remove
  chain terminating ddNMPs
• One or more TAMs may influences the
  susceptibility of HIV to ZDV, D4T, TFV, ABV (and
  possibly ddI).

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The HIV Viral Life Cycle
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Structure of HIV Protease
no drug
with PI
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HIV Protease Inhibitors
45
EM of Virus Production
no drug
with PI
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Protease Inhibitor Mutations
IAS-USA 2005
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Structure of HIV Protease
no drug
with PI
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