HCV: Basic virology, drug targets and -resistance Volker Lohmann - - PowerPoint PPT Presentation

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HCV: Basic virology, drug targets and -resistance Volker Lohmann - - PowerPoint PPT Presentation

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HCV: Basic virology, drug targets and -resistance Volker Lohmann http://www.molecular-virology.uni-hd.de Genetic diversity of HCV Genetic diversity of HCV 7 genotypes, subtypes quasispecies 20-25% difference 31-33% difference Simmonds

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SLIDE 1

HCV: Basic virology, drug targets and

  • resistance

Volker Lohmann

http://www.molecular-virology.uni-hd.de

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SLIDE 2

Genetic Genetic diversity diversity

  • f HCV
  • f HCV

20-25% difference 31-33% difference

Simmonds et al., 2005

7 genotypes, subtypes quasispecies

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SLIDE 3

Functions Functions

  • f HCV Proteins
  • f HCV Proteins

Cytosol

Virus particle Ion channel Assembly Protease Assembly Protease/ Helicase NS3- cofactor Membrane vesicles Phosphoprotein RNA Binding Assembly RdRp Replicase complex ‚assembly module‘ Non-structural proteins Structural

ER

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SLIDE 4

HCV HCV replication replication cycle cycle

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SLIDE 5

Model Systems to Model Systems to Study Study HCV in HCV in Cell Cell Culture Culture infection

  • f cultured

cells (PHH; cell lines)

very inefficient

HCV replicons

RNA replication

HCVcc

whole life-cycle

HCV pseudoparticles

Early steps

  • f infection

HCVpp

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SLIDE 6

Con1 (gt1b) gt 1a gt 2a gt 3 (HCV 2011) gt 4 (EASL 2012)

HCV HCV Cell Cell Culture Culture Models Models

HCV replicons HCVcc

RNA

+

  • Wakita et al.: NatMed (2005)

Lindenbach et al.: Science (2005) Zhong et al.: PNAS (2005) Pietschmann et al.: PNAS (2006) Lindenbach et al.: PNAS (2006) Lohmann et al.: Science (1999) Blight et al.: Science (2000) Lohmann et al., J. Virol (2001, 2003) Krieger et al., J. Virol (2001)

JFH1 (gt2a) J6/JFH1 (gt2a) wt adapt NS3/4A: gt 2a, 3a, 5a, 6a NS5A: gt 1-7

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SLIDE 7

HCV DRUG TARGETS 2012:

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Targets for direct-acting antivirals (DAAs) Targets for indirect-acting antivirals (host cell factors)

NS3/4A pro NS3 hel NS5B pol NS2 pro

p7

NS5A NS4B CD81 SR-BI PI4KIIIα EGFR NPC1L1

HCV Replicon HCVcc HCVpp

Cyclophill. miR-122

Targets w/Clinical P.O.C. Targets w/o Clinical P.O.C.

DGAT-1 MTP CD81

R.de Francesco

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SLIDE 8

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 9

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 10

Sarrazin et al., J. Hepatol. 2012

Current SOC gt1: pegIFN/Rbv + NS3 protease inhibitor

Standard of Standard of Care Care 2012 2012

Poynard T et al. Lancet 2003;362:2095-2100

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SLIDE 11

Resistance to PIs in vivo Resistance to PIs in vivo vs vs in vitro in vitro

  • Low barrier to resistance
  • Single mutations → resistance
  • Potential for broad cross-resistance to all protease inhibitor classes (R155, A156)
  • Broader mutation spectrum in vivo vs. in vitro
  • Limited viral diversity in cell culture
  • Double amino-acid mutations observed in vivo

– R155K+V36M: variant most frequently associated with viral breakthrough – Compensatory mutations needed to restore viral fitness of high-resistance variants

V36 A/M T54 A R155 K/T V36 A/M +R155K/T A156 S/T/V A36A/M+A156V/T V36 A/L/M F43 C/S V55A T 54 A/S V36M+T54S R155 K/T/P V36A+R155K T54S/A+R155K A156 S T54S+A156S V170A/T/L

Telaprevir Boceprevir

Clinical

Telaprevir Boceprevir

Replicon

T54 A/T R155 R/K A 156 A/S/T/V T 54 A/S R155 K A156 S/T V170A

  • R. de Francesco, INGM
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SLIDE 12

Reasons for treatment failure Reasons for treatment failure

  • Critical Factors for Treatment Failure (Telaprevir triple therapy):

PROVE 1. McHutchison JG, et al. N Engl J Med. 2009; PROVE 2. Hézode C, et al. N Engl J Med. 2009

SVR

(~60-70%)

Discontinuation (12-21%) Relapse (7-20%) Breakthrough (3-7%)

  • High discontinuation rates due to adverse effects
  • Emergence of resistant virus
  • Preexisting resistant variants (high frequency for gt

3)

  • Linked to poor response to PEG-IFN/RBV

→virtual monotherapy

  • Severe rash, anemia
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SLIDE 13

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 14

Drug Drug-

  • resistance to non

resistance to non-

  • nucleoside inhibitors

nucleoside inhibitors

  • ABT-333; ANA598

Cys 316; Met 414; Tyr 448; Gly 554; Asp 559

  • VX-222; VX-759; Filibuvir

Leu 419; Met 423; Ile 482; Val 494

  • BI 207127; MK-3281

Pro 495; Pro 496; Val 499

  • HCV-796

Leu 314; Cys 316; Ile 363; Ser 365; Met 414

  • GS-9190

Cys 445; Tyr 448; Tyr 452

A B D E C

  • Cross-resistance among different NNI classes
  • R. de Francesco, INGM

–Restricted spectrum of action (most active on genotype 1b) –Low barrier to resistance in vitro and in vivo, viral breakthrough –Cross-resistance among different NNI classes –Resistant variants/polymorphisms pre-exist in patient population (e.g., C316Y→ N in genotype 1b)

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SLIDE 15

No resistance to nucleoside analog No resistance to nucleoside analog

  • R. de Francesco, INGM

GS-7977 (Former PSI-7977; Pharmasset/Gilead) –Uridine analog, active across genotypes –NO resistance in vivo due to high fitness cost of S282T –100% SVR (10/10) in gt2/3 patients with PSI-7977+RBV (AASLD, 2011) –Atomic: >90% SVR with pegIFN/RBV (EASL 2012) –Potential to substantially increase SVR when combined with SOC –Potential for SVR by combining nucleosides with other DAAs!

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SLIDE 16

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 17

BMS BMS-

  • 790052: the most potent DAA

790052: the most potent DAA

M Gao et al. Nature 465, 96-100 (2010)

Picture from: Schmitz U, Tan SL. “NS5A--from obscurity to new target for HCV therapy”, Recent Pat Antiinfect Drug Discov. 2008

HCV RNA

  • Identified in a HTS on subgenomic replicon cells
  • Active across HCV genotypes 1-6, pM to nM
  • Drug resistance

maps to NS5A

  • Direct

binding

  • f drug

to NS5A

  • Mechanism?
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SLIDE 18

Placebo (n = 2) BMS 790052 1 mg (n = 6) BMS 790052 10 mg (n = 5) BMS 790052 100 mg (n = 5)

  • 1
  • 2
  • 3
  • 4

0 6 1624 36 48 72 144 Time (hours) HCV RNA level (log IU/ml)

1M Gao et al. Nature 465, 96-100 (2010); 2Pol S et al. EASL 2010, Abs 1189 3EASL 2012

  • Resistant variants detected with low fitness cost, but significant residual activity

(nM) against resistant mutants is likely to control emergence of resistance

  • Low genetic barrier but high “pharmacological”

barrier

  • Promising results in ongoing PhaseII

triple combination therapy2

  • NS5A inhibitor + Protease Inhibitor trial: 90% SVR in previous null-responders3
  • NS3+NS5A resistant variants in breakthrough patient

Summary BMS Summary BMS-

  • 790052/Daclatasvir

790052/Daclatasvir

single

  • ral dose!1
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SLIDE 19

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 20
  • Cyclophilins

are molecular chaperones with peptidyl-proline isomerase activity

  • Inhibition by Cyclosporin

A (CsA) and derivatives thereof, like DEBIO- 025 (Alisporivir)

  • Inhibition of HCV replicons

by CsA (Watashi et al., 2003)

  • Cyclophilin

A is essential for HCV replication

Cylophilin Cylophilin A A is is required required for for HCV HCV replication replication

Kaul et al., PLoS Path. 2009

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SLIDE 21

Debio Debio-

  • 025 (

025 (Alisporivir Alisporivir) )

Flisiak et al., Hepatology 2009, *EASL 2012

  • No resistant variants detected in vivo
  • Selection of resistance in vitro difficult. High genetic barrier?
  • Mutations conferring partial resistance in vitro map primarily to NS5A and NS5B
  • Multiple targets?
  • VITAL-1 (phase 2b): SVR ALV-Mono: 32%; ALV+RBV: 90% SVR (gt2/3)*
  • Trials with pegIFN/RBV, recently halted due to adverse effects (pancreatitis)
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SLIDE 22

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

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SLIDE 23
  • Cellular

micro RNA, expressed to high levels in the liver

  • Direct

interaction miR-122 – HCV

  • Two

seed-sequences near 5‘end

  • Essential for

replication

  • Sequestration
  • f miR-122 inhibits

HCV replication

  • Miravirsen: LNA-modified

anti-miR122 oligonucleotide (SC)

  • Phase 2a trial*: 4/9 HCV negative in monotherapy

(high dose)

  • no Resistance! (host

target)

miR miR-

  • 122

122

Jopling et al., Science 2005 Henke et al., EMBO J. 2008 Lanford et al., Science 2010 Norman and Sarnow, J. Virol. 2010 Jangra et al., J. Virol. 2010

IRES

INTRO

*EASL 2012

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SLIDE 24

The The Two Two Faces Faces

  • f Drug
  • f Drug Resistance

Resistance

  • Fitness cost
  • Identification
  • f Drug target
  • Mechanism
  • Loss
  • f therapeutic

potential

  • Viral

breakthrough

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SLIDE 25

G418 selection cell clone expansion

Selection for drug resistance Selection for drug resistance

double selection drug-resistant clones cells with persistent replicon Identification

  • f conserved

mutations Phenotypic analysis

  • Degree
  • f resistance
  • Fitness cost

G418 + antiviral drug

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SLIDE 26
  • R7128: 10x and 15x IC50 eliminate HCV replicon

within 3 weeks no resistance detected

  • HCV-796: 10x and 15x IC50 do NOT eliminate HCV replicon

C316Y and S365S/A

  • Telaprevier: 10x and 15x IC50 do NOT eliminate HCV replicon

A156T/S and T54T/A

Telaprevir

1X IC50 10X IC50 15X IC50

HCV-796

1X IC50 10X IC50 15X IC50

NS5B NI NS5B NNI NS3/4A PI

R7128 Active Moiety (PSI-6130)

Higher genetic barrier of nucleoside analogues as compared to non-nucleosidic inhibitors and protease inhibitors

Untreated 1X IC50 10X IC50 15X IC50

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SLIDE 27

Silibinin Silibinin

Silibinin:

  • Extracted

from milk thistle

  • Intravenous

formulation Legalon-SIL: 1:1 mixture

  • f succinylated

Silibinin A and B (water soluble)

  • Prevention
  • f reinfection
  • f transplanted

liver in chronic HCV patients  SVR after SIL monotherapy!

(Beinhardt

  • et. al. 2010, Neumann et. al. 2010)
  • Mechanism
  • f action

under debate

(RdRP-inhibition, entry-inhibition)

  • Aim: Analyse HCV resistance

to SIL in vitro and in vivo to clarify mechanism

  • f

action

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SLIDE 28

selection selection for for SIL SIL resistance resistance in in vitro vitro

  • isolation of total RNA
  • long template PCR
  • sequence analysis
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SLIDE 29
  • in

in vitro vitro SIL SIL resistance resistance-

  • NS4B mutation Q1914R confers

SIL resistance to gt1b in vitro

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SLIDE 30
  • in vivo

in vivo SIL SIL resistance resistance-

  • viral breakthrough
  • RNA isolation from patient sera
  • long template nested PCR, direct sequencing
  • sequence comparison before and after treatment
  • gt1a
  • several polymorphisms before, resolved

after treatment

  • 6 new mutations
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SLIDE 31
  • in vivo

in vivo SIL SIL resistance resistance-

  • gt1a reporter

replicon Luc H77S NS4B mutation D1939N confers SIL resistance in vivo (additive effect of F1809L)

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SLIDE 32

NS3 Proteaseb NS5B Polymerasee NS3 Helicasec NS2-3 Proteasea

aLorenz et al., Nature 2006 bKim et al., Cell 1996 cKim et al., Structure 1998 dTellinghuisen et al., eBresanelli et al.,PNAS, 1999 Nature 2005

NS5Ad

Important Important targets targets for for antiviral antiviral therapy therapy

Cyclophilin A Host Factors: miR-122

NS4B

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SLIDE 33

Phenotypic assays

Infection with primary isolates too inefficient too variable requires primary cell cultures Replicons cover complete replicase, but lack core to p7/NS2 authentic replication, predicting major clinical resistance mutations limited to some HCV isolates (1a, 1b, 2a) replicon vector backbone required for each geno-/subtype incompatibility between replicon fragments Infectious cell culture grown HCV covers all steps

  • f HCV replication

cycle limited to only

  • ne

isolate (JFH-1) BSL3 lab required most compounds target replicase

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SLIDE 34

Approach for Phenotypic Resistance Assay

Tripathi et al., Antiviral Res. 2007

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SLIDE 35

Phenotypic (replicon-based) assays

Kwong et al., Gastroenterology 2011

1b (Con1) 1a (H77) 1b (N)

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SLIDE 36

– Monotherapy leads in most cases to emergence of resistance and treatment failure  omitted from most trials – Higher genetic barrier to viral resistance observed with nucleosides versus non- nucleosides, NS3-4A protease or NS5A inhibitors

  • non-nucleosides, protease inhibitors , NS5A inhibitors→ frequent mutants,

high resistance, good replication fitness

  • nucleosides →

infrequent mutants, low to moderate resistance, poor replication fitness

  • pM NS5A inhibitors → pharmacological barrier to resistance?
  • Inhibition of cellular targets, e.g. Cyclophilin A, might provide promising

alternatives with high genetic resistance barriers

  • Silibinin represents a novel class of NS4B inhibitors with unique resistance profile

and clinical prove of concept

  • Combination of pegIFN/RBV with a DAA can partially suppress the emergence of

resistance  higher SVR rates  shorter treatment duration

  • Future therapies without pegIFN/RBV in sight, will resistance be

an issue?

Resistance to HCV Resistance to HCV Antivirals Antivirals

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SLIDE 37

Error prone replication of HCV Error prone replication of HCV

Kieffer T L et al. J. Antimicrob. Chemother. 2010;65:202-212

  • error

prone polymerase

  • 1012

variants daily

  • genetic

barrier ≥ 4 mutations required

  • combination therapy of 2-4 DAAs?
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SLIDE 38

Ines Romero-Brey

Ralf Bartenschlager

CNRS, Lyon

  • F. Penin

University of Heidelberg

  • T. Longerich, P. Schirmacher
  • C. Eisenbach, T. Ganten, W. Stremmel

University of San Francisco

  • T. Balla

Twincore, Hannover

  • T. Pietschmann

University of Freiburg

  • R. Thimme

University of Lausanne

  • J. Gouttenoire, D. Moradpour

Novartis, Cambridge

  • K. Lin, B. Wiedmann

Rockefeller University, New York C.M. Rice INGM, Milan

  • P. Neddermann, R. de Francesco

Katharina Esser-Nobis Christian Harak Rahel Klein Danijela Radujkovic Simon Reiss Philipp Schult

Volker Lohmann

SFB/TRR77

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SLIDE 39

Kwong et al., Gastroenterology 2011

HCV-specific DAAs

slide-40
SLIDE 40

pegIFN

Ribavirin

pegIFN

Ribavirin NS3-4A inhibitor

Evolution of HCV therapies

pegIFN

Ribavirin 2nd GEN NS3-4A inhibitor NS5B Pol inhibitor NS5A inhibitor

+ + + + + and/or

Ribavirin ??? DAA-1 DAA-2 DAA-3

+

and/or

+

until 2010 2011 2020? DAA-1 DAA-2 DAA-3 … Once a day pill

and/or

R.de Francesco