HCV replicative mechanism

and potential targets

Massimo Levrero
Dipartimento di Medicina Interna e Specialita Mediche
Laboratorio Life-Nanoscience
EAL INSERM U795
Sapienza Universita di Roma

Hepatitis C virus replication cycle

RNA Virus with high viral turnover and error prone replication

Virus entry and replication in

hepatocytes

Different HCV geno- and subtypes

with 10-30% sequence difference

major
population based
sequencing (~25%)
minor
clonal (~5%)
deep (~0.5%)

RNA virus without integration

into host genome / stable DNA
intermediate

RNA polymerase without

proofreading activity leads
to generation of multiple
variants (quasispecies)

Treatment endpoints
in HIV, HBV and HCV infections

HBV1,2

HIV1
Host cell

Host cell

Host cell
cccDNA

Host DNA

H
Proviral DNA

Host DNA

HCV1,3

Host DNA

HCV RNA

Integrated DNA

Nucleus

Nucleus

Life-long suppression
of viral replication

Long-term suppression
of viral replication

Nucleus

Definitive viral clearance

and SVR

Adapted from 1. Sorriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F.
Antiviral Therapy 2010;15 (suppl 3):3-14. 3. Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.

Evolution of HCV cell culture system

Replicon systems (1999-2002)

Huh7
Hepatoma cells

* * *

Adaptive changes

Huh7.5 : highly permissive cell line

HCVpp (pseudoparticle) (2003)

HCVE1E2

Reporter gene

HIV nucleocapsid

HCVcc (JFH-1 cell culture) (2005)

Complete life cycle

Rice C HepDart 2011

Effects of IFN
treatment on HBV replication and
transcription in humanized uPA/SCID mice

woodchuck,
tupaia, human
hepatocytes

In vivo
HBV infection

Petersen, PNAS 1998, Dandri, Hepatology 2001, 2002, 2008, J Hepatol 2005, Petersen, Nature Biotech.2008

HCV entry

Virus particle

Assembly and release

Membranous web
RNA replication

Genome organization
Poly-protein processing

Topology of HCV proteins

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

Bartenschlager et al., J.Hepatol., 2010

HCV genome structure and functions of viral proteins

SPP, signal peptide peptidase;
SP, signal peptidase

HCV pipeline by mechanism of action

DAA combinations (17)

Preclinical
Abbott
Pharmasset

Phase II
BI

Cyclophilin I
(2)

ACH-2928
(Acillion)

BI

Phase I

Gilead

Others (6)

Nucleotide NS5B
Polymerase
Inhibitors (4)

Vitamine D
Vertex
Nitazoxamide
ROCHE
(Romark)
BMS
Celgosivir
IFN
Bavituximab
(Zymogen/Novartis)

Phase III
Filed

Japon Tonbacco

R0622 (Roche)
Medivir (Tibotec)
IDX 184
(Idenix)

GLS9393 (GSK)

PSI 938
R7128 (Pharmasset)
(Roche /pharmasset)
PSI 7977
pharmasset)

Debio025/ Taribavirin
Silibinine
NIM811
(Valeant)
SCY-835
(Novartis)
BMS 790052
(BMS)
AZD-7295 ABT267
(ABT)
(AZN)
PPI-461
MSD
GS-5885
Idenix719

NS5A inhibitors

Nucleoside
NS5B Polymerase
Inhibitors (8)

PSI-661
(Pharmasset)
Biocryst
INX 189 (Inhibitrex)

BMS791325
Filibuvir (BMS)
(PFE)
GS9190
IDX 375
(Gilead)
Boceprevir
Telaprevir
(Idenix)
Vx222
ANA598
(MSD)
(Vertex/JJ)
(Vertex)
RG7348
BI201127 (Anadys)
BI201335
(Roche)
TMC-435
(BI)
ABT333
(BI)
TMC
647055
(Tibotec/JJ)
ABT072
Vertex
ITMN191/R7227
(Tibotec)
MK7009
BMS 824393
(ABT)
(Roche/Intermune)
PPI-1301 (BMS)
(MSD)
BMS650032
A837093
GSK
(BMS)
(Abbott)
ABT450
EDP-239
(ABT)
VX-916
VBY-376
GS9256 GS9451
(Enanta)
VX-759
IDX 077
ACH2684
MK5172 (Gilead) (Gilead)
(Idenix)
VX-985 (MSD)
(Achillion)
(Vertex)
IDX 079
(13)
VX-813
AVL-192
Non
(Idenix) (Vertex)
AVL-181
(Avila)

(Avila)

NS3/4A Protease inhibitors

(19)

Nuc NS5B
Polymerase inhibitors (12)

Main targets of direct antiviral agents (DAA)

NS5A
inhibitors
NS3/4A-proteaseinhibitors

E1

E2

p7

4A

polymeraseinhibitors

4B

5A

5B

RNA-dependent
NS3-protease
RNA-polymerase
cofactor
membrane regulation of
serine- NTPase/
anchor
replication
protease helicase

metallo-/
cysteine-protease
capsid

NS3/4A Protease
inhibitors (19)

envelope

NS5A inhibitors
(13)

Nucleotide NS5B Polymerase Inhibitors (4)

Nucleoside NS5B Polymerase Inhibitors (8)
Non Nuc NS5B Polymerase inhibitors (12)

HCV Replication Cycle:

Host Targets for Intervention

Bartenschlager et al., J.Hepatol., 2010

HCV Replication Cycle: Entry

ITX 5061 (SRB-I)

Pan-genotype Env-neutralizing Abs
CD81 antibodies
Erlotinib (EGF-R)

SRBI Mode-of-Action
and Targeting by ITX 5061
Contributing to lipoprotein binding
Direct binding of E2
Altering local membrane composition
- facilitate receptor complex formation
- enhance signalling

ITX 5061

Contribute to release of apoC-I

Primary human hepatocytes HCVpp;

24h pre-incubation

adapted from Dao Thi et al., 2011

Role of RTKs in HCV entry pathway and their

potential as therapeutic targets.

Erlotinib modulates HCV kinetics and

inhibits infection in vivo in chimeric uPASCID mice repopulated with PHHs

Lupberger et al, Nature Medicine 2011

Important Role of miR-122 for HCV

IIIb

HCV RNA + miR-122

II
IIIa
IIIc

IRES
IIId

IIIe

Machlin et al. PNAS 2011

IIIf
I

Replication

IV

Honda et al., J. Virol. 1999

Stimulates RNA translation

Enhances RNA replication
Stimulates mevalonate pathway
Masking 5 triphosphate end?
Jopling et al., 2005, 2008; Henke et al.; 2008;
Machlin et al., PNAS 2011

Mode-of-action of Miravirsen
Low Amounts of Mir122
Limit HCV Replication in HepG2 Cells

Narbus et al., JVi 2011;

MJ Evans, HepDart2011, Abstract 41

miR122 as Target
Phase 2a Results of Miravirsen:
4 Weeks Monotherapy
Proof of concept in chimpanzees
Jansen et al.,, AASLD San Francisco, 2011, Abstract LB-6

4 animals (gt1); once weekly i.v.;

12 weeks treatment

Dose Group

Mean HCV RNA

decline
IU/mL (SEM) at
week 10

p-value (t)
MIR vs.
placebo

Pooled
placebo

-0.01 (0.19)

---

Miravirsen
3 mg/kg

-0.57 (0.13)

0.034

Miravirsen
5 mg/kg

-2.16 (0.58)

0.007

Miravirsen
7 mg/kg

-2.73 (0.55)

<0.001

Lanford et al., Science 2010

Long-lasting suppression of viremia

No resistance
No side-effects
Down-regulation of ISGs
Improvement of liver histology

HCV Replication Cycle:

Formation of Membranous Web & RNA Amplification

PI4Kinase
(e.g. A831)

CypA
(e.g. Alisporivir,
NIM811, SCY-635)

HCV and PI4K-III

PI4K-III identified in multiple siRNA screens

Required for replication of HCV, but not Dengue

virus
Kinase activity required for HCV replication for
integrity of the membranous web
Tai et al., 2009; Vaillancourt et al., 2009;
Berger et al., 2009; Borawski et al., 2009;
Li et al., 2009 Trotard et al., 2009;
Reiss, Rebhan et al., 2011

PI4K:
210 kDa; primarily ER resident
controlling PIP(4)P level at PM
generates PIP(4)P
two main PIP(4)P effectors
- adapter and coat complexes
- lipid-transfer proteins
Reiss, Rebhan et al., 2011

HCV Replication Cycle:

Formation of Membranous Web & RNA Amplification

PI4Kinase
(e.g. A831)

CypA
(e.g. Alisporivir,
NIM811, SCY-635)

Cyclophilins and Inhibition of HCV by CsA

CyPs are chaperones with peptidyl-prolyl isomerase activity

Effect of CsA on HCV infection in bone marrow

transplant recipients (Akiyama et al., 1997)

Discovered as specific ligand for cyclosporin A

Abundant cytosolic protein (0.1% of total cellular proteins)
Ubiquitously expressed in eukaryotic cells
Multiple functions, depending on target protein

Controlled trial

of IFN2b and CsA in HCV patients

(Inoue et al., 2003)

Pilot study of CsA and IFN in previous

nonresponders (Cotler et al., 2003)

Inhibition of HCV replicons by CsA

(Watashi et al., 2003)

Structure of CsA and CsA-Derivatives

Structure of Cyclophilin Inhibitors

Sanglifehrin

0.3nM

0.02M

adapted from Gallay, Clin Liv Dis 2009

Clinical Trials: Debio-025

active against the 4 major HCV genotypes

Flisiak et al., 2009

Cyclophilins and Inhibition of HCV

CypC, F, H, 40

Essential Role of CypA

for RNA Replication & Assembly
CypA
Nakagawa et al., 2005; Yang et al.,
2008; Fernandes et al., 2007;
Chatterji et al., 2009; Hanoulle
et al., 2009; Kaul et al.,
2009; Gaither et al., 2009;

CypB
Nakagawa et al., 2005;
Watashi et al., 2005;
Liu et al., 2009;
Fernandes et al., 2007;

Path. 2009

Kaul et al., PLoS Path. 2009

Two Groups of NS5A Mutations Reducing HCV CypA-Dependency

& Decreasing Debio-025 Sensitivity

Direct Interaction between NS5A and CypA In Vitro

Interaction site is centered around the active site of CypA

Coincides with CsA binding surface
Interaction abolished by CsA
Extended interaction surface in D2
Interaction also found with CypB
Interaction also found between CypA and NS5A D3

Hanoulle et al., 2009

Model for the Mode of Action of CypA:

Kinetic Control of HCV Replicase Activity
CypA

[CypA]

5A

[CypA]

5B

NS3/4A

5B

5A

5B

5B

5A
Time

5A

active replicase
(enhanced RNA binding?)

5B

V464L

5B

V464L: Delayed cleavage

gives more time to bind
CypA or to fold w/o CypA

dependent on CypA
5A

impaired replicase

5A

DEYN

DEYN: NS5A
folding less

5B

[CypA]

[CypA]

5A

5A

5A

5B

5A

5B

5B

Kaul et al., PLoS Path. 2009

HCV Replication Cycle:

Assembly and Release of Infectious Particles

VLDL

Bartenschlager et al., J.Hepatol., 2010

Lipid Droplets and HCV Assembly

Bartenschlager et al., Trends Microbiol 2011

Lipid Droplets and HCV Assembly

Efficient block of HCV assembly by inhibitors of:

Microsomal triglyceride transfer protein (MTP)
Diacylglycerol acyltransferase-1 (DGAT-1)
ApoE

Gastaminza et al., JVi 2006; Huang et al., PNAS 2007; Nahmias et al., Hepatology 2008;
Jiang et al. JVI 2009; Merz, Long et al., JBC 2010; Herker et al., Nat Med 2011;

Host Cell Targets for Antiviral Therapy

Host polymorphisms
More prone to cytotox than DAAs?

High genetic barrier for resistance

Possibly faster drug development
Increases otherwise limited number of targets
Higher potential for broad spectrum

Evolution of HCV Therapy

2001

2011

PegIFN/RBV
Protease inhibitor
Nucleos(t)ide polymerase inhibitor
Nonnucleoside polymerase inhibitor
NS5A inhibitor
Host targeting agent

Future

Next

?
?

Majority of protease inhibitors have

targeted genotypic coverage but
MK-5172, TMC435 have broad coverage[1,2]

Most nucleos(t)ide analogue polymerase

inhibitors are pan-genotypic
1. Brainard DM, et al. AASLD 2010. Abstract 807.
2. Fried M, et al. AASLD 2010. Abstract LB-5 .

 The next generation of protease inhibitors are in

development (expected to be available in 2015 or even
earlier)
Different DAA combination therapies are being evaluated in
early phase trials
Whether Peg-IFN and/or RBV will be needed in all patientsis
unclear
Final aim for a DAA combination regimen
All oral
QD
Safe and well tolerable
Pan-genotypic
IL28-independent
Limited resistance

Collaborations:

Laboratory of Gene Expression

Laura Belloni
Francesca Guerrieri
Natalia Pediconi
Cecilia Scisciani
Letizia Cimino
Rossana De Iaco
Valeria Schinzari
Barbara Testoni

Lena Allweiiss
Tassilo Voltz

Dept. of Internal Medicine

University of Messina
Giovanni Raimondo
Teresa Pollicino
Giuseppina Raffa
Giovanni Squadrito

Technische Universitt Munchen

Helmholtz Zentrum Munchen
Ulrike Protzer
Julie Lucifora

Massimo Levrero
Collaborations:

INSERM U761 -Lyon

Fabien Zoulim

Anna Tramontano

Julie Lucifora
David Durantel

Andrea Sbardellati,
Daniel DAndrea
Francesco Cicconardi

With the support of

University Medical Hospital Hamburg

Jorg Petersen
Maura Dandri

Fondazione
Andrea
Cesalpino