WORKING LUNCHEONE

FUTURE THERAPIES

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Brass et al. PNAS 2008

Structure of Membrane-Associated NS3/4A

• chymotrypsin-like enzyme

• activation by NS4A

• cleaves viral and cellular proteins

Polyprotein processing

innate responses (TLR3, RIG-I)

proteasedomain

helicasedomain

NS4A proteasecofactor

Boceprevir

Structures of NS3-Specific Drugs

Telaprevir

‚linear‘

TMC435 MK5172

‚macrocyclic‘

1 (+) RNA > 1.000 polyproteins

RV

LDTranslation Assembly

Mode-of-Action of NS3-specific DAAs

Blockage of polyprotein cleavage

block of new formation of replication vesicles

no effect on established replication vesicels

Restoration of innate immune response?

RIG-I (MAVS)

TLR3 (TRIF)

EC50 > 4-fold

Resistance against PIs:2nd Generation

Halfon & Locarnini, J. Hepatol. 2011

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Form of a right hand ‘closed’ active site

Thumb Palm Fingers

The NS5B RNA-Dependent RNA Polymerase

Nucleosidic and Non-nucleosidicNS5B-Specific Drugs

Non-nucleosidic Inhibitors

Benzimidazole derivative Thiophene derivative Thiadiazine derivative

2'-C-methyl cytidine

Nucleosidic Inhibitors

PSI-7977 INX-189

1 (+) RNA > 1.000 polyproteins

RV

LDTranslation Assembly

Mode-of-Action of NS5B-Specific Inhibitors

Block of RNA synthesis

direct effect also on established replication complexes

direct inhibition of NS5B (non-nucs)

block of elongation (nucs)

• R7128: 10x and 15x IC50 eliminated HCV replicons within ~3 weeks

no resistance detected

• HCV-796: 10x and 15x IC50 did not eliminate the replicon

C316Y and S365S/A

• Telaprevir: 10x and 15x IC50 did not eliminate the replicon

A156T/S and T54T/A

Telaprevir1X IC50 10X IC50 15X IC50

HCV-796

1X IC50 10X IC50 15X IC50

NS5B Nuc NS5B Non-Nuc NS3/4A PI

R7128 Active Moiety (PSI-6130)

Higher Genetic Barrier of Nucsas compared to Non-Nucs and PIs

Untreated 1X IC50 10X IC50 15X IC50

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A-831:4-NH2-quinazolines (Arrow/AZ)BMS -790052

R

R

Resistance mutations in NS5A domain I:L31V, Y93H (gt 1b)M28T, Q30H/R, L31M/V, Y93C (gt 1a)

Resistance mutations inNS5A domain I, II and III:L199F, T200P, E212D, P299L, I302T,V362A, S370P, V388D, S390GNS4B: S258TNS5B: S76A

target NS5A

PI4K-IIIαSchmitz & Tan, Rec Pat Antiinfect Drug Discov, 2008; Delang et al., Viruses 2010

2 classes of antivirals reported as „NS5A inhibitors“R. DeFrancesco et al., 18th international Symposium on Hepatitis C virus and related viruses, Seattle, 2011

Structure of NS5A

D1

D2

D3

membrane

(polyU)

Basicgroove

F. Penin F. Penin

Targett-Adams et al., JVi 2011

X

X

NS5A inhibitors are dominant negative

(1 inhibitor per 100 – 1.000 NS5A molecules)

block of 5A oligomerization?

block PI4K-IIIα activation?

block NS5A hyperphosphorylation?

Mode-of-action of NS5A inhibitors

IRBM/Merck

Genelabs Bristol Myers Squibb

A92VY93HR157W

L28VL31VP58LY93H

M28T (1a); L28V (1b)Q30H/RL31V/F/MY93H/C/W

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

HCV RNA

Schmitz Recent Pat Antiinfect Drug Discov. 2008

Resistance against NS5A inhibitorsin replicon studies

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• CyPs are chaperones with peptidyl-prolyl isomerase activity

• Abundant cytosolic protein (0.1% of total cellular proteins)

• Ubiquitously expressed in eukaryotic cells

• Multiple functions, depending on target protein

• Discovered as specific ligand for immunosuppressive drug cyclosporin A

Cyclophilins

Structure of CsA and CsA-Derivativesa

da

pte

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Ga

llay,

Clin

Liv

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Inhibition of HCV replication by CsA

Watashi et al., Hepatology 2003

Structure of Cyclophilin Inhibitorsa

da

pte

d f

rom

Ga

llay,

Clin

Liv

Dis

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Sanglifehrin 0.3nM 0.02µM?

Alisporivir

Mode-of-Action of Cyp Inhibitors?

5B

5A

3

HCV replication

5B

5A

3

HCV replication

CypA

CypA

CypA

CypA

CsA CypA

CsA

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Virus

IFN-

dsRNAvRNA

IFN-

TLR3

TRIF

RIG-I

MAVS

IFN-

adapted from Haller et al., Virology, 2005

ISGISRE

IFNAR

JAK / Stat

ISGF-3

dsRNA

antiviral genes(~ 400)

The Interferon System

• IFN-α and IFN-λ share the same signalling pathways

• Very similar set of ISGs induced

But: Receptor distribution very different

IFN-α receptor on most cells

IFN-λ receptor primarily on hepatocytes & airway epithelia

faster and prolonged ISG induction

SOC-based treatment of HCV infection:correlation with IL28B polymorphism

*Ge et al., Nature 2009; Suppiah et al., Nat Gen 2009; Tanaka et al., Nat Gen 2009Thomas et al., Nature 2009;

High positive correlation with therapy outcome

High correlation with outcome of acute therapy

A special role of IFN-lambda to combat HCV?

ISGs impairing HCV replication

Schoggins et al., Nature 2011; Han et al., 2002; Gale et al., 1997; Helbig et al., 2005; Taylor et al., 2005

RIG-IMDA-5IRF-1IRF-2IRF-7MAP3K14

OASLRNaseLPKR RNA translationIFI44L GTPaseNT5C3 nucleoside dephosph.?Viperin LDs? membrane curv?ADAR RNA editingDDIT4 ?

signal transduction

RNA degradationMultiple Attack Strategies Against HCV