WORKING LUNCHEONE
FUTURE THERAPIES
Nature 474, S6, 2010
The Hep C Drug Pipeline
Structure of Membrane-Associated NS3/4A
helicase
domain
• chymotrypsin-like enzyme
• activation by NS4A
• cleaves viral and cellular proteins
protease
domain
Polyprotein processing
innate responses (TLR3, RIG-I)
NS4A protease
cofactor
Brass et al. PNAS 2008
Structures of NS3-Specific Drugs
‚linear‘
Boceprevir
Telaprevir
‚macrocyclic‘
MK5172
TMC435
Mode-of-Action of NS3-specific DAAs
Translation
1 (+) RNA
Assembly
> 1.000 polyproteins
RV
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)
LD
Resistance against PIs:
2nd Generation
EC50 > 4-fold
Halfon & Locarnini, J. Hepatol. 2011
Nature 474, S6, 2010
The Hep C Drug Pipeline
The NS5B RNA-Dependent RNA Polymerase
‘closed’ active site
Form of a right hand
Thumb
Palm
Fingers
Nucleosidic and Non-nucleosidic
NS5B-Specific Drugs
Non-nucleosidic Inhibitors
Benzimidazole derivative
Thiophene derivative
Thiadiazine derivative
Nucleosidic Inhibitors
PSI-7977
INX-189
2'-C-methyl cytidine
Mode-of-Action of NS5B-Specific Inhibitors
Translation
1 (+) RNA
Assembly
> 1.000 polyproteins
RV
Block of RNA synthesis
direct effect also on established replication complexes
direct inhibition of NS5B (non-nucs)
block of elongation (nucs)
LD
Higher Genetic Barrier of Nucs
as compared to Non-Nucs and PIs
NS5B Nuc
NS5B Non-Nuc
NS3/4A PI
R7128 Active Moiety (PSI-6130)
HCV-796
Telaprevir
Untreated
1X IC50
10X IC50
15X IC50
1X IC50
10X IC50
15X IC50
1X IC50
10X IC50
• 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
15X IC50
Nature 474, S6, 2010
The Hep C Drug Pipeline
2 classes of antivirals reported as „NS5A inhibitors“
R. DeFrancesco et al., 18th international Symposium on Hepatitis C virus and related viruses, Seattle, 2011
R
R
BMS -790052
Resistance mutations in NS5A domain I:
L31V, Y93H (gt 1b)
M28T, Q30H/R, L31M/V, Y93C (gt 1a)
A-831:4-NH2-quinazolines (Arrow/AZ)
Resistance mutations in
NS5A domain I, II and III:
L199F, T200P, E212D, P299L, I302T,
V362A, S370P, V388D, S390G
NS4B: S258T
NS5B: S76A
target NS5A
PI4K-IIIα
Schmitz & Tan, Rec Pat Antiinfect Drug Discov, 2008; Delang et al., Viruses 2010
Structure of NS5A
membrane
(polyU)
D1
D2
Basic
groove
D3
F. Penin
F. Penin
Mode-of-action of NS5A inhibitors
NS5A inhibitors are dominant negative
(1 inhibitor per 100 – 1.000 NS5A molecules)
block of 5A oligomerization?
block PI4K-IIIα activation?
block NS5A hyperphosphorylation?
X
X
Targett-Adams et al., JVi 2011
Resistance against NS5A inhibitors
in replicon studies
HCV
RNA
IRBM/
Merck
Genelabs
Bristol Myers
Squibb
A92V
Y93H
R157W
L28V
L31V
P58L
Y93H
M28T (1a); L28V (1b)
Q30H/R
L31V/F/M
Y93H/C/W
Schmitz Recent Pat Antiinfect Drug Discov. 2008
Gao et al. Nature 465, 96-100 (2010)
Nature 474, S6, 2010
The Hep C Drug Pipeline
Cyclophilins
• 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
Structure of CsA and CsA-Derivatives
adapted from Gallay, Clin Liv Dis 2009
Inhibition of HCV replication by CsA
Watashi et al., Hepatology 2003
Structure of Cyclophilin Inhibitors
adapted from Gallay, Clin Liv Dis 2009
Alisporivir
Sanglifehrin
?
0.3nM
0.02µM
Mode-of-Action of Cyp Inhibitors?
CsA
CsA
CypA
CypA
3
3
CypA
5A
5A
CypA
5B
HCV replication
HCV replication
5B
CypA
Nature 474, S6, 2010
The Hep C Drug Pipeline
The Interferon System
IFN-
Virus
IFNAR
vRNA share
dsRNA the same signalling pathways
dsRNA
• IFN-α and IFN-λ
• Very similar setTLR3
of ISGs induced
antiviral genes
JAK / Stat
RIG-I
MAVS
(~ 400)
TRIF
IFN-
But: Receptor distribution very different
IFN-α receptor on most cells
ISGF-3
IFN-λ receptor primarily on hepatocytes & airway epithelia
ISRE
IFN- prolonged ISG induction
faster and
adapted from Haller et al., Virology, 2005
ISG
SOC-based treatment of HCV infection:
correlation with IL28B polymorphism
High positive correlation with therapy outcome
High correlation with outcome of acute therapy
A special role of IFN-lambda to combat HCV?
*Ge et al., Nature 2009; Suppiah et al., Nat Gen 2009; Tanaka et al., Nat Gen 2009
Thomas et al., Nature 2009;
ISGs impairing HCV replication
RIG-I
MDA-5
IRF-1
IRF-2
IRF-7
MAP3K14
OASL
RNaseL
PKR
IFI44L
NT5C3
Viperin
ADAR
DDIT4
signal transduction
RNA degradation Against HCV
Multiple Attack Strategies
RNA translation
GTPase
nucleoside dephosph.?
LDs? membrane curv?
RNA editing
?
Schoggins et al., Nature 2011; Han et al., 2002;
Gale et al., 1997; Helbig et al., 2005; Taylor et al., 2005