Hepatitis C Virus: Basics & Antiviral Therapy Ralf Bartenschlager AREVIR-GenaFor-Meeting Bonn, May 5-6, 2011
Hepatitis C Virus Replication 1. Entry 6. Virus assembly and release 2. Genome organization 4. „Membranous Web“ 5. RNA replication 3. Topology of HCV proteins
SOC therapy of hepatitis C Poynard T et al . Lancet 2003;362:2095-2100
The type 1 interferon system IFN- Virus IFNAR JAK / Stat vRNA dsRNA dsRNA RIG-I TLR3 antiviral genes (~ 400) MAVS TRIF IFN- ISGF-3 ISRE ISG IFN- adapted from Haller et al., Virology, 2005
ISGs impairing HCV replication RIG-I MDA-5 IRF-1 signal transduction IRF-2 IRF-7 MAP3K14 Multiple attack strategies against HCV: little/no escape from effector mechanisms OASL RNA degradation RNaseL little/no inhibition of IFN-induces signalling (cell culture) PKR RNA translation IFI44L GTPase NT5C3 nukleoside dephosph.? Viperin LDs? membrane curv? ADAR RNA editing DDIT4 ? Schoggins et al., Nature 2011; Han et al., 2002; Gale et al., 1997; Helbig et al., 2005; Taylor et al., 2005
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;
Mode-of-Action of Ribavirin • broad spectrum virostatic • reduces relapse in combination with peg-IFN in combination with NS3 protease inhibitors • little/no direct antiviral effect • possible mechanism: inhibition of IMPDH (no effect of direct IMPDH inhibitors) mutagenesis (‚error catastrophe‘) Th2 Th1 shift enhancement of IFN-response
HCV-specific targets Host factors: p7 4A C E1 E2 2 3 4B 5A 5B Cyclophilin A NS2-3 protease NS3 protease NS3 helicase NS5A NS5B polymerase Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005
HCV-specific DAAs Kwong et al., Gastroenterology 2011
The NS3/4A protease • chymotrypsin-like enzyme • activation by NS4A • shallow and exposed substrate binding pocket • cleavage of viral and cellular proteins polyprotein processing inhibition of innate immunity (TLR3, RIG-I) • substrate specificity D/E - X 4 - C/T S/A - X 3 • end product inhibition peptidomimetics
Development of NS3/4A protease inhibitors P6-P5-P4-P3-P2-P1 -- P1'-P2'-P3'-P4' P1'-P2'-P3'-P4' P6-P5-P4-P3-P2-P1 Vx950 Ac-Asp-Glu-Met-Glu-Glu-Cys-OH Ingallinella et al., Biochemistry 1998
Mode-of-action of NS3/4A protease inhibitors LD Translation Assembly 1 (+) RNA > 1.000 polyproteins RV blockage of polyprotein processing disrupt formation of replication vesicles no effect on already established replication vesicles Restoration of signal transduction in innate immunity pathways RIG-I TLR3
Inhibition of IFN-production by NS3/4A protease IFN- virus IFNAR NS3/4A cleavage also in vivo? JAK / Stat vRNA dsRNA dsRNA RIG-I TLR3 antiviral genes (~ 400) MAVS TRIF IFN- Yes for MAVS ? for TRIF ISGF-3 Reconstitution of immune ISRE ISG IFN- response? adapted from Haller et al., Virology, 2005
Resistance against NS3/4A protease inhibitors Telaprevir Boceprevir Telaprevir Boceprevir clinic Replicon V36 A/M V36 A/L/M T54 A/T T 54 A/S F43 C/S R155 R/K R155 K V55A A 156 A/S/T/V A156 S/T T54 A T 54 A/S V36M+T54S V170A R155 K/T R155 K/T/P V36 A/M +R155K/T V36A+R155K T54S/A+R155K A156 S/T/V A156 S A36A/M+A156V/T T54S+A156S V170A/T/L V170 T54 • broader spectrum of mutations in vivo vs. in vitro V36 D168 limited viral diversity in vitro NS4A R155 • double mutations in vivo A156 F43 R155K+V36M: most frequently associated with ‘break through’ compensatory mutations necessary to restore fitness R. de Francesco, INGM
HCV-specific targets Host factors: p7 4A C E1 E2 2 3 4B 5A 5B Cyclophilin A NS2-3 protease NS3 protease NS3 helicase NS5A NS5B polymerase Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005
The NS5B RNA-dependent RNA polymerase right-hand shape closed active site thumb palm fingers
Nucleosidic and non-nucleosidic NS5B inhibitors Nucleoside analogs NM283 2'-C-methyl cytidine 7-deaza-2'-C-methyl-adenosine Non-nucleosidic inhibitors Benzimidazole derivative Thiophene derivative Thiadiazine derivative De Francesco & Migliaccio, Nature 2005
Mode-of-action of HCV NS5B polymerase inhibitors LD Translation Assembly 1 (+) RNA > 1.000 polyproteins RV Block of RNA synthesis affect already established replication vesicles direct inhibition of NS5B (NNI) block of elongation (NI)
Higher genetic barrier of nucleoside analogues as compared to non-nucleosidic inhibitors and protease inhibitors NS5B NI NS5B NNI NS3/4A PI R7128 Active Moiety (PSI-6130) HCV-796 Telaprevir 1X IC 50 10X IC 50 15X IC 50 Untreated 1X IC 50 10X IC 50 15X IC 50 1X IC 50 10X IC 50 15X IC 50 • 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
Mutations conferring NS5B NNI resistance - ABT-333; ANA598 Cys 316; Met 414; Tyr 448; Gly 554; Asp 559 - GS-9190 - BI 207127; MK-3281 Cys 445; Tyr 448; Tyr 452 Pro 495; Pro 496; Val 499 A E B C D - VX-222; VX-759; Filibuvir Leu 419; Met 423; Ile 482; Val 494 - HCV-796 Leu 314; Cys 316; Ile 363; Ser 365; Met 414 • cross resistance against different NNI classes R. de Francesco, INGM
HCV-specific targets Host factors: p7 4A C E1 E2 2 3 4B 5A 5B Cyclophilin A NS2-3 protease NS3 protease NS3 helicase NS5A NS5B polymerase Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005
BMS-79005: The most potant DAA • discovered via high-throughput screen with subgenomic HCV replicons • active against genotype 1 – 6 replicons (chimeras) Replicon EC 50 (pM) HCV subtype 50 1a 9 1b 12-63 2a 127 3a 12 4a 33 5a M Gao et al. Nature 465 , 96-100 (2010)
Mode-of-action of NS5A inhibitors NS5A inhibitors are dominant negative (1 inhibitor per 100 – 1.000 NS5A molecules) block of 5A oligomerization? block NS5A recruitment to LDs? block NS5A hyperphosphorylation? (polyU) Basic groove 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 NS5A recruitment to LDs? block NS5A hyperphosphorylation? X X Targett-Adams et al., JVi 2011
Resistance against NS5A inhibitors in replicon studies IRBM/ Genelabs Bristol Myers Merck Squibb A92V L28V M28T (1a); L28V (1b ) Y93H L31V Q30H/R R157W P58L L31V/F/M Y93H Y93H/C/W HCV RNA Schmitz Recent Pat Antiinfect Drug Discov. 2008
Resistance against NS5A inhibitors in replicon studies Gao et al. Nature 465 , 96-100 (2010)
HCV and therapy resistance: a few remarks in advance • HCV persistence requires continuous replication no latency, no integration, no cccDNA continuously vulnerable to DAAs • Resistant virus variants are not archived most often impaired fitness require compensatory mutations non-adapted variants are out-competed
HCV and therapy resistance: a few remarks in advance How much of a concern will DAA resistance be in the future? Preexisting resistance DAAs combined with SOC IFN-free therapy
Pre-existing PI-resistant HCV variants: of clinical relevance? Boceprevir + IFN/R Deep sequencing of NS3 protease at baseline and during treatment (n=29, SPRINT-1 • Resistant variants detectable also with SVR patients • SVR upon triple therapy depends on sensitivity to IFN/R and type of PI Courtesy C. Sarrazin, Ffm
Persistence of resistance? Telaprevir + IFN/R Population-based sequencing, treatment failures, pooled phase 3 studies 1.0 1a: 84% 0.9 (225/269) 0.8 Probability of being WT 0.7 1b: 54% 0.6 (64/119) median 0.5 0.4 0.3 0.2 0.1 0.0 0 2 4 6 8 10 12 14 16 18 Time after failure (months) Sullivan et al., EASL 2011 • Reduction of frequency of resistant variants after cessation of therapy Courtesy C. Sarrazin, Ffm
HCV and therapy resistance: a few remarks in advance How much of a concern will DAA resistance be in the future? Preexisting resistance clinically probably little relevance DAAs combined with SOC IFN-free therapy
High potency of DAA combination therapy with/without SOC Genotype 1 NULL RESPONDERS BMS-790052 (NS5A) and BMS-650032 (PI) for 24 weeks +/- IFN/R 5A and PI alone: 4/11 achieved SVR12 and SVR24 6/11 had breakthrough 4/6 had SVR after addition of IFN/R HCV infection can be cured w/o IFN/R quadruple (5A, PI, IFN, R): 10/10 achieved SVR12 9/10 achieved SVR24 high rate of cure in this difficult to treat population A. Lok et al., EASL 2011
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