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

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 et al., 2005

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

HCV replication replication cycle HCV cycle

Model Systems to Study Study HCV in Cell Cell Culture Model Systems to HCV in Culture infection of cultured cells (PHH; cell lines) very inefficient HCV pseudoparticles Early steps of infection HCVpp HCV replicons RNA replication HCVcc whole life-cycle

HCV Cell Cell Culture Models HCV Culture Models HCVcc HCV replicons JFH1 (gt2a) Con1 (gt1b) gt 1a J6/JFH1 (gt2a) gt 2a gt 3 (HCV 2011) gt 4 (EASL 2012) NS3/4A: gt 2a, 3a, 5a, 6a NS5A: gt 1-7 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) wt adapt RNA Lohmann et al.: Science (1999) Blight et al.: Science (2000) Lohmann et al., J. Virol (2001, 2003) Krieger et al., J. Virol (2001)

HCV DRUG TARGETS 2012: HCV Replicon HCVpp HCVcc 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 NS5B pol NS5A NS4B NS2 pro p7 NS3/4A NS3 hel pro Targets for direct-acting antivirals (DAAs) DGAT-1 CD81 miR-122 MTP CD81 EGFR SR-BI Cyclophill. PI4KIII α NPC1L1 Targets for indirect-acting antivirals (host cell factors) Targets w/Clinical P.O.C. Targets w/o Clinical P.O.C. R.de Francesco

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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

Standard of Care Standard of Care 2012 2012 Current SOC gt1: pegIFN/Rbv + NS3 protease inhibitor Poynard T et al . Lancet 2003;362:2095-2100 Sarrazin et al., J. Hepatol. 2012

Resistance to PIs in vivo vs vs in vitro Resistance to PIs in vivo in vitro Telaprevir Boceprevir Telaprevir Boceprevir Clinical 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 • 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 R. de Francesco, INGM

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 Discontinuation - High discontinuation rates due to adverse effects (12-21%) • Severe rash, anemia Breakthrough (3-7%) - Emergence of resistant virus Relapse (7-20%) • Preexisting resistant variants (high frequency for gt 3) • Linked to poor response to PEG-IFN/RBV → virtual monotherapy SVR (~60-70%)

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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

Drug- -resistance to non resistance to non- -nucleoside inhibitors nucleoside inhibitors Drug - ABT-333; ANA598 Cys 316; Met 414; Tyr 448; Gly 554; Asp 559 - - BI 207127; MK-3281 GS-9190 Cys 445; Tyr 448; Tyr 452 Pro 495; Pro 496; Val 499 – Restricted spectrum of action (most active on genotype 1b) A – Low barrier to resistance in vitro and in vivo, viral breakthrough – Cross-resistance among different NNI classes E B C – Resistant variants/polymorphisms pre-exist in patient population ( e.g. , C316Y → N in genotype 1b) 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 among different NNI classes R. de Francesco, INGM

No resistance to nucleoside analog No resistance to nucleoside analog 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! R. de Francesco, INGM

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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

BMS- -790052: the most potent DAA 790052: the most potent DAA BMS •Identified in a HTS on subgenomic replicon cells HCV RNA •Active across HCV genotypes 1-6, pM to nM •Drug resistance maps to NS5A •Direct binding of drug to NS5A •Mechanism? 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

Summary BMS- Summary BMS -790052/Daclatasvir 790052/Daclatasvir HCV RNA level (log IU/ml) Placebo (n = 2) BMS 790052 1 mg (n = 6) BMS 790052 10 mg (n = 5) 0 BMS 790052 100 mg (n = 5) -1 single oral dose! 1 -2 -3 -4 0 6 1624 36 48 72 144 Time (hours) • 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 therapy 2 • NS5A inhibitor + Protease Inhibitor trial: 90% SVR in previous null-responders 3 • NS3+NS5A resistant variants in breakthrough patient 1 M Gao et al. Nature 465 , 96-100 (2010); 2 Pol S et al. EASL 2010, Abs 1189 3 EASL 2012

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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

Cylophilin A is is required for HCV replication replication Cylophilin A required for HCV • 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 Kaul et al., PLoS Path. 2009

Debio- -025 ( 025 (Alisporivir Alisporivir) ) Debio • 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) Flisiak et al., Hepatology 2009, *EASL 2012

Important Important targets targets for for antiviral antiviral therapy therapy Host Factors: miR-122 Cyclophilin A NS2-3 Protease a NS3 Protease b NS3 Helicase c NS5A d NS5B Polymerase e 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