Non-Nucleoside Inhibitors of Viral RNA Polymerases;

Non-nucleoside inhibitors of viral RNA polymerases;

scaffolds for rational drug design

Auda Abdelsalam Eltahla

A thesis submitted in fulfilment of the requirements for the degree of Doctorate of Philosophy (Biochemistry and Molecular Genetics)

School of Biotechnology and Biomolecular Sciences The University of New South Wales

March 2014

ABSTRACT

The hepatitis C virus (HCV) and norovirus (NoV) are significant human pathogens posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of these viruses through the development of both vaccines and antivirals has proven to be difficult, partly because of the refractoriness in growing these viruses in cell culture. The current standard treatment for HCV is expensive, poorly tolerated, has a long duration, and is only partly effective. Although new direct-acting antivirals (DAA) are entering clinical treatment, these emerging molecules have been mainly targeted against one of six HCV genotypes, namely G1. Conversely, there are no antivirals for the treatment of chronic NoV infections, or for use as a prophylactic measure in an outbreak setting, which typically affects hospitals, nursing homes and other enclosed environments. The viral RNA-dependent RNA polymerases (RdRp) of HCV and NoV are prime targets for antiviral development, given their crucial role for the viral replication, and the absence of a homologous human enzyme. This study describes the discovery and characterisation of the first non- nucleoside inhibitors (NNI) specifically targeted against the RdRp of HCV G3a, a neglected but increasingly important genotype of HCV. Chemical scaffolds and derivatives were identified with low micromolar inhibitory activity against recombinant RdRp, and the HCV replicon cell culture model. Furthermore, the efficacy of previously identified inhibitors against the HCV G3a RdRp was examined, and a novel mechanism of enhancement of de novo transcription was discovered for a subclass of these antivirals; the T2 and P-β binding NNIs. This study also describes the high-throughput identification of the first four NoV-directed NNI scaffolds, which provide a strong platform for future rational drug design for antivirals against another pathogen with thus far limited control measures.

ACKNOWLEDGEMENTS

To my supervisor Pete, thank you for being a great mentor. You have been very generous and patient for the past four years. I have been granted many opportunities under your guidance, and this PhD would have been a big mess without you. To JS, your assistance at the beginning of my PhD was invaluable, and you showed me how to be meticulous in my work. Thanks for answering all those questions I had, and the many coffee breaks. To Kun Lee, thank you for your persistence in the face of adversity, and listening to my complaints when things went wrong. To the current (Andrew Kelly and Jennifer Lun) and previous lab members (Philip Bebek, Sean Pham, Rowena Bull, Melanie Walker and Camelia Quek) thanks for making the lab enjoyable and fun. To my family, especially my parents, thank you for your endless support and love throughout my graduate studies, I couldn’t have done any of this without you. To Eser, thanks for simply always being there, and making my days brighter. This work would not have been possible without the help of many people with various technical expertise. To Chris Marquis, Scott Minns and Helene Lebhar from the Recombinant Protein Facility, UNSW, for all the assistance and with the protein methodology. To Jonathon Morris and Jaqueline Liu from the School of Chemistry, UNSW, for assistance with the chemical side of things. To Dai Hibbs and Jennifer Ong from the Faculty of Pharmacy, University of Sydney, for help with the molecular docking experiments, and to Kurt Lackovic from the Walter and Eliza institute, thanks for your help with the high-throughput screening.

LIST OF PUBLICATIONS

Research articles  Eltahla, A. A., Lackovic, K., Marquis, C., Eden, J. S., & White, P. A. (2013). A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase. Journal of Biomolecular Screening, 18(9), 1027-1034.  Eltahla, A. A., Lim, K. L., Eden, J. S., Kelly, A. G., Mackenzie, J. M., & White, P. A. (2014). Non-nucleoside inhibitors of the norovirus RNA polymerase; scaffolds for rational drug design. Antimicrobial Agents and Chemotherapy, 58(6), 3115-3123

 Eltahla, A. A., Tay, E., Douglas, M. W., White, P. A. (2014). Cross-genotypic examination of the hepatitis C virus polymerase inhibitors reveals a novel mechanism of action for thumb binders. Antimicrobial Agents and Chemotherapy. Submitted.

Select conference publications  Eltahla, A. A., Lim, K., Kelly, A. G., Mackenzie, J. M., White, P. A. Non-nucleoside inhibitors of the norovirus RNA polymerase; scaffolds for rational drug design (The 7th Australasian Virology Society Meeting [AVS7] 2013).  Eltahla, A. A., P. A. White. Cross genotypic inhibition of hepatitis C virus by leading polymerase inhibitors (The 20th International Symposium on Hepatitis C Virus and Related Viruses 2013).  Eltahla, A. A., Lackovic, K. Eden, J. S., White, P. A. Identification and initial characterisation of inhibitors targeting hepatitis C virus genotype 3a RNA polymerase NS5B (The 20th International Symposium on Hepatitis C Virus and Related Viruses 2013).  Eltahla, A. A., White, P. A. Cross genotypic inhibition of hepatitis C virus by leading polymerase inhibitors (The 9th Annual Workshop of the Australian Centre for HIV and Hepatitis Virology Research [ACH2] 2013).

iii

 Eltahla, A. A., Lackovic, K. Eden, J. S., White, P. A. A high throughput screen for the identification of Norovirus targeted RNA-dependent RNA polymerase inhibitors (Positive Strand RNA Viruses meeting 2013).  Eltahla, A. A., Lackovic, K. Eden, J. S., White, P. A. Identification and initial characterisation of inhibitors targeting hepatitis C virus genotype 3a RNA polymerase NS5B (Positive Strand RNA Viruses meeting 2013).  Eltahla, A. A., Lackovic, K. Marquis, C., Eden, J. S., White, P. A. A Fluorescent-based High Throughput Assay for Screening of inhibitors of Hepatitis C Virus RNA Polymerase Activity (The 8th Annual Workshop of the Australian Centre for HIV and Hepatitis Virology Research [ACH2] 2012).

TABLE OF CONTENTS

1 General introduction part I: hepatitis C virus ...... 1

History and background ...... 1

Epidemiology, transmission and pathogenesis of HCV ...... 1

Molecular Biology of HCV ...... 2

1.3.1 Structure and genome organisation ...... 3

1.3.2 Genetic diversity of HCV...... 4

1.3.3 The HCV RNA-dependent RNA polymerase ...... 7

1.3.4 Cell culture models for studying HCV replication ...... 9

HCV Therapy ...... 10

1.4.1 Interferon-based therapy ...... 10

1.4.2 Direct-acting antivirals ...... 11

1.4.3 Host-targeting agents ...... 17

1.4.4 DAA combinations ...... 18

Genotype 3 HCV; an understudied virus...... 18

2 General introduction part II: Norovirus ...... 21

Background and history of NoV ...... 21

Transmission and pathogenesis ...... 22

Molecular biology of NoV ...... 22

2.3.1 NoV classification and genome organisation ...... 22

2.3.2 Genetic diversity of NoV ...... 23

2.3.3 NoV Genome replication and the RNA-dependent RNA polymerase...... 25

2.3.4 Molecular epidemiology and diversity of NoV – the GII.4 pandemic lineage .... 26

2.3.5 Models of NoV replication ...... 27

Management of NoV infections ...... 28

2.4.1 NoV vaccines ...... 28

2.4.2 Emerging NoV antivirals ...... 30

3 General Materials and methods ...... 35

Materials ...... 35

General methods ...... 38

3.2.1 Nucleic acid purification and reverse transcription ...... 38

3.2.2 DNA sequencing ...... 38

3.2.3 Recombinant protein expression and purification ...... 38

3.2.4 Protein electrophoresis and western blotting ...... 39

3.2.5 Quantitative RdRp assays ...... 40

3.2.6 Gel-based RdRp assays ...... 41

3.2.7 High-throughput screening ...... 41

Cell culture general methods ...... 43

3.3.1 Cells and replicons ...... 43

3.3.2 Cell maintenance ...... 44

3.3.3 Sub-genomic replicons assays ...... 45

3.3.4 Cell viability assay ...... 45

4 A fluorescence-based high throughput screen to identify small compound inhibitors of the genotype 3a hepatitis C virus RNA polymerase ...... 47

Introduction ...... 48

Materials and Methods ...... 49

4.2.1 HCV G3a RdRp expression and purification ...... 49

4.2.2 RdRp activity assay development ...... 49

4.2.3 Kinetics of RdRp activity ...... 49

4.2.4 Assay miniaturisation and validation ...... 50

4.2.5 Pilot HTS ...... 50

4.2.6 Secondary screening and IC50 determination ...... 50

4.2.7 Structure-activity relationship ...... 51

4.2.8 Replicon selection and testing for cross-resistance ...... 52

Results ...... 53

4.3.1 Expression of HCV G3a RdRp and fluorescent assay development ...... 53

4.3.2 Assay optimisation and characterisation of the HCV G3a RdRp kinetics...... 54

4.3.3 Development of a high-throughput RdRp assay ...... 57

4.3.4 Pilot high-throughput screen ...... 60

4.3.5 Secondary screening of HTS hits and IC50 determination ...... 61

4.3.6 Confirmation of HAC chemical structures; the HAC02 story ...... 64

4.3.7 Inhibitory activity of HAC molecules using the HCV replicon model ...... 70

4.3.8 Structure-activity relationship studies ...... 71

4.3.9 Primer-extension activity of HAC molecules ...... 74

4.3.10 Selection of drug resistant replicons ...... 76

Discussion ...... 79

4.4.1 PicoGreen compared to available assays ...... 79

4.4.2 Replicon activity of the identified HAC molecules ...... 80

4.4.3 Photocatalytic activation of HAC02-01 ...... 81

4.4.4 Structure-activity relationship ...... 82

4.4.5 Binding site of the HAC molecules ...... 83

5 Mechanistic examination of HCV polymerase inhibitors reveals a novel mechanism for thumb binders...... 85

Introduction ...... 86

Materials and Methods ...... 88 vii

5.2.1 Compounds ...... 88

5.2.2 Recombinant RdRps and quantitative assays ...... 89

Results ...... 90

5.3.1 Inhibitory activity of NNIs against HCV replicons ...... 90

5.3.2 Inhibitory activity of HCV NNIs against recombinant RdRps ...... 94

5.3.3 Gel Based examination of RdRp inhibition ...... 97

5.3.4 Analysis of resistant mutations in HCV RdRp sequences ...... 99

Discussion ...... 101

6 Non-nucleoside inhibitors of the norovirus RNA polymerase ...... 105

Introduction ...... 106

Materials and Methods ...... 108

6.2.1 Recombinant RdRp expression, purification and comparison ...... 108

6.2.2 Biochemical RdRp assays...... 108

6.2.3 High-throughput screening ...... 108

6.2.4 Mode of RdRp inhibition ...... 109

6.2.5 Inhibition of murine norovirus replication ...... 109

6.2.6 Quantitative reverse transcriptase polymerase chain reaction ...... 109

6.2.7 Structure-activity relationship analysis ...... 110

Results ...... 111

6.3.1 Identification of RdRp inhibitors by high-throughput screening (HTS) ...... 111

6.3.2 Mode of Inhibition ...... 115

6.3.3 Activity of identified NNIs across related calicivirus RdRps ...... 117

6.3.4 Inhibition of the NoV GI Replicon ...... 119

6.3.5 Inhibition of infectious murine norovirus GV.1 ...... 121

6.3.6 Preliminary structure-activity analysis ...... 124

viii

Discussion ...... 125

7 General discussion ...... 128

Findings and implications ...... 128

7.1.1 Quinoxalines and quinolinones as HCV G3a inhibitors ...... 128

7.1.2 Only palm II HCV NNIs are cross-genotypic ...... 129

7.1.3 Thumb II NNIs enhance the de novo RdRp activity...... 131

7.1.4 NoV NNIs: scaffolds for rational drug design ...... 132

Limitations and future directions...... 133

7.2.1 Binding sites of the identified NNIs ...... 133

7.2.2 Model systems used in the study ...... 134

7.2.3 Past hit identification ...... 135

Final remarks ...... 136

8 Appendix ...... 139

9 References ...... 171

LIST OF FIGURES

Figure 1.1. Genome organisation of the hepatitis c virus...... 3 Figure 1.2. The genetic diversity of HCV...... 6 Figure 1.3. Crystal structure of the HCV RNA-dependent RNA polymerase...... 9 Figure 1.4. Direct-acting antivirals in clinical development for the treatment of HCV. . 16 Figure 2.1. The genome organisation of NoV...... 23 Figure 2.2. The diversity of noroviruses infecting a range of hosts...... 24 Figure 2.3. The crystal structure of the norovirus RNA-dependent RNA polymerase. .. 26 Figure 3.1. Schematic representation of HCV and NoV sub-genomic replicons in this study ...... 44 Figure 4.1. SDS-PAGE analysis of fractions from the purification of HCV G3a RdRp. .... 53 Figure 4.2. Assessment of fluorescent dyes for the detection of RdRp products ...... 54 Figure 4.3. Characterisation of biochemical factors influencing HCV G3a polymerase activity ...... 55 Figure 4.4. The effect of divalent salts on RdRp activity and fluorescence signal output...... 56 Figure 4.5. Steady-state kinetics of the HCV G3a RdRp measured using the PicoGreen assay...... 57 Figure 4.6. Titration curve of the reference compound 3′dGTP...... 58 Figure 4.7. Optimisation of the PicoGreen assay for HTS...... 59 Figure 4.8. Pilot RdRp inhibitor screening for inhibitors of the HCV G3a RdRp...... 61 Figure 4.9. Activity and Titration curves for the top HCV RdRp hits from the pilot HTS.63 Figure 4.10. Chemical structures of HCV G3a RdRp inhibitors identified in this study.. 64 Figure 4.11. Inhibitory activity of different lots of HAC molecules against the G3a RdRp...... 65 Figure 4.12. Time-dependent light activation of HAC02-01...... 66 Figure 4.13. Liquid chromatography–mass spectrometry (LC-MS) analysis of HAC02-01...... 68 Figure 4.14. Tandem mass spectrometry of HAC02-01...... 69 Figure 4.15. Inhibitory activity of the identified HACs against the HCV replicon...... 71

Figure 4.16. Activity of HAC01 and HAC02 derivatives using the HCV replicon model. 74 Figure 4.17. Inhibition of the primer-dependent activity of HCV RdRp by the HAC molecules...... 75 Figure 4.18. Selection of resistant HCV replicons with the identified HAC molecules. . 77 Figure 5.1. Chemical structures of HCV NNIs examined in this study...... 88 Figure 5.2. Inhibitory activity of HCV NNIs against sub-genomic replicons...... 92 Figure 5.3. Effect of HCV NNIs on the de novo activity of recombinant RdRps...... 96 Figure 5.4. Analysis of the effect of HCV NNIs on different modes of RdRp activity. .... 98 Figure 5.5. Amino acid substitutions conferring resistance to HCV inhibitors in this study...... 100 Figure 6.1. Outline of the pathway to identify NoV RdRp inhibitors in this study ...... 111 Figure 6.2. HTS for inhibitors of the NoV GII.4 RdRp...... 112 Figure 6.3. Chemical structures and inhibitory activity of NoV inhibitors in this study...... 114 Figure 6.4. Differential mechanism of RdRp inhibition by the four NIC compounds ... 116 Figure 6.5 Amino acid sequence analysis of RdRps from representative viruses...... 117 Figure 6.6. Inhibitory profiles of lead NoV inhibitors against RdRps from related caliciviruses...... 119 Figure 6.7. Inhibition of the Norwalk sub-genomic replicon by lead norovirus NNIs .. 120 Figure 6.8. MNV plaque reduction by lead NoV NNIs...... 121 Figure 6.9. Effect of NIC compounds on the replication of murine norovirus...... 123 Figure 7.1. Pathways towards pre-clinical development of antivirals...... 137 Figure 8.1 Nanospray mass spectrometry analysis of HAC molecules ...... 141 Figure 8.2 Alignment of the RdRp amino acid sequences for HCV genotypes 1b, 2a and 3a ...... 148 Figure 8.3. Effect of the HCV NNI Lomibuvir on the de novo RdRp activity...... 149 Figure 8.4 Kinetics of NoV GII.4 RdRp activity in the presence of NIC 2 inhibitors...... 150 Figure 8.5 Core structures used for SAR analysis of the NoV RdRp inhibitors...... 151

LIST OF TABLES

Table 1.1. Summary of size and function of HCV-encoded proteins...... 4 Table 1.2. Clinical trials of DAAs involving G3a HCV...... 20 Table 3.1. General reagents and kits used in this study...... 35 Table 4.1. Details of Purchased compounds for structure-activity relationship studies...... 52 Table 4.2 Control measurements of pilot HTS assay performance...... 60 Table 4.3. Activity of HCV inhibitors against HAC-selected replicons...... 78 Table 4.4. Amino acid substitutions detected upon selection of HCV replicons...... 78 Table 5.1 Cross-genotype activity of HCV inhibitors against sub-genomic replicons .... 93 Table 5.2 Inhibitory activity of HCV NNIs against recombinant RdRp...... 97 Table 6.1 Amino acid identity between the RdRp enzymes in this study...... 117 Table 6.2 Summary of the inhibitory activity of top NoV inhibitors in this study ...... 120 Table 8.1 Inhibitory activity of HAC01 and HAC02 derivatives in the recombinant enzyme and replicon models* ...... 142 Table 8.2 Inhibitory activity of NoV NNI analogues ...... 152

xii

ABBREVIATIONS

ALT Alanine aminotransferase bp Base pair BSA Bovine serum albumin cDNA Complementary DNA CI Confidence interval CsA Cyclosporin A CTB CellTiter-Blue CV Coefficient of variation Da Dalton DAA Direct Acting Antivirals DdDp DNA-dependent DNA polymerase DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate ds double-stranded

EC50 Half maximal effective concentration EDTA Ethylenediaminetetraacetic acid EMCV Encephalomyocarditis virus ER Endoplasmic reticulum FBS Foetal bovine serum GTP Guanosine triphosphate HBGA Histo-blood group antigens HBV Hepatitis B virus HCC Hepatocellular carcinoma HCV Hepatitis C virus HCVcc HCV cell culture system HIV Human immunodeficiency virus HRP Horseradish peroxidase HSV Herpes simplex virus HTA Host-targeting agents HTS High-throughput screening Huh-7 Hepatocellular carcinoma cells

IC50 Half maximal inhibitory concentration IDU Injection drug use IFN Interferon IRES Internal ribosome entry site JFH Japanese fulminant hepatitis xiii kb Kilo base LB Luria-Bertani LC/MS Liquid chromatography/mass spectrometry LHS Left hand side MEM Minimum essential media miRNA microRNA MNV Murine norovirus mRNA messenger RNA neo neomycin phosphatase NI Nucleoside inhibitors NNI Non-nucleoside inhibitors NoV Norovirus NS Non-structural protein ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction PDB Protein data bank PEG-IFN/BV Pegylated interferon-α and ribavirin PI Protease inhibitors qRT-PCR Quantitative reverse transcriptase PCR RdDp RNA-dependent DNA polymerase RdRp RNA-dependent RNA polymerase RHS Right hand site RNA Ribonucleic acid rRNA Ribosomal RNA RV Rotavirus RVR Rapid virological response S/B Signal-to-background ratio S/N Signal-to-noise ratio SDS Sodium dodecyl sulphate SGR Sub-genomic replicon SOC Standard of care ss single-stranded SVR Sustained virological response UTR Untranslated region VLP Virus-like particles

xiv

1 General introduction part I: hepatitis C virus

History and background

Nearly 3% of the world’s population are infected with hepatitis C virus (HCV), a leading cause of morbidity and mortality worldwide. HCV was discovered in 1989 as the causative agent of non-A, non-B hepatitis (NANBH) using HCV infected chimpanzee plasma screened with sera from infected patients.1, 2 This “agent” was identified as a novel RNA virus, related to flaviviruses, with a genome of nearly 10,000 bases.1 Today HCV accounts for 366,000 deaths per year from infection-related complications such as cirrhosis, liver failure and hepatocellular carcinoma (HCC).3 There is no vaccine for HCV and, until recently, antiviral treatments have been limited by side-effects and variable response rates amongst patients. Although research in the HCV field has come a long way since 1989, many basic questions about the viral replication and pathogenesis are yet to be addressed, and there remains an urgent need for effective measures to manage and treat an increasing number of HCV infected individuals.

Epidemiology, transmission and pathogenesis of HCV

The prevalence of HCV infection varies widely between different geographical regions of the globe. Countries like the United States and Canada have a relatively low (1.3%) prevalence, whereas 2.4-2.9% of the population in Europe are HCV positive.4 Central and East Asia are estimated to have an HCV prevalence exceeding 3.5%.4 In Australia, between 1% and 1.9% of the population are infected with HCV5, with almost 10,000 new infections each year.6 The highest prevalence of HCV worldwide is documented in Egypt, with nearly 20% seropositivity in the population.7, 8 HCV is mainly transmitted parenterally, and infections occur principally through exposure to contaminated blood or blood-derived products.9 Risk factors for HCV acquisition therefore include injection drug use (IDU), blood transfusions, renal dialysis and organ transplantations.9 In developed countries, the risk for HCV infection by blood-transfusions was lowered after the discovery of the virus, and the application of screening approaches to minimise the medical use of HCV contaminated blood and tissues.10 Nearly 70% of infections in the developed world are now acquired through 1

IDU10, 11 and HCV is a major problem in correctional facilities.11 In developing countries, however, HCV transmission occurs through unscreened blood transfusions and unsafe injection practices.12 In Egypt, where the highest HCV prevalence has been reported, a large proportion of infections were attributed to parenteral antischistosomal therapy using contaminated, poorly sterilised needles.7 The majority of HCV infections are asymptomatic during the acute phase.13 Several weeks after the initial infection, serum alanine aminotransferase (ALT) levels begin to increase14 and, in 25% of acute infections, symptoms may include malaise, nausea, and right upper quadrant pain.14 Chronic infection with HCV, which is defined as viral RNA persistence for >6 months, occurs in 60-80% of patients.15, 16 Approximately 20-30% of chronically infected patients develop liver cirrhosis within 20 years9, 17, and about 3% of all HCV patients eventually develop HCC. Chronically infected patients may be asymptomatic, or have only mild nonspecific symptoms as long as cirrhosis is not present.9, 17, 18 The most frequent complaint in chronically infected patients is fatigue, and less common manifestations are nausea, weakness, myalgia, arthralgia, and weight loss.18

Molecular Biology of HCV

HCV belongs to the family Flaviviridae, and used to be the only member of the genus Hepacivirus. However, several viruses have been recently identified and added to this genus, including the GB virus B19 as well as canine20, horse21, rodent22, and bat23 hepaciviruses. The origin of HCV, however, remains unclear. No evidence has yet been obtained for a human immunodeficiency virus (HIV)-like zoonotic origin, such as infection of HCV-like viruses in either apes or Old World monkey species (reviewed in reference 24).

1.3.1 Structure and genome organisation

HCV is a small enveloped virus, 50–80 nm in diameter, with a positive sense, single stranded RNA genome (ssRNA) of ~9,600 nucleotides. Similar to other positive-strand RNA viruses, the HCV genome can serve as a messenger RNA (mRNA) for the translation of viral proteins in the cytoplasm. The RNA molecule contains a single open reading frame (ORF) but lacks a 5´ cap. Instead, translation is initiated through an internal ribosome entry site (IRES).25, 26 A single 3011 amino acid precursor polyprotein is translated at the endoplasmic reticulum (ER) and is then cleaved by host and viral proteases into 10 structural and non-structural (NS) proteins (Figure 1.1 and Table 1.1). The structural proteins of HCV are encoded by the 5´ terminus of the genome and include the core protein as well as the two glycoproteins, E1 and E2. These are followed by the NS proteins which include p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.27 A summary of the properties and function of HCV-encoded proteins is shown in Table 1.1. In addition to its role as an mRNA, the HCV genome serves as a template for RNA replication, as well as new genomes to be packaged for nascent virion production. The NS5B, or the RNA-dependent RNA polymerase (RdRp) has been identified as the key enzyme involved in “copying” the viral genome. Upon infection, the NS proteins involved in genome replication (the replicase complex; NS3, 4A, 4B, 5A and NS5B) induce complex membrane remodelling near the ER, producing membranous vesicles known as the membranous web, a marker for active HCV replication.28

5’UTR 3’ UTR IRES Core E1 E2 p7 NS2 NS3 4A 4B NS5A NS5B

Capsid Envelope Cysteine Serine Membrane RNA-dependent glycoproteins protease protease alterations RNA polymerase RNA Ion Serine helicase Phosphoprotein channel protease cofactor

Figure 1.1. Genome organisation of the hepatitis c virus. The HCV genome is ~9.6 kb and contains a single open-reading frame and an internal ribosome entry site at the 5´ terminus to mediate translation. The 5’ end of the genome encodes for the structural proteins (shown in green) which make up the virion, while the 3´ terminus encodes seven non-structural proteins (grey), which are mainly involved in viral replication and assembly. Modified from 27 3

Table 1.1. Summary of size and function of HCV-encoded proteins. Protein Size (kDa) Function* C 21 Core protein: nucleocapsid-formation and particle assembly, interacts with cellular factors and interferes with intracellular signalling. E1 35 Transmembrane glycoproteins 1 and 2: adsorption and receptor-mediated endocytosis. E2 70

p7 7 Unknown: forms an ion-channel in the endoplasmic reticulum and essential for infectious virion assembly. NS2 23 Cysteine protease: portion of the NS2-3 protease which catalyses cleavage at the NS2/NS3 junction, assembly factor. NS3 70 Serine protease (N-terminal), NTPase/RNA helicase (C-terminal) cleavage of the downstream HCV proteins and viral assembly. NS4A 8 Cofactor of the NS3-NS4A protease.

NS4B 27 Assembly of functional replication complex, induction of membranous web near the ER. NS5A 56 Multi-functional phosphoprotein, RNA binding, phosphorylation-dependent regulation of RNA replication and viral assembly, interaction with host factors and regulation of the cell-cycle. NS5B 67 Viral RNA-dependent RNA polymerase.

*Summarised from 29

1.3.2 Genetic diversity of HCV

The polymerase of HCV, the RdRp, exhibits a high mutation rate of approximately 10-4 substitutions per site.30, 31 This is combined with a very high rate of virion production in infected individuals (1012 virions per day32). As a result, the HCV genome exhibits natural genetic diversity, with the virus classified into six different genotypes (G1-G6) differing by approximately 35% at the nucleotide level.33 These genotypes are further classified into “subtypes” (a, b, c, etc.), with about 20% inter-subtype divergence33, 34 across the genome (Figure 1.2). Even within an individual infected with a particular subtype, HCV exists as a heterogeneous RNA population known as quasispecies.35 This heterogeneity contributes to a significant evolutionary advantage, and provides the

4 virus with the means to adapt to the host immune response and persist as a chronic infection in humans.36 The distribution of different HCV genotypes is variable between different geographic regions. The most common HCV genotype, G1, accounts for 50% - 80% of infections worldwide, with the exception of the middle-east and Africa.34, 37, 38 G2 is also detected globally, and is the predominant genotype in western Africa, and accounts for 42% of infections in Finland and Taiwan.39 Conversely, G4 is mainly found in the Middle East and central Africa, and almost all (90%) of HCV infections in Egypt are with this genotype. 7, 8, 39 G5 is predominantly found in southern Africa, whereas G6 is almost exclusively found in south east Asia.40 Around 20-30% of all HCV infections are caused by G3, with several countries demonstrating a high prevalence of G3 infections including; India (up to 67% of HCV infections)41, Brazil (40%)42, the UK and Finland (40%)39 and Australia (34%).43 HCV genotypes pose significant implications with regards to the viral replication, pathogenesis, and for reasons not fully understood, response to antiviral therapy (discussed in section 1.4).

Figure 1.2. The genetic diversity of HCV. Phylogenetic representation of 60 full length HCV sequences. The tree and was generated using the Neighbour-Joining, best-fit method in MEGA5.44 The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site, indicated by the scale bar. Genotype numbers are shown to the right of the tree. Bootstrap values are provided as percentages of 1000 replicates for values ≥ 70%.

1.3.3 The HCV RNA-dependent RNA polymerase

Soon after the discovery of HCV, the analysis of its amino acid sequence predicted the existence of a “replicase” protein based on the identification of a Gly-Asp- Asp (GDD) motif, a signature conserved sequence for RdRps of other RNA viruses.45 The RdRp, was later characterised as a 66 kDa protein that associates with cellular membranes by means of the hydrophobic C-terminal amino acid tail, like most of the HCV NS proteins.46 However, the deletion of this hydrophobic “anchor” had little effect on the enzymatic activity in vitro, and recombinant, soluble RdRp could be produced efficiently in both insect cells and E. coli.47, 48 This is turn allowed for extensive structural and functional studies of the enzyme, perhaps becoming the most characterised of the HCV enzymes. The crystallisation of the HCV RdRp revealed a canonical right-hand like structure, where the active site (GDD motif, known as motif C) in the palm subdomain is fully encircled by an extensive interaction between the fingers and thumb subdomains (Figure 1.3).49-51 The HCV RdRp shares some structural homology with other viral RdRps and reverse transcriptases (RTs), including the RdRp from the RNA bacteriophage ϕ6.52 The protein also harbours a conserved aspartic acid motif that coordinates the binding of metal ions like Mg2+, known as motif A. The thumb subdomain of the HCV RdRp contains a β-hairpin loop insertion, which protrudes into the active site cavity (Figure 1.3). This loop is thought to influence the orientation of the newly synthesised RNA, and its position discriminates between different modes of RdRp activities.13 Interestingly, an allosteric guanosine-5´-triphosphate (GTP)-binding pocket has also been identified in the interface between the fingers and thumb subdomains.53 The binding of GTP to this site has been implicated in facilitating conformational changes required for a processive RdRp.54 The RdRp was originally crystallised in a “closed” conformation, which can only accommodate a single strand of RNA in the active site.49- 51 However, this did not explain how the enzyme could accommodate the double- stranded RNA being synthesised. In vitro experiments using recombinant RdRp show that the enzyme is capable of two biochemical activities. Firstly, the enzyme can catalyse RNA synthesis in a primer-dependent manner, extending from the 3´ end of an RNA molecule.55, 56 However, the RdRp of HCV, like other members of the Flaviviridae family,

7 is also able to catalyse transcription through a primer-independent, or de novo mechanism, where the polymerase catalyses the formation of a dinucleotide molecule at the 3´ end of the template, which could then be used as a primer.57-59 This mechanism is non-deleterious for the viral genome, that is, the whole genome is copied from start to finish, and this mode of activity for the RdRp is believed to be how genome replication is initiated in vivo.58 The two modes of RdRp activities were later shown to correspond to different conformations of the enzyme. A “closed” conformation, facilitated by the interactions between the fingers and the thumb domains, is too narrow to allow for the template–primer RNA duplex formed during replication. This conformation, however, facilitates the de novo formation of a dinucleotide complementary to the 3´ end of template RNA.60 The more relaxed “open” conformation, on the other hand, is formed by the displacement of the β-hairpin loop as well as a C-terminal segment, upstream of the transmembrane domain, called the linker (Figure 1.3).13, 61 This allows the nascent double-stranded RNA into the active site and is thought to represent the primer- extension activity of the HCV RdRp.

Fingers Thumb

Linker

Palm

Figure 1.3. Crystal structure of the HCV RNA-dependent RNA polymerase. The secondary structure of the HCV G2a polymerase [Protein Data Bank (PDB) accession number 2XHW] is shown. The enzyme has a typical RdRp “right hand” structure with fingers (red) and thumb (blue) subdomains encircling the active site within the palm subdomain (green). The enzyme is typically crystallised in the shown “closed” conformation, thought to be associated with de novo RdRp activity, with both the β-hairpin from the thumb domain, and the C-terminal “linker” extending into the palm domain.

1.3.4 Cell culture models for studying HCV replication

For a decade following the discovery of HCV, attempts to grow the virus in cell culture largely failed, and chimpanzees were the only animal model found to support viral replication in vivo. In 1999, however, a major breakthrough in HCV research was the development of a sub-genomic replicon (SGR) model by Lohmann et al.62 The HCV replicon was based on a particular G1b strain (Con1, GenBank accession number AJ238799), and was constructed by the removal of the structural genes of the HCV genome (C, E1 and E2) as well as NS2 (Figure 1.1), which are not essential for RNA replication. Both 3’ and 5’ untranslated regions (UTRs) were retained, and the structural genes were replaced with a neomycin phosphotransferase (neo) gene and another internal ribosome entry site (IRES) from the encephalomyocarditis virus (EMCV).62 When this recombinant RNA was transfected into human hepatoma cells (Huh-7), it was capable of autonomous replication, and conferred resistance to neomycin (G418) in replicon bearing cells.

Further studies to improve the replication of the HCV replicon in cell culture identified several adaptive mutations; amino acid substitutions that were shown to increase the replication efficiency of the HCV SGR in cell culture.63-66 Initially, replicon models were limited to genotypes 1 and 2, in particular, G1b63, 67, G1a65, 68 and G2a.69, 70 Very recently (2013), however, replicons for genotypes 3 and 4 were developed.71, 72 This delay has therefore had a large impact on the study of these HCV genotypes, with research into vaccine and antiviral development lagging significantly behind compared to G1 viruses (section 1.4.2). Another milestone in the field of HCV research was the development of the first fully infectious cell culture model (HCVcc) in 2005.73-75 Isolated from the serum of a Japanese patient with fulminant hepatitis (hence the name JFH-1), this particular G2a strain replicated efficiently in cell culture without the requirement of any adaptive mutations in the genome.73-75 A recombinant virus was also constructed by replacing the structural genes and NS2 from JFH-1 with those of another G2a strain (J6), and was shown to replicate 100 to 1,000 more efficiently in cell culture.73 Since then, several groups have also described cell culture models of other G2 subtypes76, 77 and G1 viruses.78, 79 These, however, were all adapted to cell culture by introducing known amino acid substitutions, and were limited to genotypes G1 and G2. Alternatively, chimeric infectious viruses from all genotypes have been developed to study individual HCV proteins, including the protease and polymerase, using the “backbone” of the original G2a HCVcc.80-85 However, until now, the JFH-1 system remains the only culturable strain that does not require adaptation to cell culture.

HCV Therapy

1.4.1 Interferon-based therapy

The aim of treatment for HCV infected patients is to achieve a sustained virological response (SVR), deﬁned as the absence of detectable HCV RNA from the blood 24 weeks after treatment is discontinued.86 For all genotypes other than G1, the standard of care (SOC) involves a combination of pegylated interferon-α and ribavirin (PEG-IFN/RBV) for 24 to 48 weeks.86, 87 Patients’ HCV RNA level, known as viral load, is monitored throughout the duration of the treament in order to determine the pattern

10 of response to therapy. This has also become essential for individualising the treatment regimen; for example, the duration of the dual therapy could be shortened to 12-24 weeks for patients with a rapid virological response (RVR), defined as undetectable HCV RNA four weeks into treatment. 80, 88 The response to IFN-based therapy varies greatly amongst HCV patients, and is largely affected by the diagnosed genotype, although other factors are also important. These include the viral load, fibrosis stage, gender, age, ethnic background and alcohol intake.89, 90 Overall, patients infected with G2 and G3 HCV are better respondents to the IFN-based therapy, with up to 80% SVR rates.91 In contrast, the more common G1 is associated with a poorer prognosis, and less than 50% of patients achieve SVR.92 The PEG-IFN/RBV treatment is also expensive and almost all patients experience adverse effects.86 The most common of these are influenza-like symptoms including fever, fatigue and headaches. Other common side-effects of the treatment include depression, irritability, insomnia (psychiatric) as well as neutropenia and anaemia (clinical abnormalities).86 The poor tolerance to the PEG-IFN/RBV treatment is a major reason for the discontinuation of therapy, with up to 14% of discontinuation rates among patients.93 Recently, the SOC for G1b has been modified, with the approval of the first generation direct-acting antivirals, administered in combination with PEG-IFN/RBV as a triple therapy (Section 1.4.2), however the PEG-IFN/RBV dual therapy is still given in countries where these DAAs have not been approved.2

1.4.2 Direct-acting antivirals

The limited efficacy of IFN-based therapies, combined with their poor tolerability, meant that more efficient antivirals were needed to treat HCV. These antivirals are ideally ones that are potent, effective against all six genotypes, and have minimal undesirable side-effects. For over a decade, extensive efforts by pharmaceutical companies and researchers were dedicated to the development of direct-acting antivirals (DAAs); defined as compounds that specifically bind to and inhibit one of the virus-encoded proteins, impeding the replication of the virus. Aided by a deeper understanding of the viral life-cycle, and the characterisation of the crystal structure of several viral proteins, the first reward for those efforts, in terms of the first approved DAAs, was in 2011 when two protease (NS3) inhibitors were approved for the treatment 11 of G1 HCV patients in the USA, Europe and Australia. Telaprevir (Incivek) and Boceprevir (Victrelis) were shown to increase SVR rates for G1b patients by 20-30% when administered with PEG-IFN/RBV.94-96 The largest developments in the field of DAAs occurred very recently with the approval of the first cross-genotypic inhibitor of HCV. Sofosbuvir (GS-7977), an RdRp nucleoside inhibitor, has shown very promising SVR rates amongst HCV patients of all genotypes (90%), although less effective against G3a.97-99 Sofosbuvir has been FDA approved for use in all HCV genotypes in combination with PEG-IFN/RBV.

1.4.2.1 NS3/4A protease inhibitors

The first protease inhibitor (PI) to be investigated in clinical trials was BILN-2061.100 Due to cardiac toxicity in animal models, however, further development was stopped101 and the compound has since been mainly used as a research tool in the development of new antivirals. BILN-2061 belonged to the first generation PIs; structurally diverse molecules which bind covalently to the active site of NS3, including the α-keto amides Telaprevir and Boceprevir. First generation PIs have displayed potent activity against G1b HCV, both in vitro and in vivo, however, the genetic “barrier” to resistance has been shown to be relatively low; single amino acid substitutions in NS3 are sufficient to confer resistance to these compounds.95 Resistant viral variants emerged upon treatment with both Telaprevir and Boceprevir, and were associated with viral breakthrough96, 102 defined as the rebound of HCV RNA and loss of response during antiviral therapy. A “second wave” of the first generation PIs was also developed. Unlike the first compounds described, these did not require covalent binding to the active site of the target enzyme and were therefore expected to have better tolerability features.103 These compounds include linear (Faldaprevir, Asunaprevir, Sovaprevir and GS-9451) and macrocyclic molecules (Simeprevir, Danoprevir, ABT-450, Vaniprevir and GS-9256), and are presently in phase II and III clinical trials (Figure 1.4). The most advanced compound, Simeprevir, has been recently approved to treat G1 patients, in combination with the PEG-IFN/RBV SOC. Molecules in this class appear to share similar resistance profiles, with mutations at amino acid R155 and D168 conferring resistance to almost all compounds within this family (reviewed in95). 12

Lastly, the term “second generation” was used to describe yet another wave of PIs which have recently been reported.103 These are distinct from previous inhibitors in that they have a higher barrier to resistance, and a wider coverage of HCV genotypes. Furthermore, these compounds are active against known PI resistant variants. Two of these compounds, MK-5172104, 105 and Neceprevir (ACH-2684)106 are undergoing early phase clinical trials (Figure 1.4).

1.4.2.2 NS5A inhibitors

NS5A is perhaps the most complex HCV-encoded protein in terms of functionality and structure. This “phosphoprotein” has been implicated in the modulation of a variety of roles, including RNA replication107, 108, virus assembly109, 110, interaction with a wide range of host factors111, and even regulation of the cell-cycle and hepatocarcinogenesis.112, 113 The discovery of the first NS5A inhibitors was somewhat unexpected. The lack of a defined enzymatic activity for NS5A meant different approaches than those used for NS3 and NS5B were needed to identify inhibitors, including assays using cells harbouring the HCV replicon. Specificity to NS5A was determined by the selection of resistant HCV replicon variants to these molecules, which had mutations in the NS5A coding sequence.114, 115 Although the mechanism of action for NS5A inhibitors remains poorly understood116, they have demonstrated the most potent antiviral activity amongst all HCV DAAs. For example, the most advanced NS5A inhibitor, Daclatasvir, displayed picomolar activity against HCV in cell culture, and was the first proof-of-concept for the clinical use of an NS5A inhibitors.117 Daclatasvir is currently in phase III clinical trials for the treatment of G1 HCV infections (Figure 1.4). However, this molecule has a very low barrier to resistance84, and displays loss of potency against other HCV genotypes. For instance, the EC50 value of Daclatasvir in cell culture was 9 pM for G1b HCV, 290 pM for G1a and 910 pM against G3a.84 This loss of efficacy was also shown when Daclatasvir was administered to G1a patients and compared to those infected with G1b HCV.118 Ledipasvir and ABT-267 are other NS5A inhibitors of the same generation that are also in phase III trials (Figure 1.4). Similar to PIs, a new generation of NS5A inhibitors have been described which are inhibitory against all known resistant variants, and are potent inhibitors of all HCV genotypes. These include GS-5861, ACH-3102 and MK-8742 13

(reviewed in116 ) which are all in phase II clinical trials (Figure 1.4). These drugs are among the most promising of the DAAs so far developed, and are likely to be a key component of a future HCV SOC.

1.4.2.3 NS5B RdRp inhibitors

Due to its vital role in the replication of HCV, the RdRp has long been a prime target for antiviral development. Furthermore, no mammalian enzyme homologue has been identified, reducing the possibility of off-target, undesirable effects for NS5B-binding molecules. Inhibitors of the HCV RdRp have been described which fall into two main categories; nucleoside and non-nucleoside inhibitors. Nucleoside inhibitors (NI) are nucleotide analogues which are incorporated into the nascent genome by the RdRp, and prevent further incoming nucleotide being incorporated, thus causing RNA chain termination. These inhibitors, including the recently approved HCV NI Sofosbuvir, are usually developed as “prodrugs”, requiring the phosphorylation by cellular kinases in order to produce the active triphosphate form. The difference between nucleoside and nucleotide inhibitors is that the latter are specifically targeted to the liver, requiring the activation by hepatic enzymes to produce their active form, therefore reducing systematic side-effects.29 Because all NIs target the active site of the polymerase, which is highly conserved, these inhibitors tend to be cross-genotypic, at least in vitro.119 Although single amino acid mutations are sufficient to confer resistance to this class of inhibitors, these mutations appear to have a fitness cost on the polymerase.120, 121 The NI Valopicitabine (NM283) was the first to demonstrate a proof-of-concept for the use of a polymerase inhibitor in the clinic for the treatment of HCV122, although its development was later halted due to gastrointestinal side-effects. However another cytidine derivative, Mericitabine is currently in phase II trials123, 124 (Figure 1.4). A large number of NIs have also been developed, but halted mainly due to toxicity.95, 121 Unlike NIs, non-nucleoside inhibitors (NNI) are allosteric inhibitors of the RdRp which are non-competitive with regards to the nucleotide substrate. Instead, the binding of these compounds to the RdRp inhibits conformational changes required for polymerase activity.125 HCV NNIs have been identified encompassing a diverse range of chemical scaffolds, reviewed in 126. These however have been found to bind the RdRp at 14 one of at least five allosteric sites. Two binding sites lie within the thumb subdomain; thumb I, to which compounds like benzimidazole and indole derivatives bind (e.g. Deleobuvir, BMS-791325 and TMC647055), and thumb II, which is the target site for thiophene-2-carboxylic acids127 like VX-222 (Lomibuvir) and GS-9669, as well as dihydropyranones such as PF-00868554 (Filibuvir).128 Two other allosteric sites have been characterised within the palm subdomain, distinct but in close proximity to the RdRp active site. The palm I site has been targeted by proline129, benzodiazepine130 and benzothiadiazine131 derivatives such as RG7790 (Setrobuvir), ABT-333 and ABT-072, whereas compounds that bound to palm II included benzofurans such as HCV-796 (Nesbuvir).132 Imidazopyridines, including the NNI GS-9190 (Tegobuvir) are another class of compounds that have been identified which uniquely bind to the palm site of the polymerase in close proximity to the active site, however in contrast to other palm binder, the binding involves an interaction with the β-hairpin which extends from the thumb into the palm domain (site P-β).133, 134 The first proof-of-concept for the clinical use of HCV NNIs was with the palm II inhibitor Nesbuvir (ViroPharma/Wyeth).132 However the development of Nesbuvir was later halted due to abnormal liver enzyme elevation. Other NNIs are now undergoing clinical trials mainly for the treatment of G1 infections, with limited data on their activity in non-G1 infections. The most advanced of these NNIs are the thumb I inhibitor Deleobuvir and the palm I inhibitor ABT-333, both of which are in phase III clinical trials (Figure 1.4). First generation HCV NNIs have a relatively low barrier to resistance; single mutations in the NS5B region have been associated with resistance to all identified HCV NNIs in vitro.103, 132, 135 Furthermore, mutations conferring resistance to NNIs were detected in patients infected with all HCV genotypes, even without NNI treatment.132 The influence of the existence of these viral variants on antiviral therapy remains to be examined.

Figure 1.4. Direct-acting antivirals in clinical development for the treatment of HCV. A summary of DAAs in different stages of clinical trials along with the developing companies is shown. Compounds targeting the NS5B inhibitors are divided into nucleoside (NI) and non-nucleoside inhibitors (NNI). Compounds that have been approved for clinical use are shown as green spheres. Red circles indicate compounds that are on hold or halted from development. Molecules in this study are shown as orange spheres.

1.4.3 Host-targeting agents

Rather than targeting HCV proteins, host-targeting agents (HTA) bind to one of the cellular proteins required for the entry/replication of the virus. Cyclophilins (Cyps), also known as peptidyl-prolyl cis-trans isomerases, are a group of proteins that falls under this category. Cyps were identified as cellular cofactors essential for HCV replication, most likely via interactions with the RdRp and the NS5A protein.136-138 The immunosuppressive drug cyclosporin A (CsA) has been shown to have anti-HCV activity139, which was linked to binding to Cyps. Non-immunosuppressive derivatives of CsA, including Alisporivir, were also shown to have antiviral activity both in vitro126, 127 and against multiple genotypes in vivo.140-142 143 Alisporivir has progressed to phase III clinical trial, however, its development has been put on hold due to a number of acute pancreatitis cases in clinical trials, including one fatality. Another HTA in clinical development is Miravirsen, an antagonist for microRNA (miR-122), a liver-specific miRNA involved in the regulation of cholesterol synthesis genes.144, 145 Two miR-122 binding sites were identified in the 5´ UTR of the HCV genome146 and HCV was found to utilise miR-122 for several purposes, including viral protein translation147, 148 and the stabilisation of genomic RNA to prevent its degradation.149 By targeting miR-122, Miravirsen inhibited recombinant viruses which contained the 5´UTR from all HCV genotypes in vitro.150 The compound also reduced vireamia in HCV-infected chimpanzees151 and in infected patients152, and the antiviral is currently in phase II clinical trials. Since these compounds target host factors, resistance attributed to the genetic diversity and high mutation rate of HCV could be overcome. Furthermore, HTAs are more likely to have pan-genotypic HCV activity.153 The obvious caveat, however, is the likely undesirable side-effects accompanied by targeting a host factor to inhibit viral replication and, therefore, the development of HTAs will need to be evaluated through further clinical studies.154

1.4.4 DAA combinations

The treatment of HCV infections is likely to change significantly over next 5-10 years. With the availability of new therapeutic molecules in 2011, the SOC for G1 has already been modified to include new DAAs, and changes in the SOC for the remaining genotypes will likely follow as the new DAAs target a wider range of genotypes. However, PEG-IFN remains an essential part of all current regimens. Due to its side effects, the ultimate goal for HCV therapy is an IFN-free based SOC. Several DAA combinations, containing protease, polymerase and NS5A inhibitors are currently in late stage clinical trials, with and without ribavirin, and the recently published data from these trials is encouraging (reviewed in 155). However, issues including drug resistance and drug interactions need to be addressed, and their efficacy for the treatment of non- G1 genotypes also remains largely unexamined.

Genotype 3 HCV; an understudied virus

HCV G3 represents nearly a fifth of all infections worldwide (section 1.3.2), and growing evidence suggests that this particular genotype is clinically distinct from other HCV variants. It is well established that infections with HCV G3 are associated with a significant increase in the incidence of hepatic steatosis (fatty liver)156-158, and the accelerated progression to fibrosis.158-160 Although this association is not fully understood, several mechanism have been suggested.161 For instance, HCV core protein has been shown to interfere with lipid metabolism in cell cultures162, and amino acid substitutions in the G3 core have been suggested to have higher affinity for lipids than other genotypes.163 Despite its prevalence and clinical significance, G3a remains relatively understudied compared to G1 and G2 HCV for a number of reasons. Firstly, G1 remains the predominant genotype in most developed countries (~75%), particularly in developed countries (section 1.2).164 Secondly, for decades HCV G3 was regarded an easy to treat genotype, as G3 infected patients were better respondents to the IFN- based therapies (near 80% SVR rate), compared to G1 patients (~50% SVR).91 Thirdly, until very recently (2013) models for studying HCV were largely limited to the G1 and G2 replicon models, and the G2 infectious model (section 1.3.3). For the most part, the

relative lack of G3 targeted studies had significantly impacted the development of G3 DAAs. For example there are no reports describing DAAs where a G3 has been the main target for the inhibitor rather than G1. Instead, antiviral development has focused on the “harder to treat” G1 infections, with the hope that the newly discovered molecules will have cross-genotypically inhibitory activity. This in turn has been a major factor in the poor response rates of G3 patients towards developed antivirals, at least to the first generation PIs, NIs and NS5A inhibitors (Table 1.2). This was not surprising given that these DAAs demonstrate weak inhibition of G3 in vitro. Of the newly approved protease inhibitors, Telaprevir was shown to be significantly less active against G3a in vitro83, 165 and had negligible activity when administered to G3a infected patients.166 There is limited clinical data on the activity of the other approved PIs, Boceprevir and Simeprevir, in G3a patients; however, in vitro studies indicate that they are significantly less effective against G3 HCV, along with sister molecules in clinical development such as Danoprevir and Vaniprevir.83, 165 Daclatasvir, the most advanced NS5A inhibitor, was less active against G3a when compared to other genotypes both in vitro84, 167 and in vivo168, although improved SVR rates when combined with the Peg-IFN/RBV as a triple therapy.168 Perhaps the biggest surprise however was the significant reduction in response to the most advanced NI, Sofosbuvir, in G3a patients. Although in vitro data suggested that Sofosbuvir, similar to other NIs, was cross-genotypic169, when given to patients Sofosbuvir was significantly less effective against G3 compared to all other genotypes.98 G3 patients were even less responsive than patients with G2 HCV infection (62% SVR rate for G3 vs 94% for G2170, and 56% for G3 vs 97% for G298). However, like the NS5A inhibitor Daclatasvir, the addition of Sofosbuvir to the current IFN-based SOC in a small trial of 10 patients improved response rates for G3 patients (Table 1.2). In a time where IFN-free regimens are the next development of HCV therapies, the options for the treatment of G3 patients remain limited. Therefore, there remains an urgent need for new antivirals for G3 infections that account for an estimated 33.1 million people171, which is around the same number of HIV infections worldwide (reviewed in 161).

Table 1.2. Clinical trials of DAAs involving G3a HCV. Treatment SVR rate Reference Telaprevir monotherapy 50% 166 Telaprevir + PEG-IFN + RBV 67% PEG-IFN + RBV 44% Daclatasvir + PEG-IFN + RBV 69% (12 weeks) 168 70-78% (16 weeks) PEG-IFN + RBV 52-59 % (24 weeks) Sofosbuvir + RBV 56% 98 PEG-IFN + RBV 63% Sofosbuvir + RBV (POSITRON) 61% 170 Sofosbuvir + RBV (FUSION) 30% (12 weeks) 62% (16 weeks) Sofosbuvir monotherapy 60% 97 Sofosbuvir + PEG-IFN + RBV (10 patients) 100% Alisporivir + RBV 81-94% 143 Alisporivir + PEG-IFN 77-86% PEG-IFN + RBV 58-74%

2 General introduction part II: Norovirus

Background and history of NoV

Gastroenteritis is one of the most common causes of morbidity and mortality worldwide. Every year, nearly 4.6 billion cases of diarrhoeal illness are recorded globally, causing up to 2.2 million deaths.172 Gastroenteritis accounts for 18% of deaths in children under 5 years of age172, 173, as many as those caused by respiratory diseases. In the developed world, the burden of gastroenteritis is mainly economic.174-176 The total costs for gastroenteritis-related illness is over $340 million in Australia174, and around $3 billion in the United States177, in direct healthcare expenses and loss of productivity. Approximately 70% of acute gastroenteritis cases are caused by viral pathogens, whereas bacteria and parasites account for 20% and 10% of this illness, respectively.178 The two most prevalent viral pathogens are rotavirus (RV) and norovirus (NoV).178 However, since the introduction of the rotavirus vaccine, a sharp decline in RV infections and RV-related hospitalisations has been observed.179-181 For this and other reasons, NoV has become the leading cause of gastroenteritis globally (Approximately 50%).182 Furthermore, NoV is estimated to be the cause of over 200,000 deaths every year, the majority of which are children in developing countries.183 Before the discovery of NoV in 1968, the exact aetiology for what was described as “winter vomiting disease” was not fully understood. Several transmissibility studies, in which stool filtrate of infected patients was orally administered to volunteers, established that a filterable, non-bacterial agent was a cause of transmissible acute gastroenteritis.184-186 A similar study was then conducted by analysing samples collected from an outbreak that had occurred in 1968 at a school in Norwalk, Ohio, USA in 1972. Using electron microscopy, Kapikian et al. observed a 27-nm particle, later called the Norwalk virus, which became the prototype strain of the genus Norovirus.187

Transmission and pathogenesis

NoV transmission occurs primarily from person-to-person through the faecal-oral route, ingestion of aerosolised vomitus, or by exposure to contaminated objects and surfaces.188 Transmission through contaminated food and water has also been documented.189 NoV is highly infectious; as few as 18 virions have been reported to establish an infection190, although more recent estimates of the NoV infectious dose are between 1,320 and 2,800 virions.191 This is combined with high levels of shedding during acute illness192; approximately 100 million virions per gram of faeces.193 Viral shedding in NoV infected individual is not limited to the symptomatic phase of the illness, and patients continue to shed for four (up to eight) weeks after infection.192, 194 Viral shedding could also occur in the absence of any symptoms, and has been documented in up to 26% of surveyed individuals.195 Because of its rapid spread, NoV is a major problem in semi-closed environments like hospitals, nursing homes, schools, restaurants and cruise ships.188, 196 Following an incubation period of 1-2 days, the clinical features of NoV infection include acute onset of nausea, vomiting, abdominal cramps, headaches and diarrhoea that generally last for 2-4 days.197 However, chronic infections are increasingly recognised as a subset of NoV infections, where severe and prolonged illness is observed in immunocompromised patients, although the prevalence of chronic NoV infections in the population is unknown.198-200

Molecular biology of NoV

2.3.1 NoV classification and genome organisation

NoV is classified into the family Caliciviridae under its own genus; Norovirus, along with the genera; Sapovirus, Lagovirus, Nebovirus and Vesivirus. The genome of NoV was cloned from the stool of volunteer patients infected with the filtrate containing the virus.201, 202 This work revealed a positive sense, single-stranded RNA genome of approximately 7.5 kb in length. The genome in uncapped and lacks an internal ribosome entry site. However, a 15 kDa protein (VPg) is covalently linked to the 5´ end of the genome which is thought to facilitate the initiation of translation and replication of the NoV RNA193, 194 (Figure 2.1). The genome contains three ORFs; ORF1 encodes a 200 kDa polyprotein which is cleaved by the viral protease203 into seven non-structural (NS) 22

proteins (NS1-NS7), including VPg (NS5), a 3C-like protease (NS6) and an RdRp (NS7). ORF2 and ORF3 encode the major capsid protein (VP1) and a minor basic protein (VP2), respectively202 (Figure 2.1). Translation of genomic and subgenomic RNA is facilitated by protein-protein interaction with the host proteins eIF3 and eIF4 which help recruit the cellular translation machinery.204, 205 Furthermore, the genome contains short untranslated regions at the 5’ end, and at the 3’ end the genome is polyadenylated. Another feature of caliciviruses genomes is the presence of a conserved 20-base sequence near the 5’ terminal end of the genome that is repeated internally at the ORF1/ORF2 overlap (Figure 2.1). This sequence has been suggested to serve as a promoter for subgenomic RNA production by the RdRp.206

Figure 2.1. The genome organisation of NoV. The genome of NoV contains three open-reading frames (ORFs), with a 20-base overlap between ORF1 and ORF2. ORF1 (grey) encodes for a polyprotein which is cleaved into non-structural proteins NS1-NS7, whereas ORF2 and ORF3 (green) encode the major (VP1) and minor (VP2) capsid proteins, respectively.197

2.3.2 Genetic diversity of NoV

Like HCV, the error-prone RdRp results in extensive genetic diversity across the Norovirus genus. NoV has therefore been classified into six different genogroups (GI-GVI, Figure 2.2) based on amino acid sequences of the major capsid protein (VP1).207 VP1 sequences of the genogroups differ by approximately 45% at the amino acid level. The genogroups have been further classified into genotypes, with around 14% divergence in the VP1 sequence.208 Only three genogroups have so far been associated with human NoV infections (GI, GII and GIV).208, 209 Genogroup III infects cows and sheep 210, 211, GV is a murine virus212 whilst GVI is canine213 (Figure 2.2). Genogroup II, which is associated with human infections, has also been found infect pigs210, 214, and GIV was also associated with enteritis in lions.215 23

Figure 2.2. The diversity of noroviruses infecting a range of hosts. Amino acid sequence analysis of the NoV capsid region was performed using MEGA44 and the tree was generated using the Neighbour-Joining method The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site, indicated by the scale bar. Bootstrap values are provided as percentages of 1000 replicates for values ≥ 70%. NoV genogroup numbers and their typical hosts are indicated.

2.3.3 NoV Genome replication and the RNA-dependent RNA polymerase

Like HCV, the replication of the NoV genome is thought to be associated with host membranes, and studies with murine norovirus (MNV) suggest that viral replication occurs in the perinuclear region, in association with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes.216 Like other RNA viruses, the genome is first transcribed into a negative-sense RNA, which serves as a template for the production of positive-sense genomic and subgenomic RNA molecules. Double stranded RNA intermediates could be observed throughout the course of the infection in association the NoV RdRp in infected cells.216 The mechanism by which the NoV RdRp initiates the replication remains unclear. A number of studies have used recombinant NoV RdRp to characterise its biochemical properties in vitro.217-219 Like the HCV RdRp, the NoV RdRp is capable of both de novo and primed extension of nascent RNA. However, is has also been shown to uridylate the VPg protein, which could then be used to prime the transcription from the polyadenylated RNA.220 The X-ray crystal structures of RdRps from human GI, GII and the GV mouse NoV have been solved.217-219 Like the HCV RdRp (section 1.3.3) the NoV polymerase has a typical right hand conformation where the fingers and thumb domains encircle the active site in the palm domain (Figure 2.3). Unlike its HCV homologue, however, the NoV RdRp does not have the β hairpin extending from the thumb into the palm subdomain. Instead, the NoV RdRp possesses a C-terminal tail which, in the absence of an RNA template, extends into the active site of the RdRp.218 However, it was later found that displacement of the C‐terminal tail away from the active site is triggered by dsRNA binding.221 These structural changes resemble the HCV RdRp rearrangements upon switching from the “closed” de novo conformation to the open primer-extension one (see section 1.3.3). The NoV RdRp likely functions as a dimer, where the interaction of monomers increases the overall polymerase activity.219, 222 More recently, the enzyme from human GII.4 pandemic NoVs was shown to be phosphorylated by the cellular kinase Akt.223 While the effect of this phosphorylation in vivo remains unclear, the phosphorylation occurs at a site which appears to be critical for the enzymatic activity in vitro, and is interestingly absent in non-pandemic NoV RdRps.223

Fingers Thumb

C-terminus

Palm

Figure 2.3. The crystal structure of the norovirus RNA-dependent RNA polymerase. The NoV RdRp structure is shown, highlighting the fingers (red), thumb (blue) and palm (green) subdomains. The enzyme is crystallised in a conformation where the C-terminal tail extends into the palm subdomain, although thought to be displaced away from the active site upon binding to dsRNA221 (enzyme PDB 1SH0).

2.3.4 Molecular epidemiology and diversity of NoV – the GII.4 pandemic lineage

Although the GI.1 Norwalk prototype was the first identified NoV, it has become clear that viruses which belong to the GII.4 genotype are of particular significance. Variants of the GII.4 genotype have caused all major NoV pandemics of acute gastroenteritis in the last two decades, and account for over 80% of all reported human NoV infections.192, 224, 225 Furthermore, analysis of the NoV genome from archival samples suggests that GII.4 strains have been in circulation for at least three decades.226 Similar to the epidemiological patterns of influenza A, new variants of NoV arise every two to three years and are associated with global epidemics.227 To date, six NoV pandemics have been recorded which were all associated with GII.4 variants. The first one to be fully characterised was in 1996 and the virus was termed US 1995/96.228 It was followed by the variant Farmington Hill in 2002229, Hunter virus in 2004230, Den Haag 2006b virus between 2007–2008192 and New Orleans virus from 2009 to 2012.231 More recently, a new GII.4 pandemic strain termed Sydney 2012

was identified.224, 232, 233 Sydney 2012 was associated with a pandemic of acute gastroenteritis in late 2012, early 2013 and remains the dominant NoV to-date.231 Two genetic mechanisms are thought to contribute to the evolution of NoV and the emergence of new epidemic strains. Firstly, genetic drift occurs by mutations within the capsid region of the genome, mainly within the protruding P2 domain, which contains several antibody neutralising epitopes (A–E) as well as the histo-blood group antigen (HBGA) binding domain.234-237 Recombination is another well documented drive of NoV evolution233, 238, and mostly occurs within the ORF1/ORF2 overlap (Figure 2.1), which allows the exchange of non-structural and/or structural genes between different NoV lineages. At least two of the six pandemic strains are known to have arisen from recombination events within the GII.4 lineage; namely New Orleans 2009 and the recent Sydney 2012.224, 233 The continuous evolution of NoV is mainly attributed to herd immunity and the high mutation rate of the NoV RdRp235, 236, and poses significant challenges for the development of control measures such as vaccines and DAAs (section 2.4).

2.3.5 Models of NoV replication

Significant effort has been made to identify a permissive cell line for human NoV, however these attempts have largely failed.239 The lack of a cell culture system for human NoV has hindered replication studies and the identification of human NoV therapeutic agents. In 2003, MNV was identified212, which led to the development of the first cell culture system and small animal model for NoV infection.240 Wobus et al. demonstrated that MNV replicates in cells of mononuclear origin, such as primary dendritic cells and macrophages.240 Building from this work with MNV, recently it has been shown that BALB/c Rag-γc-deficient mice could also support the replication of human GII.4 NoV.241 Another important breakthrough towards the establishment of a human NoV cell culture model was the development of a human sub-genomic replicon based on the prototype NoV GI strain, Norwalk virus.242 The replicon consists of the complete ORF1, encoding the replicative enzymes (NS1-7), while ORF2, which encodes the major structural protein, is disrupted by the insertion of a neomycin resistance gene. This

allows replication of autonomous RNA in Huh-7 cells without the expression of viral structural genes and, therefore, no virions are produced.242 The inability to grow human NoV in cell culture is mainly attributed to the lack of understanding of its tropism197, and the absence of an identified receptor for viral entry. Although HBGAs are implicated as entry factors for NoV (Section 2.4.1), over-expression in human hepatoma cells was not sufficient for a full infectious cycle243, suggesting that other factors/co-receptors remain to be unidentified. Undoubtedly though a GII replicon, or a fully infectious NoV GII cell culture system, would drive further research into the NoV life-cycle and provide invaluable tools with which to identify and develop antiviral agents against the virus.

Management of NoV infections

There are no vaccines or antiviral agents for the prevention and/or control of NoV infections. The current recommendations for NoV prevention include the implementation of good sanitation and hygiene measures, such as hand-washing and surface decontamination.188 NoV particles, however, have been shown to be resilient to multiple commonly used disinfectants.244, 245 The main intervention for acutely infected individuals consists of fluid and electrolyte therapy246, and isolation of patients to prevent further person-to-person transmission.188 There are no control strategies for chronically infected patients, and therapies based on antibody immunisation and commonly used antivirals have largely failed.198 Given the global health burden of NoV, and the continuous emergence of pandemic strains, there is an urgent need for specific approaches to control NoV infections either through vaccines or antivirals.

2.4.1 NoV vaccines

In the absence of an efficient cell culture or animal model, vaccine development efforts for NoV had to rely on recombinant subunit protein expression to produce antigens as vaccine candidates, particularly focusing on the capsid protein. The expression of recombinant NoV VP1 directs the self-assembly of virus-like particles (VLPs), which can induce NoV-specific antibodies in animal models247, and in human volunteers.248 Similar to other caliciviruses, NoV VLPs can agglutinate human and chimpanzee red blood cells by binding to HBGAs249, 250, and sera from NoV patients can 28

block the ability of VLPs to bind HGBAs in vitro.251 One of the enzymes responsible for the expression of HGBAs on the surface of epithelial cells and in mucosal secretions is fucosyltransferase 2 (FUT2). Interestingly, a naturally occurring genetic mutation, which ablates the activity of FUT2, was demonstrated to confer complete resistance against NoV infection in challenged volunteers.252 Taken together these findings suggest that interfering with the NoV binding might be an effective way to protect against infection. When VLPs generated from GI and GII NoVs were administered to chimpanzees, all animals were protected against a following challenge for up to 18 months after vaccination.253 Today, two VLP-based vaccine candidates are in early phases of human clinical trials. The first is an intranasal monovalent GI.1 vaccine (Takeda Pharmaceuticals Co.)254 where volunteers were given the vaccine and then challenged with the virus or given a placebo. The results, however, were not significant; 37% of vaccinees developed gastroenteritis compared to 69% of the placebo recipients. Takeda is also developing an intramuscular bivalent (GI.1/GII.4) vaccine; however, this was less effective than the intranasal monovalent vaccine (52% infections in vaccinees compared to 60% in placebo recipients).255 A more recent approach to NoV vaccine development has been the use of NoV P particles; multimeric structures that assemble spontaneously upon expression of the protrusion (P) domain of the VP1 protein in vitro.256 Although not trialled in humans as of yet, these particles were shown to illicit innate and adaptive immunity against NoV in mice257 and sera from vaccinated animals blocked NoV VLP binding to HBGA.256 Despite these advances, there remain further challenges for the prospects of an effective NoV vaccine; namely, the extensive genetic diversity and the continuous evolution of human NoVs. There is evidence of evolutionary changes within the antibody epitopes of the pandemic variants of GII.4 NoVs, and antibodies that bind to one GII.4 variant have poor cross-reactivity to different variants.235 This is also true for NoVs that belong to different genotypes or genogroups.258 The continuous evolution of NoV, and the emergence of new pandemic strains every 2-3 years, implies that one vaccine for all will not be plausible, and that a new formulation will be required at least every few years, in a similar approach to influenza A vaccine strategies.237

2.4.2 Emerging NoV antivirals

The search for antivirals against NoV is still in its infancy, and there are only a handful of publications reporting NoV DAA development (reviewed in reference 259). The most advanced pre-clinical studies have so far focussed on protease inhibitors260, 261 and on repurposing available RdRp inhibitors developed to treat other viral262-264 and non-viral infections.297 Using structure-based design approaches, Tiew et al. synthesised a series of peptidyl protease inhibitors.261 Derivatives were later developed from these scaffolds with inhibitory activity (EC50 values), between 0.2 µM and 2.8 µM, as assessed using the NoV GI.1 replicon model. These compounds were shown to have broad antiviral activity, inhibiting homologous proteases of coronaviruses and picornaviruses.260 Two classes of Nis, originally developed for the treatment of other viral infections262-264, have also been shown to inhibit the NoV RdRp. The nucleoside analogue 2´-C-Methylcytidine (2CM), developed as an inhibitor of viruses within the Flaviviridae family including HCV, has been shown to inhibit NoV replication in vitro (replicon and

MNV infectious cell culture system) with EC50 values between of 1.3 µM and 18 µM, respectively.263, 264 Recently, the antiviral activity of 2CM has also been demonstrated in mice infected with MNV, where it prevented NoV-induced diarrhoea, mortality, and even protected mice against a rechallenge of NoV.265 The use of 2CM in humans however remains in some doubt as clinical trial using Valopicitabine, an oral prodrug of 2CM, were halted due to adverse gastrointestinal effects in HCV infected patients (section 1.4.2.3). T-705 (Favipiravir) is another nucleoside analogue with broad inhibitory activity against viral RdRps, although originally developed as an influenza inhibitor.266 T-705 was found to inhibit MNV replication262, but only at high concentrations (EC50 of 250 µM compared to 0.2 µM for influenza). Using available crystal structures, Mastrangelo et al. 297 targeted the active site of the NoV RdRp with an in silico approach to identify inhibitors from a panel of commercially available compounds. Two molecules were identified as potential NoV NNIs; Suramin, a drug used in the treatment of sleeping sickness caused by the protozoan Trypanosoma, and its analogue NF023. These relatively large compounds inhibited the NoV RdRp at nanomolar concentrations, with IC50 values of 24.6 nM and

71.5 nM for Suramin and NF023, respectively. 297 The studies described above have so far been limited to the use of known viral polymerase inhibitors repositioned against a new viral target – the NoV RdRp, or alternative uses for existing drugs. Therefore, no new compounds have yet been explored as potential inhibitors of the NoV RdRp. Compounds targeting other factors necessary for NoV entry/replication have also been described.267 Since the identification of HGBAs as attachment factors for NoV, several compounds have been shown to target the HGBA-binding site on the NoV capsid.268-270 However the inhibitory activity of these compounds against an infectious virus is yet to be examined. Targeting host factors is also a potential strategy for NoV antiviral development. Perry et al.271 recently showed that the cellular deubiquitinase (DUB) is essential for NoV replication, and DUB inhibitors significantly reduced NoV replication in vitro and even protected mice from MNV infection.271 Other host factors which have been implicated in NoV replication provide potential targets for antiviral therapies, although none have been tested for NoV (reviewed in 267).

Aims:

HCV and NoV are significant human pathogens posing a substantial health and economic burden in developing and developed countries. Controlling the spread of these viruses through the development of both vaccines and antivirals has proved to be difficult, partly because of the refractoriness in cultivating these viruses in cell culture. The standard treatment for HCV (PEG-IFN/RVN) is expensive, poorly tolerated, and is only partly effective. Furthermore, despite recent advances in HCV drug discovery, little is known about the potential use of most of the new antivirals for the treatment of HCV G2-G6 infections. Initial data is indicative of poor response by G3 patients to PIs developed for G1, and the NI Sofosbuvir when administered without IFN. The traditionally “easy to treat” HCV G3 is now becoming the most difficult to treat genotype with antiviral therapy, and the treatment options remain limited to IFN-based therapies. A better understanding of the antiviral activity of G1 HCV DAAs against other HCV genotypes is therefore required. Furthermore, new effective molecules are needed for the treatment of G2-G6 HCV infections, for which antiviral development lags significantly behind. The highly infectious nature of NoV, its ability to cause outbreaks in hospitals, age-care facilities and cruise ships, highlights the need for specific approaches to control NoV infections either through vaccines or antivirals. The development of a NoV vaccine has so far been limited by poor protection rates following rechallenge.272 Conversely, thus far no novel molecules or scaffolds have been identified as the first step towards specific NNIs to the NoV RdRp, a key target for antiviral development.

The overall aims of this study are:  To develop a simple, inexpensive method for the detection of HCV G3a RdRp activity in vitro that can be utilised for the high-throughput identification of novel NNIs, and to evaluate these inhibitors and their derivatives using enzyme and replicon systems (chapter 4).  To examine the cross-genotype activity of representatives from five classes of HCV NNIs, and to understand the mechanism of action for each class against the RdRp (chapter 5).  To conduct a high throughput screen for the identification of small molecule inhibitors of the GII.4 NoV RdRp, and to examine the identified NNIs and their derivatives using enzyme, replicons systems, and a fully infectious MNV cell culture system (chapter 6).

3 General Materials and methods

Materials

Table 3.1. General reagents and kits used in this study.

Reagent Source and details Bacterial strains E. coli BL21 Bioline (BIO-85031) E. coli α-Select Gold Bioline (BIO-85027)

Kits BigDye Terminator sequencing kit Applied Biosystems (4337458) CellTiter-Blue cell viability assay Promega (G8080) ExoSAP-IT PCR product clean-up Affymetrix (78250) HisTrap HP purification columns GE Healthcare (17-5247-01) Immobilon Western HRP Substrate Kit Millipore (P36599A) iProof High Fidelity PCR kit Bio-Rad (172-5330) iTaq Universal SYBR Green Supermix Bio-Rad (172-5120) Luciferase assay System Promega (E1500) QIAamp Viral RNA kit Qiagen (52904) QIAprep spin plasmid miniprep kit Qiagen (27104) Quant-iT™ PicoGreen dsDNA assay kit Life Technologies (P11496) Quant-iT™ RiboGreen RNS assay kit Life Technologies (R11490) RNeasy Mini RNA extraction kit Qiagen (74104) Slide-A-Lyser dialysis cassettes Thermo Scientific (66380) SuperScript III One-Step RT-PCR System with Life Technologies (12574030) Platinum Taq High Fidelity SuperScript VILO™ cDNA Synthesis Kit Life Technologies (11754050)

Reagents [8-3H]-Guanosine-5'-triphosphate ([3H]GTP) PerkinElmer (NET1201) 2’-C-Methylcytidine (2CM) Sigma-Aldrich (M4949) 2-Mercaptoethanol Sigma-Aldrich (M3148) 3'-Deoxyguanosine-5'-Triphosphate (3' dGTP) TriLink (N-3002) Agarose for nucleic acid electrophoresis Bio-Rad (161-3100)

Reagent Source and details Ampicillin Sigma-Aldrich (A9518) Benzonase Nuclease (250 U/µL) Sigma-Aldrich (E1014) Bovine serum albumin (BSA) Sigma-Aldrich Dimethyl Sulfoxide (DMSO) Sigma-Aldrich (D4540) Diothiothreitol (DTT) Promega (P1171) Dulbecco's Modified Eagle Medium (DMEM) Life Technologies (11965-118) Foetal Bovine Serum (FBS) Sigma-Aldrich (12003C) G418 (Geneticin) Life Technologies (10131035) GelCode Blue protein stain Thermo Scientific (24594) GlutaMAX media supplement (100×) Life Technologies (35050-061) Glycerol Sigma-Aldrich (G5516) Herring sperm DNA (10 mg/mL) Promega (D1811) Imidazole Sigma-Aldrich (I5513) Isopropyl-β-D-thiogalactopyranoside (IPTG) Sigma-Aldrich (I6758) Luria Bertani (LB) broth Life Technologies (12795-027) Lysozyme Sigma-Aldrich (L7651) Methanol Univar (A2314) Microscint Scintillation Fluid O PerkinElmer (6013611) Nickel sulphate Sigma-Aldrich (656895) n-octyl glucopyranoside Sigma-Aldrich (O8001) Penicillin-Streptomycin Life Technologies (15140-122) Polycitidylic acid (poly C) Sigma-Aldrich (P4903) Precision Plus protein standard Bio-Rad (161-0375) Protease inhibitor cocktail Sigma-Aldrich (P8849) Ribonucleotide triphosphates (rNTPs, 10 mM) Promega (P1221) SOC medium Bioline (BIO-86033) Trichloroacetic acid (TCA) Sigma-Aldrich (T6399)

Buffers Tris/Glycine/SDS buffer (10×) Bio-Rad (161-0732) Tris/Glycine (10×) Bio-Rad (161-0734) Tris-HCl (pH 7.5) (1M) Life Technologies (15567-027) Phosphate-buffered saline (PBS) Life Technologies (14080-055)

Reagent Source and details Tris/Borate/EDTA (TBE) buffer Life Technologies (15581-044) Tris/Acetate/EDTA (TAE) buffer Life Technologies (24710-030) Bacterial lysis buffer (CelLytic B, 10×) Sigma-Aldrich (C8740) Passive lysis buffer for mammalian cells (5×) Promega (E1941)

Protein binging buffer 50 mM Mono-Sodium Phosphate, 50 mM Di-Sodium Phosphate, 500 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol and 0.1% n-octyl glucopyranoside

Protein storage buffer 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 20% (v/v) glycerol and 0.1 mM DTT

SDS gel sample buffer (2×) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 20% (v/v) glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol

General methods

3.2.1 Nucleic acid purification and reverse transcription

Plasmids were purified from bacterial cultures grown overnight in Luria-Bertani (LB) broth with 50 μg/mL ampicillin using QIAprep spin plasmid miniprep kit (Qiagen). Total RNA was extracted from replicon-bearing cells using the RNeasy Mini kit (Qiagen), and from virus-infected cells using QIAamp Viral RNA Mini kit (Qiagen). For sequencing, cDNA was synthesised using SuperScript® III One-Step RT-PCR System (Life Technologies), whereas first-strand cDNA for use in quantitative RT-PCR (qRT-PCR) was performed using a SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies) following the manufacturer’s instructions.

3.2.2 DNA sequencing

Sequencing of plasmids or PCR products was carried out using BigDye® Terminator v3.1 cycle sequencing kit (Life Technologies). Briefly, 100 ng – 500 ng (plasmid) or 20 ng – 50 ng (PCR) DNA was added to a 20 µL sequencing reaction which also contained 1 µL BigDye® Terminator v3.1 reaction mix, 3.5 µL 5× sequencing buffer and 3.2 pmol primer. The reaction was run for 25 cycles at 96°C for 10 secs, 50°C for 5 secs and 60°C for 4 mins. Products were precipitated with 5 µL EDTA (125 mM) and 60 µL 95% ethanol, Incubated at room temperature for 15 mins and centrifuged for 20 mins at 14,000 g. Supernatant was removed and pellets were washed with 70% ethanol, centrifuged for 15 mins at 14,000 g and submitted to The Ramaciotti Centre for Gene Function Analysis at the University of New South Wales (Sydney, Australia) where it was run on an ABI 3730 DNA Analyser (Applied Biosystems).

3.2.3 Recombinant protein expression and purification

Recombinant HCV and NoV RdRps were expressed in E. coli and purified using affinity chromatography. All RdRps were previously cloned into the expression vector pTrcHis2C (Invitrogen), and contained a hexahistidine tag at the C-terminus.222, 273 The HCV RdRps were cloned removing the 21 amino acid residues (∆21) from the hydrophobic C-terminal region.273 Overnight cultures of E. coli BL21 (5 mL, grown in LB broth containing 50 μg/mL ampicillin) were diluted 1:100 in LB broth containing ampicillin and incubated for 3-4 h at 37°C, or until the OD600 reached 0.5. Expression of 38

the RdRp was then induced with 1 mM isopropyl- β-D- thiogalactopyranoside (IPTG) and the culture incubated for 16 h at 22°C. Cells were collected by centrifugation at 6,000 g for 15 min at 4°C, washed with phosphate-buffered saline (PBS) and the bacterial pellet stored at -80°C until use. For RdRp purification, bacterial pellets were re-suspended in CelLytic B lysis reagent (Sigma-Aldrich) prepared with RdRp binding buffer (section 3.1) containing 50 mM (HCV) or 10 mM (NoV) imidazole. This was followed by the addition of 0.2 mg/mL Lysozyme, 1:200 (vol/vol) protease inhibitor cocktail and 50 U/mL benzonase nuclease (Sigma-Aldrich). Resuspended cells were incubated at room temperature for 20 mins to allow for chemical lysis before the lysate was clarified by centrifugation at 16,000 g for 30 min at 4°C. The supernatant was passed through a 0.22 µM polyethersulfone filter, then applied to a Ni2+ charged HisTrap HP column (GE Healthcare). Once bound, the column was washed with binding buffer containing 50 mM (HCV) or 20 mM (NoV) imidazole. The RdRp was then eluted by increasing the imidazole concentration to 500 mM and collecting multiple elution fractions. The purity of RdRp was assessed by sodium dodecyl sulphate (SDS) PAGE (section 3.2.4) Fractions containing the recombinant protein were pooled and dialysed against storage buffer using Slide-A-Lyser dialysis cassettes with a 10 kDa cut-off membrane. Dialysis cassettes were placed in 1 L storage buffer and kept at 4°C overnight while stirring. Storage buffer was refreshed on the following day and the protein was dialysed for further 2 h. Samples were then removed from the dialysis cassettes and the protein concentration was quantified via spectrophotometry. The RdRp was then aliquoted and stored at -80°C.

3.2.4 Protein electrophoresis and western blotting

Protein samples were resolved using SDS-PAGE. Samples were mixed with equal volumes of sample loading buffer and loaded onto a 10% SDS PAGE (Bio-Rad). Gels were run in Tris/Glycine/SDS buffer for 90 min at 110 V. For staining, gels were washed with distilled water for 40 minutes and stained with GelCode Blue (Thermo Scientific). Western blotting was performed for the detection of the histidine-tagged recombinant proteins. Proteins were transferred from the gel onto a nitrocellulose membrane by electrophoresis (60 min at 200 V) in Tris/Glycine buffer containing 20% (v/v) methanol. The membrane was blocked at room temperature for 1 hr in 5% (w/v) skim milk, 39

dissolved in PBS with 0.1% (v/v) and Triton X-100 (PBS-T). The membrane was washed with PBS-T for 30 min, and a 1:20,000 dilution of hexahistidine antibody conjugated to horseradish peroxidase (HRP) was added. After 1 h the membrane was with PBS-T for 30 min and the HRP substrate was added (Immobilon Western Chemiluminescent HRP Substrate, Millipore). Detection was performed using Amersham Hyperfilm ECL (GE Healthcare).

3.2.5 Quantitative RdRp assays

Standard quantitative RdRp reactions contained 6-40 μg/mL poly(C) RNA

(average length 300 nucleotides, Sigma-Aldrich), 2.5 mM MnCl2, 5 mM DTT in and 0.5 mM rGTP (Promega) in 20 mM Tris-HCl (pH 7.5), unless stated otherwise. Reactions were initiated by the addition of 300 nM (HCV) or 13.3 nM (NoV) RdRp. For the fluorescent-based quantitative assay, the synthesis of double stranded (ds) RNA was determined using the commercially available fluorescent dye PicoGreen (Invitrogen) which was developed for the quantitation of dsDNA, but found to also preferentially bind dsRNA compared to ssRNA (chapter 4). Reactions were stopped with 5 mM EDTA and PicoGreen, freshly diluted in TE buffer (pH 7.5), was added in 50 μL of 1:230 dilution for a 384-well plates, or 165 μL of 1:680 dilution for a 96-well plates. The mixture was incubated for 5-8 mins at room temperature protected from light. Fluorescence intensity was then measured on a POLARstar Omega microplate reader (BMG Labtech) at excitation and emission wavelengths of 485 nm and 520 nm, respectively. Separate control reactions with heat-inactivated RdRp, GAA mutant RdRp or reactions that were stopped with EDTA at 0 min were used to quantify background fluorescence. For the radioactive-nucleotide incorporation assays, reaction conditions were similar to those described above, with the addition of 0.04 µCi/µL [3H]GTP. Reactions were terminated with the addition of 37 mM EDTA, 33 mg/mL herring sperm DNA and 170 μL of 20% (v/v) ice-cold trichloroacetic acid to a final volume of 200 µL. The mixture was incubated on ice for 30 min to allow for RNA precipitation, then filtered through a UniFilter-96 GF/C filter plate (PerkinElmer). The filter plate was washed with 800 µL/well of absolute ethanol, then by the same volume of nuclease-free water. The plates were

dried at 80°C, and 30 µL of Microscint-O was added to each well before measuring radioactivity using a liquid scintillation counter (TopCount, PerkinElmer).

3.2.6 Gel-based RdRp assays

Polyacrylamide gel-based assays were used to examine the two mechanisms of RdRp activity; primer-extension and primer-independent (de novo) using the method developed by Yi et al.274 The RNA template PE46 was designed to direct primer- extension activity through a stable hairpin at the 3´ end, whereas the template LE19p can only direct de novo RdRp activity by the addition of puromycin at the 3´end of the RNA.274 Reactions were performed using either 1 µM of PE46 or 0.5 µM of LE19p. Each reaction contained 240 nM RdRp, 0.2 mM rGTP, 0.1 mM of each of rCTP, rATP and rUTP,

5 mM dithiothreitol (DTT), 2.5 mM MnCl2 and 20 mM Tris-HCl in a 25 µL final volume. Reactions were incubated for 5 h at 30°C in the presence of test compounds or the compound vehicle DMSO (0.5% vol/vol). PE46 products were separated using 15% denaturing polyacrylamide gels containing 7M Urea, whereas LE19p products were separated using 15% Urea-free gels (Bio-Rad, Hercules, USA). Gels were stained with SYBR Green II (Invitrogen) and visualised on a Molecular Imager Gel Doc (Bio-Rad).

3.2.7 High-throughput screening

High-throughput screening (HTS) was performed to identify small molecule inhibitors of HCV and NoV RdRps. Molecules were randomly selected from the ~ 114,000 WECC lead-like compound libraries (Parkville, Victoria, Australia). An extensive battery of functional group filters was developed to remove poorly optimisable and assay interference compounds in creating this compound library. All compounds had molecular weights between 150-450 daltons, between 1-4 rings, CLogP values <5, a maximum of 10 rotatable bonds, a maximum of 3 chiral centres, a maximum of 5 hydrogen bond donors and between 1-8 hydrogen bond acceptors. Compounds, dissolved in DMSO at 5 mM concentration, and were assayed at a 10 µM final concentration. To that end, 50 nL of each compound were transferred into 384-well plates containing the RdRp, while DMSO was aliquoted into control wells. Plates were incubated for 10 min at room temperature and the reaction was started with the addition of the remaining components (rGTP, poly(C) RNA, MnCl2, DTT, BSA and Tween-

20 in 20 mM Tris-HCl pH 7.5). Each test plate contained sixteen wells with control (no compound) and heat-inactivated RdRp for positive and negative control measurements, respectively. Once reactions were finished, PicoGreen dye was added and fluorescence quantified on an EnVision Multilabel plate reader (PerkinElmer, Waltham, USA). Screening data were analysed in ActivityBase (IDBS, Guilford, UK) and visualised using Spotfire (TIPCO, Palo Alto, USA). Compound activity, equivalent to RdRp inhibition, was calculated as follows: Percentage inhibition = 100 - [100 × (Sample - Mean NSA) ÷ (Mean

TA – Mean NSA)], where NSA = Non Specific Activity, TA = Total Activity. Assay quality for

HTS was determined based on signal to background S/B = Mean TA ÷ Mean NSA], signal to noise [S/N = (Mean TA – Mean NSA) / SD NSA], coefficient of variation [CV = 100 × SD ÷

Mean] and finally Z´ values (a measure of assay robustness) Z´ = 1 – [(3SD TA + 3SD NSA) /

275, 276 (Mean TA – Mean NSA)], where a Z´ ≥ 0.5 is considered acceptable for HTS. Each parameter was calculated on a plate-by-plate basis using internal control RdRp reactions.

Cell culture general methods

3.3.1 Cells and replicons

The bicistronic HCV G1b replicon (strain Con1, GenBank accession number AJ238799), which contains a luciferase reporter gene277, was kindly provided by Dr Ralf Bartenschlager (University of Heidelberg, Germany). The bicistronic G3a HCV replicon (strain S52, GenBank accession number GU814264), containing a chimeric gene encoding firefly luciferase fused with neomycin phosphotransferase (Feo)72, was kindly provided by Dr. Charles M. Rice (The Rockefeller University, New York). The plasmid for a tricistronic HCV G2a replicon278 (strain JFH-1, GenBank accession number AB047639) was kindly provided by Dr. John McLauchlan (The University of Glasgow, Scotland). HG23 cells, a human hepatoma (Huh-7) cell-line bearing the Norwalk virus (GI.1) sub-genomic replicon242, were kindly supplied by Dr Kim Green (NIAID, NIH, Bethesda, USA). A schematic representation of all replicon models used in this study is shown in Figure 3.1. Murine norovirus (MNV) strain CW1240 was kindly provided by Dr Herbert W. Virgin, (Washington University, Saint Louis, USA). For the HCV G2a replicon, RNA transcripts were generated using the MEGAscript T7 transcription kit (Life Technologies) following the manufacturer’s instructions. Two micrograms of replicon RNA were electroporated into 2 × 106 Huh-7 cells in a 40 mm cuvette using a Gene Pulser Xcell electroporation systems (Bio-Rad) set at 340 V and 975 µF. Cells were allowed to recover in complete media for 24 h, then grown in the presence of 750 µg/mL of neomycin for 3 weeks. Surviving colonies were isolated and characterised for HCV NS5a expression by western blotting (section 3.2.4).

Figure 3.1. Schematic representation of HCV and NoV sub-genomic replicons in this study Structures are shown for replicon models used in this study which included (A) HCV G1b (B) HCV G2a (C) HCV G3a, and (D) NoV GI.1 sub-genomic replicons. Genes encoding the non-structural proteins for HCV and NoV are shown in green. The selection marker neomycin phosphotransferase (neo) and the gene encoding firefly luciferase (FLuc) are shown in red. HCV replicons contain an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV). Introduced cell culture adaptive mutations and their position are shown for HCV G1b (A) and G3a (C).

3.3.2 Cell maintenance

All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), Glutamax (Life Technologies) and 100 U/mL penicillin-streptomycin (Life Technologies), from here on referred to as complete DMEM. Cells bearing sub-genomic replicons (HCV and NoV) were also maintained with the addition 750 µg/mL neomycin (Geneticin®, Life Technologies). When cells reached a semi-confluent state, they were washed with PBS and split by trypsinisation (Huh-7 cells) or by scraping (RAW 264.7 cells), pelleted and resuspended in fresh, complete media. 44

3.3.3 Sub-genomic replicons assays

To assess the replication of HCV and NoV replicons, Huh-7 cells harbouring the replicons were seeded in 96-well plates at a density of 5,000 cells/well (HCV) or 10,000 cells/well (NoV) in antibiotic-free, complete DMEM. On the next day test compounds were freshly diluted in DMEM, added to the cells and incubated for 72 h at 37°C. Untreated cells were incubated 0.5% to 1% (v/v) DMSO, the compound vehicle. The nucleoside 2CM was used as a positive control for both HCV and NoV replicon inhibition. For the quantitation of replicon levels, cells were washed twice with PBS (100 µL/well) before lysis. For HCV replicons, which contained firefly luciferase reporter genes (Figure 3.1), cells were lysed with 50 µL of ice-cold passive lysis buffer (Promega) and incubated for 10 min at room temperature. Luminescence was then measured using the luciferase assay system kit (Promega) on a FLUOstar OPTIMA microplate reader (BMG Labtech), following the manufacturer’s instructions. For the NoV replicon, which does not contain a reporter gene242, total RNA was isolated for using the RNeasy kit (Qiagen), followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Inhibition of HCV replicon replication was calculated as a percentage of replication compared to untreated cells. Half maximal effective concentration (EC50) values were determined by nonlinear regression in GraphPad Prism, version 6.02.

3.3.4 Cell viability assay

To examine the cytotoxicity of inhibitor molecules in this study, cell monolayers (5 × 103 /well for Huh-7, 1 × 104/well for HG23 or 2.0 × 104/well for RAW 264.7) were treated with various concentrations of each compound in 96-well plates. Cytotoxicity was measured by resazurin to resorufin conversion assay (CellTiter-Blue, Promega, Madison). After 72 h, media was removed and replaced with 80 µL freshly prepared CTB reagent, diluted 1:6 (v/v) in complete DMEM. Cells were incubated for 1.5 – 2 h and fluorescence was measured on a FLUOstar OPTIMA microplate reader (BMG Labtech).

4 A fluorescence-based high throughput screen to identify small compound inhibitors of the genotype 3a hepatitis C virus RNA polymerase

Acknowledgements:

Part of the work presented in this thesis chapter was performed by the following collaborators:  Dr. David Hibbs and Jennifer Ong (Faculty of Pharmacy, University of Sydney, NSW, Australia) performed the two-dimensional quantitative structure–activity relationship analysis (section 4.2.7).  Dr. Jonathan Morris and Jacqueline Liu (School of Chemistry, University of New South Wales, NSW, Australia) performed the chemical synthesis of derivative molecules and part of the analysis of compound derivatives (section 4.2.7).

Part of the following work has been published in: Eltahla, A. A., Lackovic, K., Marquis, C., Eden, J. S., & White, P. A. (2013). A Fluorescence- Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase. Journal of Biomolecular Screening, 18(9), 1027- 1034.

Introduction

Hepatitis C virus (HCV) infection is a major cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma.15 Currently there are an estimated 185 million people infected with the virus, which causes a chronic infection in nearly 70% of patients.4, 15, 16 There is no vaccine for HCV, and current treatment regimens rely on a combination of pegylated interferon alpha and ribavirin (PEG-IFN/RBV), which are expensive and accompanied by adverse side-effects. The HCV RdRp plays a fundamental role in the viral lifecycle, and has therefore become a prime target for the development of direct acting antivirals (DAA), both as NIs and NNIs (section 1.4.2.3). High-throughput screening (HTS) campaigns involving recombinant HCV G1 enzyme have been successful in the identification of several NNIs130, 131, 279 for HCV G1, nine of which have reached phase II and III clinical trials (section 1.4.2). A major advancement for the search for antiviral agents was the development of G1b subgenomic RNA replicons62 and G2a infectious cell culture systems (HCVcc).75 Downstream selection and characterisation of NNIs using these model systems has been a vital process to advancing past pre-clinical studies. However, this strategy has been difficult to replicate for non-G1 HCVs due to the lack of cell culture systems for genotypes G3-6 these, until very recently where replicons for G3a and G4a have been developed.72 Therefore the search for non-G1 inhibitors lags far behind, with the current hope that G1 inhibitors may demonstrate cross genotype inhibition. While G3a patients are better respondents to the PEG-IFN/RBV treatment,89 the newly developed G1 DAAs show lower efficacy for the treatment of this genotype,83, 84, 280 including the recently approved PIs and NNIs, as well as NS5A inhibitors in clinical development (section 1.5). This chapter describes a simple, inexpensive, fluorescence-based HTS that was utilised for the identification of small non-nucleoside inhibitors targeting the HCV G3a RdRp. Furthermore, the activity of these NNIs in available cell culture models was examined, and their structure-activity relationship was analysed to guide future development of HCV G3a-specific antivirals.

Materials and Methods

4.2.1 HCV G3a RdRp expression and purification

The RdRp-encoding region (GenBank accession number EF189901) of HCV G3a strain VRL69b, excluding the C-terminal 21 amino acids, was previously cloned into the expression vector pTrcHis2C (Life Technologies)273 Recombinant RdRp was expressed in E. coli and purified using metal affinity chromatography (section 3.2.3) using the ÄKTAprime plus platform (GE Healthcare).

4.2.2 RdRp activity assay development

De novo RdRp activity assays were performed in 25 μl. The synthesis of dsRNA was examined using commercially available fluorescent dye PicoGreen (Life Technologies), developed for the quantitation of dsDNA (section 3.2.5). Alternatively, the fluorescent dye RiboGreen (Life Technologies), which was developed for the quantitation of total RNA, was used. Enzyme activity was optimised by a systematic examination of reaction conditions including enzyme, template, substrate and divalent cations (Mn2+ and Mg2+) concentrations, and by varying the incubation time and reaction temperature. The commercially available bacteriophage RdRp, ϕ6 (NEB) was used as a positive control.

4.2.3 Kinetics of RdRp activity

To determine the kinetic constants of nucleotide incorporation by the HCV 3a RdRp, reactions were run with 40 μg/mL poly(C) for 40 min with increasing rGTP concentrations (0 to 2.5 mM). The kinetics of template utilisation [poly(C)] were also examined with 0.5 mM of rGTP substrate and the poly(C) template concentrations ranging from 0 to 80 μg/mL. Molar amounts of double stranded product were determined from a standard curve generated with a reference 19 bp dsRNA (Sigma- Aldrich). The velocity of the reaction was plotted against substrate concentration and kinetic parameters determined by non-linear regression in GraphPad Prism, version 5.04.

4.2.4 Assay miniaturisation and validation

A pilot HTS was conducted to identify novel lead-like small molecule inhibitors of the HCV G3a RdRp. For this purpose, the assay was miniaturised to a 384-well microtitre plate format where the RdRp reaction (25 µL) was followed by the addition of 65 μL (1:130 dilution) of PicoGreen to each well (final volume 90 μL). The miniaturised assay was tested for tolerance to Tween-20 [0-0.01% (v/v)] and bovine serum albumin (BSA) [0-0.02% (v/v)], which were required for robotic liquid handling, and to minimise nonspecific protein adsorption to the plasticware utilised in the screen, respectively. Assay tolerance to the compound vehicle DMSO was also tested in the range 0-5% (v/v). For that purpose, the effect of increasing concentrations of each additive on RdRp activity was measured using a fluorescence output. The nucleotide analogue 3′dGTP (TriLink Biotech), which acts as a chain terminator, was used as a positive control to validate sensitivity for RdRp inhibition. A titration curve was generated for 3′dGTP and the half maximal inhibitory concentration (IC50) value was determined by non-linear regression using the 4-parameter, variable slope model in GraphPad Prism, version 5.04.

4.2.5 Pilot HTS

A total of 10,208 compounds (>90% purity) were randomly selected from the ~114,000 WECC lead-like compound library (Parkville, Victoria, Australia) and HTS was performed at a final compound concentration of 10 µM, as described in section 3.2.7. RdRp reactions were carried out for 1 h at 30°C and each reaction contained 240 nM

RdRp, 230 µM rGTP, 10 µg/mL poly(C) RNA, 2.5 mM MnCl2, 5 mM DTT, 0.01% BSA and 0.005% Tween-20 in 20 mM Tris-HCl pH 7.5.

4.2.6 Secondary screening and IC50 determination

Percent inhibition of RdRp activity for all 10,208 compounds screened was determined to identify leads from the pilot HTS. A frequency distribution was then generated for all screened compounds based on the number of hits at defined percent inhibition bins. Compounds were selected if they showed ≥ 16.4% inhibition of RdRp activity, which represented inhibition exceeding the mean + 2.5 × SD of the distribution. Hits were then assayed in triplicate using the same fluorescence RdRp assay, at 10 µM to confirm inhibitory activity. After excluding false positives, hits were re-confirmed

using a radioactive nucleotide incorporation assay (section 3.2.5). The non-fluorescent nature of the second confirmatory assay removed further false-positive compounds that either interfered with or enhanced the fluorescence signal.281 Finally, the remaining ‘top’ hits were titrated to a final assay concentration of 60 µM to allow IC50 determination using the radioactive RdRp assay.

4.2.7 Structure-activity relationship

In order to gain insights into structural determinants of the biological activity of the HTS hits, and to identify potent derivatives, preliminary structure-activity relationship (SAR) analysis was conducted. Firstly, the chemical space around HAC01-01 and HAC02-01 was explored by purchasing existing compounds that share the same scaffold from chemical vendors. These included HAC01-02 to HAC01-07 and HAC02-02 to HAC02-10 (Appendix Table 8.1). A second set compounds from the HAC01 scaffold were designed using two-dimensional quantitative SAR (2D-QSAR), performed by the laboratory of Dr. David Hibbs (University of Sydney, Sydney, Australia). Canvas v1.8 software in the Schrödinger Suite282 was used to screen for compounds similar to HAC01-01 from the Ambinter catalogue, accessed via the ready-to-dock compound database ZINC, which contains >5 million compounds (http://zinc.docking.org). Fingerprints were computed for each HAC compound (HAC01-01 to HAC01-07) using parameters suggested to have superior performance.283 This incorporated the use of a 32-bit MOLPRINT2D fingerprint with Mol2 atom type and Tanimoto metrics. Only hit compounds that achieved a similarity cut-off score of 0.5 or better with the compounds HAC01-01 to HAC01-07 were selected. These compounds were synthesised by the laboratory of Dr. Jonathan Morris (University of New South Wales, Sydney, Australia) and included HAC01-08 to HAC01-24 (Appendix Table 8.1). Lastly, a set of HAC02 derivatives (HAC02-10 to HAC02-17) were designed and synthesised by the laboratory of Dr. Jonathan Morris to examine the substructures of the HTS hit, and identify chemical groups important for RdRp inhibitory activity. All compounds were dissolved in 100% DMSO, and freshly diluted in nuclease-free water or DMEM on the day of the experiment. Compounds were examined for inhibitory activity using the recombinant G3a RdRp and the G1b replicon model in cell culture (section 3.3.3). The cytotoxicity of each molecule was also examined simultaneously, as described in section 3.3.4. 51

Table 4.1. Details of Purchased compounds for structure-activity relationship studies. Compound Supplier Supplier code HAC01-01 Chemdiv 7472-0021 HAC01-02 Chemdiv 7472-0040 HAC01-03 Chemdiv 7472-0207 HAC01-04 Chemdiv 7472-0014 HAC01-05 Chemdiv 7472-0238 HAC01-06 Enamine Z220408292 HAC01-07 Enamine Z275868218

HAC02-01 ASINEX ASN 13777643 HAC02-02 ASINEX ASN 07408909 HAC02-03 ASINEX ASN 07409702 HAC02-04 ASINEX ASN 07410483 HAC02-05 ASINEX SYN 13777738 HAC02-06 ASINEX LMK 07405487 HAC02-07 ASINEX ASN 13777642 HAC02-08 ASINEX ASN 07408403 HAC02-09 ASINEX ASN 13777596 HAC02-10 ASINEX ASN 07412803

4.2.8 Replicon selection and testing for cross-resistance

In order to characterise RdRp mutations which confer resistance to the antiviral compounds, cells harbouring the HCV G1b replicon were cultured in the presence of neomycin and the test compound for 50 days, as described previously284 with modifications. Briefly, cells were seeded in 6-well plates containing 750 µg/mL G418 at 150,000 cells/well. On the next day, media was refreshed and test compounds added at

20 × EC50 concentration (Lomibuvir and HAC02-05) or 5 × EC50 concentration (HAC01-01 and HAC01-03). After 72 h, cells were washed, split and seeded into 10 cm dishes containing test compounds and G418. Media containing G418 and test compound was refreshed twice weekly and cells were passaged when necessary to maintain sub-confluent levels. After 50 days, selection was removed and cells containing the HCV G1b replicon were allowed to recover for two passages before resistance was tested. The resistance of replicon bearing cells to individual inhibitors was examined by measuring EC50 values for each test compound, and compared to the mock (DMSO) treated cells. Furthermore, selected replicons were examined for cross-resistance with known HCV NNIs, which bind to all five allosteric site of the HCV RdRp.

Results

4.3.1 Expression of HCV G3a RdRp and fluorescent assay development

In order to obtain large quantities (i.e. mgs) of purified RdRp for assay development and HTS, the RdRp from the HCV G3a strain VRL69 was expressed in E. coli and purified by nickel affinity chromatography. Eluted fractions were screened for the recombinant protein by SDS-PAGE and GelCode blue staining. A 67 kDa protein was eluted with 500 mM imidazole (Figure 4.1). The identity of the eluted protein was confirmed by western blotting using an anti-hexahistidine antibody.171 The total yield of purified RdRp was 7.5 mg/L culture, with >74% purity (Figure 4.1).

Figure 4.1. SDS-PAGE analysis of fractions from the purification of HCV G3a RdRp. Purification of recombinant HCV G3a RdRp using affinity chromatography. Lane 1, Precision Plus standards protein marker (Bio-Rad); Lane 2, pre-purified whole cell lysate; Lane 3, unbound lysate; Lanes 4-5, wash fractions with 50 mM imidazole; Lanes 6-9, fractions eluted with 500 mM imidazole.

Synthesis of dsRNA by the HCV RdRp was initially examined using two fluorescent dyes; RiboGreen, which is used for the quantitation of total RNA, and PicoGreen, a dye used for the quantitation of double-stranded (ds) DNA. Using the ϕ6 bacteriophage RdRp as a control RdRp, RiboGreen bound and detected the dsRNA product (Figure 4.2). Surprisingly, however, PicoGreen also bound dsRNA, and resulted in a 7-fold increase in signal to background ratio (S/B) compared to RiboGreen (Figure 4.2). The PicoGreen

RdRp assay was therefore adopted as the main method to measure polymerase activity in this study.

Figure 4.2. Assessment of fluorescent dyes for the detection of RdRp products The two fluorescent dyes, RiboGreen and PicoGreen, were compared for sensitivity to stain the dsRNA product of the RdRp. Reactions were run using either control or heat-inactivated RdRp in order to measure S/B values. All results are the average of triplicate experiments plotted with standard deviations.

4.3.2 Assay optimisation and characterisation of the HCV G3a RdRp kinetics

A systematic examination of biochemical factors that influenced the activity of the HCV 3a RdRp was performed in order to establish the optimal HTS assay conditions. The effect of enzyme concentration on polymerase activity was examined. The activity of the HCV G3a RdRp increased linearly from 10% of maximal activity at 80 nM RdRp to 40% at 160 nM with maximal activity at 420 nM (Figure 4.3A). No increase in fluorescence was detected with increasing concentrations of the control heat-inactivated enzyme (Figure 4.3A). A functionally inactive mutant RdRp, with a GAA mutation at the active site, demonstrated similar background fluorescence to the heat-inactivated RdRp (data not shown). The optimum temperature for RdRp activity was in the range 30-33°C, with a 40% decrease in activity at 37°C (Figure 4.3B). Finally the time course examination of dsRNA formation showed linear accumulation of

product for up to 1 h, with the rate of product formation decreasing after 90 mins (Figure 4.3C).

Figure 4.3. Characterisation of biochemical factors influencing HCV G3a polymerase activity (A) The effect of enzyme concentration on dsRNA formation measured using PicoGreen fluorescence readout was examined. Solid circles represent control reactions while open circles represent reactions with heat-inactivated enzyme. (B) The optimal temperature for the HCV G3a RdRp activity was determined by measuring dsRNA formation between 22°C and 37°C (C) The HCV G3a RdRp-catalysed dsRNA synthesis over a 2 h period. Results for all graphs are the average of triplicate experiments plotted with standard deviations.

We were unable to use the fluorescent assay to quantify the effect of the divalent cation Mn2+ concentration on RdRp activity. At concentrations greater than 2.5 mM, MnCl2 significantly affected the PicoGreen signal in the presence of dsRNA, and resulted in about 80% reduction in fluorescence at 20 mM (Figure 4.4A). Alternatively, the optimal concentration of MnCl2 was determined to be 2.5 mM using the radioactive RdRp assay. The enzyme was inactive in the absence of a divalent cations, and no RdRp activity was detected using 0-15 mM Mg2+ (Figure 4.4B).

Figure 4.4. The effect of divalent salts on RdRp activity and fluorescence signal output. (A) The addition of MnCl2 to the PicoGreen assay reduced the fluorescent output in a dose- dependent manner. (B) Using the radioactive nucleotide incorporation assay, the optimal MnCl2 concentration was 2.5 mM, and no RdRp activity was detected using MgCl2. Results are shown as the average of triplicate experiments with standard deviations.

The steady state kinetics of the HCV G3a RdRp reaction were examined and the

Km of the rGTP substrate was calculated at 237 ± 40 µM, with a maximum reaction

-1 velocity Vmax of 52 ± 3 pmoles GMP.h (Figure 4.5A). The Km for the RNA template

-1 [poly(C)] was 10 ± 2 µg/mL (~33 µM) with a Vmax at 75 ± 5 pmoles GMP.h (Figure 4.5B).

Figure 4.5. Steady-state kinetics of the HCV G3a RdRp measured using the PicoGreen assay. Kinetics of the RdRp reaction were examined by titrating the substrate (A) or the RNA template (B) and constants were calculated using non-linear regression in GraphPad Prism 5.04. Results are shown as the average of triplicate experiments plotted with standard deviations

4.3.3 Development of a high-throughput RdRp assay

The fluorescent-based RdRp assay was further developed for HTS of small molecule libraries to identify inhibitors of the HCV G3a RdRp. Initially, the sensitivity of the assay to detect RdRp inhibition was tested by titrating the nucleotide analogue 3′dGTP, a known NI and chain terminator. Complete enzyme inhibition was reached at

60 µM 3′dGTP, and the IC50 value was calculated at 0.56 µM (Figure 4.6). The assay was then miniaturised for HTS in 384-well plates, and tested for tolerance to Tween-20, BSA and the compound vehicle, DMSO. No effect on RdRp activity or fluorescence signal was observed with concentrations up to 0.01%, 0.02% and 0.6% of Tween-20, BSA and DMSO, respectively (Figure 4.7). In fact, a 38% and 29% increase in the RdRp activity was observed at 0.001% (v/v) of Tween-20 and BSA, respectively (Figure 4.7A and B). Assay 57

robustness and suitability for high-throughput screening was examined by calculating signal to background and Z´ values, standard statistical measures of assay quality for HTS campaigns.275 The miniaturised assay demonstrated high sensitivity and robustness, with a mean Z´ of 0.73 and an S/B ratio exceeding 20 in validation screens (data not shown).

Figure 4.6. Titration curve of the reference compound 3′dGTP. RdRp polymerase activity was quantified using PicoGreen in the presence of increasing concentrations of 3′dGTP. Inhibition of dsRNA formation was calculated based on control RdRp reactions representing full reaction (DMSO vehicle only) and no reaction (heat inactivated RdRp). Results are the average of triplicate experiments plotted with standard deviations.

Figure 4.7. Optimisation of the PicoGreen assay for HTS. The tolerance of the fluorescent assay to Tween-20 (A), BSA (B) and DMSO (C) was examined. None of these additives reduced RdRp activities at the examined concentrations, while Tween- 20 and BSA resulted in a 38% and 29% increase in the RdRp activity at concentrations of 0.001%, respectively. Results are shown as the average of triplicate experiments plotted with standard deviations.

4.3.4 Pilot high-throughput screen

A pilot screen for NNIs of the HCV G3a RdRp was conducted using a random selection of 10,208 compounds with lead-like characteristics, at a final concentration of 10 µM. The screen was performed in two batches of 384-well plates and assay control measurements for both batches are summarised in Table 4.2. Overall, Z´ values for all test plates ranged between 0.65 and 0.78, with the exception of one plate (plate 19), which had a Z´ of 0.33, and was therefore excluded from further analysis (Figure 4.8A). The frequency distribution of RdRp inhibition, as a percentage of control, for all screened compounds showed a normal distribution (Figure 4.8B). One hundred and fifty compounds which demonstrated ≥ 16.4% inhibitory activity, or > 2.5 × SD above the mean inhibition for the HTS, were chosen for follow-up screening. The coefficient of variation (CV) for all controls was 7.8%, which was below the acceptable limit of 15% typically set in HTS assays276 (Table 4.2).

Table 4.2 Control measurements of pilot HTS assay performance.

Plates (n) Controla Mean SD CV b Z´ S/Bc Batch 1 10 Positive 187,839 11,680 8.34 0.71 10.37 Negative 18,110 281 4.15 Batch 2 19 Positive 214,945 18,868 8.79 0.71 12.81 Negative 16,831 735 10.01 a Reactions with control or heat-inactivated enzyme. b Coefficient of variation. c Signal-to-background ratio.

Figure 4.8. Pilot RdRp inhibitor screening for inhibitors of the HCV G3a RdRp. (A) Z´ values from individual screening plates in chronological screening order. Twenty nine plates were screened in two batches and Z´s were calculated based on internal controls. The acceptable limit for Z´ is represented by a dashed line. One plate failed acceptance criteria and was excluded from future analysis. (B) Frequency distribution of the calculated percent inhibition for all compounds screened on plates where Z’>0.5. Compounds demonstrating more than 16.4% inhibitory activity (2.5 × SD above the mean, represented by a dashed line) were selected for confirmation analyses.

4.3.5 Secondary screening of HTS hits and IC50 determination

Compounds selected as hits from the pilot HTS (n=150) were re-tested in triplicate using the same fluorescent-based RdRp assay. At 10 µM, 85 compounds showed no inhibitory activity against the HCV G3a RdRp, and 55 others showed weak activity (<16.4% inhibition, data not shown). These compounds were excluded from further analysis. The remaining 10 compounds were subsequently confirmed as inhibitors using both the fluorescent RdRp assay and an additional radioactive-nucleotide incorporation RdRp assay. The latter assay was used to exclude compounds that effect (e.g. quench) fluorescence rather than inhibit enzymatic activity. Six of 10 hits were confirmed in both fluorescent and radioactive assays, and the RdRp inhibition results showed a positive correlation between the two formats, with a coefficient r = 0.8 (p < 0.01, Figure 4.9A). None of the six compounds showed inhibition of a control norovirus RdRp (data not shown). The four most potent compounds 61

assessed using both polymerase assays were selected for further characterisation, and dose-response curves were generated (Figure 4.9B) to calculate IC50 values. This revealed four novel scaffolds as HCV G3a RdRp inhibitors (Figure 4.10); a quinoxaline

(HAC01-01) and a quinolinone (HAC02-01), which had IC50 values of 12.7 µM and 1.0 µM, respectively, followed by a less potent pyridazin-3-piperidine (HAC03-01) and a thiazole-4-furan (HAC04-01), with IC50 values of 21.6 µM and 20.7 µM, respectively (Figure 4.9B, Figure 4.10).

Figure 4.9. Activity and Titration curves for the top HCV RdRp hits from the pilot HTS. (A) Hits identified using PicoGreen were examined using a radioactive-based counter-assay. Percent inhibition, calculated using the two different assays for the top 10 compounds, are shown (r = 0.8, p < 0.01). (B) Reactions were carried out with increasing compound concentrations and the activity of the RdRp was calculated as percentage of control reactions (DMSO vehicle or heat-inactivated RdRp). Half maximal inhibitory concentrations were determined with non-linear regression. Results are shown as the average of triplicate experiments with standard deviations.

HAC01-01 HAC02-01

HAC03-01 HAC04-01

Figure 4.10. Chemical structures of HCV G3a RdRp inhibitors identified in this study.

4.3.6 Confirmation of HAC chemical structures; the HAC02 story

In order to further confirm the inhibitory activity of lead hits identified by the pilot HTS, the top four compounds were reordered from the chemical vendors (section 4.2.7) and retested against the G3a RdRp at 10 µM. While HAC01-01 demonstrated consistent activities across the two lots (Figure 4.11), reordered lots of HAC02-01, HAC03-01 and HAC04-01 had reduced inhibitory activity. The largest difference was observed with HAC02-01 for which the inhibitory activity was reduced from 84% to 22% between the two lots, respectively, whereas a 31% and 20% reduction in HCV G3a RdRp inhibitory activity was observed between different batches of HAC03- 01 and HAC04-01, respectively (Figure 4.11). A change in the colouration between the two HAC02-01 lots was observed. While the original lot (lot 1) of HAC02-01 had a dark yellow colour, lot 2 was colourless when dissolved in 100% DMSO. This, together with the significant drop in the inhibitory activity of the most potent HTS hit, HAC02-01, prompted further analysis of different batches of the compound. Interestingly, when an aliquot of the reordered HAC02-01 (lot 2) was incubated at room temperature overnight, the colour of the clear solution yellowed and then darkened to a colour that resembled the original compound batch (lot 1). This indicated a chemical reaction may occur that changes the chemical structure of the HAC02-01 molecule. Serendipitously, the colour change in HAC02-01

corresponded with a significant (~6-fold) increase in the RdRp inhibitory activity in vitro, from 14% (colourless) to 80% (dark yellow) when examined at 10 µM (Figure 4.12A).

Figure 4.11. Inhibitory activity of different lots of HAC molecules against the G3a RdRp. Compounds were tested at a single concentration (10 µM) using the radioactive assay. Lot numbers represent different batches of the same compounds; lot 1 are from the HTS library while lot 2 were reordered from the suppliers. Results are shown as the average of triplicate experiments plotted with standard deviations.

To examine the “activation” reaction of HAC02-01, freshly dissolved compound was incubated at room temperature, and aliquots were removed and frozen over a period of 8 days, then analysed for HCV G3a RdRp inhibition. Two compounds were also included as aged controls; the quinolinone HAC02-05, which shares the same scaffold as HAC02-01, and the quinoxaline HAC01-03, which is a derivative of HAC01-01 (section 4.3.8), both of which have no inhibitory activity against the RdRp activity at 10 µM (section 4.3.8). As a negative control, the compound vehicle DMSO was also included (Figure 4.12B). The inhibitory activity of HAC02-01 increased by 13% within 24 h, and then to 74% within 72 h, after which it plateaued with 80-86% inhibition of RdRp activity, relative control (Figure 4.12B). None of the control compounds or the vehicle demonstrated significant changes in the inhibitory activity, although an increase in RdRp inhibition was observed with HAC02-05 after 144 h incubation, with 31% inhibition at 10µM, compared to the original lot of the same compound (Figure 4.12B).

Figure 4.12. Time-dependent light activation of HAC02-01. (A) The inhibitory activity of HAC02-01 against G3a HCV RdRp was assayed at 10 mM final concentration using freshly dissolved compound (lot 2) and compared to lots 3 and 4, exposed to light for 7 and 14 days, respectively. (B) Time-course incubation of HAC molecules and the effect on RdRp inhibitory activity. Little inhibition of RdRp is observed with freshly dissolved HAC02-01. However exposure to light at room temperature results in a time-dependent increase in the inhibitory activity, which was not observed with the vehicle (DMSO) or compounds with the same (HAC02-05) or a different (HAC01-03) chemical scaffold. Results are shown as the average of triplicate experiments plotted with standard deviations.

4.3.6.1 Chemical analysis of HAC molecules

The chemical structure of all HAC molecules identified from the HTS (HAC01-01 to HAC04-01) was examined in order to elucidate the discrepancy in biological activity between different lots, particularly for HAC02-01. Chemical analysis was performed at the Bioanalytical Mass Spectrometry Facility (BMSF) at the University of New South Wales. The identity of all four molecules was initially confirmed using static nanospray mass spectrometry, and two batches (lot 1 and lot 2) of each compound were run for comparison. Two peaks were detected for each HAC molecule, corresponding to the protonated (+1) and the sodiated (+23) ions, and the mass spectra were identical between different lots of HAC01-01, HAC03-01 and HAC04-01 (Appendix Figure 8.1). For HAC02-01, however, an additional (+16) peak was detected with lot 1 but not with lot 2 of the compound (Appendix Figure 8.1B), suggesting oxidation of the molecule. In order to further analyse the formation of an oxidised product in the coloured HAC02-01 sample, liquid chromatography/mass spectrometry (LC-MS) was used. For comparison, samples were run which included lot 1, lot 2 (freshly dissolved) and an “aged” (lot 3) batch of the compound. Analysis of the chromatograms for HAC02-01 (390.4 daltons) revealed two major peaks (peaks A and B, top panel, Figure 4.13) corresponding to mass-to-charge ratios (m/z) of 391 and 407. Interestingly, the relative abundance of the two ions was different between the examined lots, as measured by UV-absorbance (bottom panel, Figure 4.13). Both ions were present with a similar abundance in the original HAC02-01 lot. However, ion A (m/z = 391) was identified as the main component of the fresh sample (lot 2), whereas ion B was (m/z = 407) was the main component of the aged sample (lot 3, Figure 4.13). Tandem mass spectrometry (MS/MS) analysis of the HAC02-01 revealed further insights into the oxidation reaction. Upon fragmentation of ions from lot 1, parent ions were observed at m/z 391 and 407. However an additional fragment ion was detected at m/z 218 (Figure 4.14). This ion was detected upon fragmentation of both the non-oxidised (Figure 4.14A) and oxidised (Figure 4.14B) ions. Incidentally, this corresponds to the molecular weight of the left hand side (LHS) of the HAC02-01 molecule (Figure 4.14C), indicating that the oxidation site is likely to be on the right hand side (RHS) of the molecule.

Figure 4.13. Liquid chromatography–mass spectrometry (LC-MS) analysis of HAC02-01. Analysis was performed using the original HAC02-01 lot 1 (left panel), a freshly dissolved lot 2 (middle panel) and an aged solution of the molecule (lot 3, right panel). The chromatograms are shown at the top for total ions (TIC, black) or single ions (red and green). The UV-absorbance for each lot is shown at the bottom. Peaks A and B correspond to the 391 and 407 ions, respectively.

Figure 4.14. Tandem mass spectrometry of HAC02-01. Product ion mass spectra were obtained for the fragmentation products of the 391 (A) and 407 (B) ions from lot 1 HAC02-01. Parent ions were detected in addition to a fragment ion with m/z 218. (C) The structure of HAC02-01 showing the molecular weights of different fragments of the molecule in daltons.

4.3.7 Inhibitory activity of HAC molecules using the HCV replicon model

Inhibition of the HCV sub-genomic replicon was examined by treating replicon- bearing Huh-7 cells with test compounds (20 µM) for 72 hours, followed by measuring the luciferase output and compared to mock (DMSO) treated cells (section 3.3.3). The viability of treated Huh-7 cells was also measured in order to examine cytotoxicity of test compounds, and the NNI Lomibuvir (VX-222, Vertex) was used as a positive control for HCV replicon inhibition. Due to the lack of a G3a replicon at the time, the G1b replicon was used. Out of the four identified hits, only HAC01-01 demonstrated inhibitory activity, with 61% inhibition of replicon luciferase output and no observable cell death at 20 µM (Figure 4.15A). No reduction in replicon luciferase levels was observed with the three remaining HACs, or the aged HAC02-01 sample (HAC02-01Y). In fact, an increase in luciferase signal was observed with 20 µM of HAC04-01, although at this concentration the compound was toxic to Huh-7 cells (Figure 4.15A). The half-maximal effective concentration (EC50) for HAC01-01 was calculated by measuring the replicon-encoded luciferase levels at increasing compound concentrations. This revealed an EC50 value of 19.5 µM, and no cytotoxic effects were detected with concentrations up to 100 µM of HAC01-01 (Figure 4.15B).

Figure 4.15. Inhibitory activity of the identified HACs against the HCV replicon. (A) Huh-7 cells harbouring the HCV replicon were treated with 20 µM of test compounds for 72 hours. Replication of the HCV replicon (red boxes), quantified by luciferase output, and cell viability (blue circles), measured using CellTiter-Blue, were compared to mock (1% v/v DMSO) treated cells. (B) The EC50 value of HAC01-01 was calculated by generating a dose-response curve and non-linear regression. Results are shown as the average of three experiments with the standard deviation.

4.3.8 Structure-activity relationship studies

Preliminary structure-activity relationship (SAR) was performed using compounds that shared similar scaffolds to the original HTS hits HAC01-01 and HAC02-01 (section 4.2.7). All compounds were examined for inhibitory activity using the

recombinant G3a RdRp at 10 µM, and the G1b replicon model in cell culture at 20 µM. The chemical structures for all examined derivatives are shown in Appendix Table 8.1.

4.3.8.1 HAC01 series

Derivatives of HAC01 were selected to have the same LHS of the molecule; the quinoxaline scaffold. These included HAC01-01 through to HAC01-07. The remaining HAC01 analogues (n=17) were selected by 2D QSAR then synthesised to replace the quinoxaline group with a benzothiazole while retaining the right-hand side (RHS) of HAC01-01. These molecules included HAC01-08 to HAC01-24 (Appendix Table 8.1). Using the recombinant florescent RdRp assay, all HAC01 derivatives displayed significant losses of inhibitory activity when examined at 10 µM, as compared to the original hit HAC01-01 (Appendix Table 8.1). Surprisingly however, when examined using the HCV G1b replicon model, several quinoxaline HAC01 derivatives demonstrated potent inhibition. These included HAC01-03 (60.7% inhibition), HAC01-05 (50.9%) and HAC01-06 (42.9%), with no significant cytotoxicity (Appendix Table 8.1). The most potent of these, HAC01-03 had an EC50 value of 22.3 µM (Figure 4.16A), compared to 19.5 µM of the original hit.

4.3.8.2 HAC02 series

Like the HAC01 series, HAC02 derivatives were divided into two subsets. The first set of compounds (HAC02-02 to HAC02-10) was purchased from commercial sources and was selected to retain the quinolinone scaffold of the LHS of HAC02-01. The second set of compounds (HAC02-11 to HAC02-17) were synthesised in order to examine and identify chemical moieties responsible for biological activity of HAC02-01 (Appendix Table 8.1). In the in vitro RdRp assays, only one compound, HAC02-09, displayed significant activity against the HCV G3a RdRp, with 45% inhibition at 10 µM, and an IC50 of 30.1 µM (Appendix Table 8.1). When the HAC02 derivatives were examined using the HCV G1b replicon, several unexpected observations were made. Firstly, HAC02-09 which inhibited the RdRp had no inhibitory effect on the replicon, in a similar fashion to the lead hit HAC02-01. Conversely, HAC02-05 which had no effect on recombinant RdRp activity, was a potent inhibitor of the HCV replicon. Treatment with HAC02-05 reduced HCV

replicon levels in a dose-dependent manner, with an EC50 value of 2.6 µM and no cytotoxicity (Figure 4.16B). Attempts to re-synthesise the original HAC02-01 have largely failed due to technical difficulties; however the imine (HAC02-11), the LHS aldehyde (HAC02-12) and the RHS amine (HAC02-13) were synthesised. These compounds had no observable antiviral activity, as examined by both RdRp and replicon experiments (Appendix Table 8.1). Surprisingly, the amides HAC02-14, 15 and 16 were potent inhibitors of the HCV G1b replicon at 20 µM, with 68%, 86% and 72% reduction of luciferase levels in treated cells, respectively. The amides HAC02-14 and HAC02-15, however, demonstrated slight cytotoxicity when examined using Huh-7 cells, with 21% and 36% cell death at 20 µM, while HAC02-16 had no observable toxic effects (Appendix Table 8.1). HAC02-14 and HAC02-15 demonstrated modest inhibition against the RdRp (16.7% and 21.4% inhibition, respectively). However, HAC02-16, like HAC02-05 did not inhibit the RdRp activity in vitro. While the examined derivatives of HAC01 and HAC02 in this study were structurally diverse, it is worth noting that a significant correlation was observed between the compound lipophilicity (CLogP) and activity in cell culture for HAC01 molecules (r 0.7, p < 0.0001). The effect was less significant for HAC02 derivatives (r 0.4, p = 0.07), while no correlation was observed between CLogP and the enzymatic activities of both series (r = 0.02 and 0.07 for HAC01 and HAC02 compounds, respectively).

Figure 4.16. Activity of HAC01 and HAC02 derivatives using the HCV replicon model. Dose-response curves were generated for two representative HAC derivatives by treating Huh-7 cells harbouring the HCV replicon with increasing concentrations of HAC01-03 (A) or HAC02-05 (B) for 72 hours and measuring replicon replication (red boxes) and cell viability (blue circles). Results are shown as the average of triplicate experiments plotted with standard deviations.

4.3.9 Primer-extension activity of HAC molecules

A number of HAC01 (n=3) and HAC02 derivatives (n=4) displayed ≥40% inhibitory activity against the HCV G1b replicon at 20 µM, with little effect on the de novo activity of recombinant RdRp in vitro (section 4.3.8). These molecules included HAC01-03, HAC01-05, and HAC01-06 from the first scaffold, and HAC02-05, HAC02-14, HAC02-15 and HAC02-16 from the HAC02 scaffold (Appendix Table 8.1). In order to elucidate the mechanism of RdRp inhibition for these compounds, HAC01-03 and HAC02-05 were taken as representatives from the two scaffolds, and their effect on the primer-extension activity of the HCV RdRp was examined using the gel based method (section 3.2.6). RdRp reactions were performed using the PE46 RNA template in the 74

presence of increasing concentrations of test compounds, and products were run on a denaturing polyacrylamide gel (section 3.2.6). HAC01-01 inhibited the primer- dependent activity of the HCV RdRp, with 33% inhibition at 10 µM and 63% at 50 µM (Figure 4.17A). In contrast, HAC01-03 and HAC02-05 did not have an effect on primed RdRp activity at the examined concentrations (Figure 4.17A and B). The control compound Lomibuvir displayed potent inhibition of the RdRp activity when examined at concentrations between 1 µM and 10 µM (Figure 4.17B).

Figure 4.17. Inhibition of the primer-dependent activity of HCV RdRp by the HAC molecules. Reactions were run using PE46 RNA template in the absence (lane 1) or presence (lanes 3 to 8) of test compounds. As a control, a reaction was run in the absence of RdRp (lane 2). (A) HAC01- 01 (lanes 3 to 5) and HAC01-03 (lanes 6 to 8) tested at concentrations 2 µM, 10 µM and 50 µM, respectively. (B) Lomibuvir (lanes 3 to 5) tested at concentrations 0.1 µM, 1 µM and 10 µM, and HAC02-05 (lanes 6 to 8) tested at concentrations 2 µM, 10 µM and 50 µM, respectively.

4.3.10 Selection of drug resistant replicons

In an attempt to map the binding site of HAC molecules on the HCV RdRp, and to characterise potential resistance mutations, replicon selection experiments were performed. Cells harbouring the HCV replicon were cultured in the presence of neomycin and test compound. The NNI Lomibuvir and the compound vehicle DMSO were used as controls. Cell death was observed in cells treated with all compounds after continuous selection for 10 days. After 3 weeks colonies began forming for cells treated with Lomibuvir, whilst cell death did not result in colony formation for the HAC molecules. After 50 days, selection was removed and cells were allowed to recover for two passages before being tested for resistance (section 4.2.8). Cells treated with HAC01-01 and HAC01-03 did not display resistance to these compounds, and no shift in the inhibitory profiles was observed with these molecules compared to mock-treated cells (Figure 4.18A and B). However, selection using HAC02-05 resulted in a 14-fold increase in the EC50 value, from 1.9 µM to 26.3 µM (Figure 4.18C). A similar loss of inhibitory activity was observed with the control compound Lomibuvir, although the effect was more significant, with a 92-fold increase in the EC50 value (350 nM) when compared to replicons treated with the compound vehicle DMSO (3.8 nM, Figure 4.18D). Replicon-bearing Huh-7 cells that were selected using HAC01-01 and HAC02-05 were also tested for cross-resistance to other HAC molecules, as well as resistance to known HCV NNIs which bind to all five allosteric sites of the RdRp. Interestingly, although replicons selected using HAC01-01 did not display resistance to that compound, these replicon cells were 3-fold less sensitive to inhibition by HAC02-05 (Table 4.3). The selected replicons generated in this study did not have cross-resistance to any of the reference HCV NNIs used in this study, and predominantly identical inhibitory profiles were observed with the mock-selected replicons (Table 4.3).

Figure 4.18. Selection of resistant HCV replicons with the identified HAC molecules. Huh-7 cells harbouring the HCV replicon were treated with HAC01-01 (A), HAC01-03 (B), HAC02- 05 (C) or Lomibuvir (VX-222, C) for 50 days in order to select for resistant replicons. Resistance was confirmed by calculating EC50 concentrations using cells treated with the corresponding compound (red boxes) and compared to DMSO-treated cells (black circles). Only treatment with HAC02-05 (66 µM) and the control Lomibuvir (140 nM) resulted in decreased potency of these compounds to inhibit replicon replication. EC50 values are shown for cells treated with test compounds (red) or DMSO (black). Results are shown as the average of triplicate experiments with standard deviations.

Table 4.3. Activity of HCV inhibitors against HAC-selected replicons.

* Antiviral HCV RdRp EC50 [nM] Compound Binding site Mock HAC01-01 treated HAC02-05 treated 2CM active site 992.3 987.7 823.9 JTK-109 Thumb I 898.4 525.7 862.7 Lomibuvir Thumb II 10.1 10.0 8.4 Filibuvir Thumb II 117.4 114.8 95.9 Setrobuvir Palm I 8.8 9.9 5.8 Nesbuvir Palm II 16.4 11.5 9.8 Tegobuvir Palm-β 7.1 3.5 2.4

HAC01-01 20,110 13,280 14,910 HAC01-03 13,860 19,830 16,060 HAC02-05 1,867 5,675 26,270 * EC50 values were calculated individually for Huh-7 cells harbouring the HCV G1b replicon and selected with HAC01-01, HAC02-05 or DMSO as a mock treatment.

Cells that demonstrated resistance to the inhibitor molecules were pooled and RNA was extracted for RT-PCR and Sanger sequencing. Lomibuvir-resistant and DMSO- treated cells were used as controls. The RdRp-encoding region of the replicon was sequenced to identify mutations that may be linked to compound resistance. Overall, no specific mutations could be identified in cells that were resistant to HAC02-05 (Table 4.4). Substitutions were observed at RdRp amino acid positions 59 and 500, but were also present in mock-treated (DMSO) cells. Conversely, three non-synonymous mutations were detected in Lomibuvir-resistant replicons. These resulted in the amino acid substitutions M423T, A486V and V494A (Table 4.4), all of which are known mutations that confer resistance to HCV NNIs which bind to the thumb II allosteric pocket, including Lomibuvir.95, 285

Table 4.4. Amino acid substitutions detected upon selection of HCV replicons. Treatment DMSO Lomibuvir HAC02-05 V59I V59I V59I M423T Detected A486V Mutations V494A W500G W500G W500G

Discussion

The HCV RdRp is an attractive target for DAA development given the crucial role the enzyme plays in the viral life cycle. Since the majority of global HCV infections are caused by HCV G1,15 most antiviral campaigns for HCV inhibitors have been developed against this particular genotype, mainly using recombinant enzyme assays130, 131, 279 or with the HCV-G1b replicon model.286 Despite the recent development of new replicon systems for G3a and G4a viruses,72 the long delay (>10 years) in their availability, compared to the G1 replicons, has hindered campaigns aimed at identifying DAAs that work against non-G1 HCV genotypes. DAAs in clinical development have already demonstrated reduced efficacy against HCV G3a, including those that target the viral proteins NS3 (protease),83 NS5A (phosphoprotein),84 and NS5B (RdRp).280, 287 These finding suggest that a “one drug for all HCV genotypes” approach may not be feasible for HCV, and that development of genotype-specific enzyme inhibitors may be essential. No HTS campaigns aimed at identifying HCV RdRp inhibitors have thus far been reported where the HCV G3a RdRp is the primary enzyme target. In this chapter, a robust in vitro fluorescence-based assay was developed for the detection of primer-independent (de novo) RdRp activity, and subsequently applied in a pilot HTS campaign against the HCV G3a RNA polymerase.

4.4.1 PicoGreen compared to available assays

Traditional methods for detecting the activity of viral RdRps have relied on the incorporation of radiolabelled nucleotides into an RNA template.131, 273 Although very sensitive, these assays have limitations for HTS because of the handling requirements and safety precautions for the use of radioactive materials, as well as the multiple filter and wash steps.273 Alternative assays for RdRp activity have been described based on fluorescent labelling of the nucleic acid template,288 chemically modified nucleotides289 and/or require the addition of other enzymes into the reaction.290 These methods are expensive and labour intensive and, therefore, not ideal for use in HTS campaigns. The fluorescent dye PicoGreen was originally developed to quantitate dsDNA in a sample. The assay described in this chapter utilises PicoGreen and a poly(C) RNA template with unmodified ribonucleotides (rGTP) to generate end-point fluorescence based

measurement of the de novo activity of the HCV G3a RdRp. Interestingly, PicoGreen was shown to be significantly more sensitive for the detection of dsRNA when compared to RiboGreen, which was developed for the quantitation of RNA (Figure 4.2). However, RiboGreen was shown to be significantly biased against poly(C) and poly(G) homopolymeric RNA291 which may explain the low efficiency binding to stretches polyC:G dsRNA formed by the RdRps in this study. The PicoGreen assay, which was miniaturised to a high-throughput format, was suitable for HTS with minimal handling requirements and small assay volumes easily accommodated. Additionally, all measurements for assay performance and sensitivity were within the recommended range for HTS assays (Z´ > 0.5, S/B and S/N > 2, Table 1).276 The assay, therefore, could easily be adapted for any positive sense RNA viruses that cannot be easily cultured. The steady-state kinetics of the HCV G3a polymerase were examined using the

PicoGreen assay in order to define optimal reaction conditions for HTS. KM and Vmax, for nucleotide incorporation by the HCV RdRp were determined (Figure 4.5). The affinity for rGTP of the HCV G3a-VRL69 RdRp (237 µM) was similar to that reported for the HCV G3a

292 strain ALB-3a RdRp at 208 µM. However, Vmax was lower for the VRL69 RdRp at 74.7 pmoles rGMP h-1 compared to 193.8 pmoles rGMP h-1 for the ALB-3a.292 The requirement of the G3a RdRp for divalent cations was consistent with previous reports, where Mn2+ but not Mg2+ facilitated de novo HCV RdRp activity in vitro (Figure 4.4).292 In this study, HTS was conducted with a relatively low concentration of compound (10 µM). This was done to avoid unfavourable assay interference caused by high compound concentrations, which is often problematic for large-scale HTS.293 Furthermore, hits from the pilot HTS (n=150) were subjected to a secondary screen followed by a counter-screen using a radioactive based assay. This resulted in a final hit rate of 0.06%. Four novel compounds were identified which inhibited HCV G3a RdRp activity in the low micromolar range; a quinoxaline (HAC01-01), a quinolinone (HAC02- 01), a pyridazin-3-piperidine (HAC03-01) and a thiazol-4-furan (HAC04-01).

4.4.2 Replicon activity of the identified HAC molecules

Out of the four identified hits, only HAC01-01 demonstrated inhibitory activity using the HCV replicon model in cell culture. Surprisingly, an apparent increase in the replicon levels was observed upon treatment with HAC04-01, an effect that was 80

reproducible. Compounds with similar activities have been reported in cell-based HTS, and were regarded as “proviral compounds”; compounds with a specific effect on host or viral factors to increase the viral replication efficiency.286 Interestingly, however, the RHS of HAC04-01 has been reported as a potential “stabiliser” of luciferase in reporter assays similar to the one used in this study, a mechanism by which apparent increase in the signal is observed.294 This is consistent with the observation of an increase in the replicon signal while the compound was inhibitory in the enzyme assays. Therefore, although this study focused on the top two compounds for SAR, HAC04-01 remains a molecule with potential for further development as an RdRp inhibitor, given its activity against the recombinant RdRp in vitro (Figure 4.9). However future cell based assays will have to utilise a system that does not rely on a luciferase output, for example by measuring replicon levels by qRT-PCR, in order to accurately measure the antiviral activity of HAC04-01 and derivative molecules.

4.4.3 Photocatalytic activation of HAC02-01

In order to confirm the inhibitory activity of the HAC molecules against the G3a RdRp, different lots of the same molecules were examined and compared. Surprisingly, when lots of the most potent hit (HAC02-01) were examined, a significant variation in compound activity was observed, which was correlated with the compound appearance (section 4.3.6). The identity of the active yellow and potentially oxidised molecule in the original batch (lot 1) remains unclear; however, biological and chemical analysis of the HAC02-01 lots revealed multiple insights. Firstly, the compound is “activated” by a photocatalytic reaction, and the process is completed within 72-96 h (Figure 4.12). Secondly, the biological activity of HAC02-01 corresponds to a chemical modification of the compound. Exposure to light catalyses the oxidation of HAC02-01. The oxidised component is present in all compound lots, although at significantly higher concentrations in the “potent” lots 1 and 3, compared to the freshly dissolved lot 2 (Figure 4.13). The oxidation site, however, remains to be identified. Analysis of the oxidised product by MS/MS is suggestive of a chemical modification in the RHS of HAC02-01. The oxidation of the LHS to form the aldehyde HAC02-12 had no effect on the inhibitory activity (Appendix Table 8.1). The amides, however, which were designed to include both the LHS and RHS (HAC02-14 and HAC02-15) displayed modest inhibitory 81

activity of the enzyme and potent HCV replicon replication inhibition. The substitution of the LHS with a heterocyclic ring retained the replicon activity and decreased the cellular cytotoxicity, but abolished the enzymatic activity (HAC02-16, Appendix Table 8.1).

4.4.4 Structure-activity relationship

Preliminary SAR analysis was performed for the two most potent compounds HAC01-01 and HAC02-01. For HAC01-01 derivatives, both sides of the original molecule (LHS and RHS) appear to be critical for the antiviral activity. For instance, the substitution of the furan on the RHS with a tetrahydrofuran (HAC01-04) abolished the activity of the compound (Appendix Table 8.1). Similarly, HAC01-07, which is similar to HAC01-01, but lacks the pyrrolidine in the LHS also had no inhibitory activity (Appendix Table 8.1). The replacement of the quinoxaline group with another benzimidazole mimic, benzothiazole (HAC01-13), but not benzoxazole (HAC01-18), retained some of the activity in the enzyme assays. Interestingly, the LHS of the molecule was more critical for the antiviral activity when examined using the HCV replicon in cell culture. None of the compounds that had quinoxaline substitutions (HAC01-07 to HAC01-24) had an inhibitory effect on the replication of the replicon, whereas structural changes on the RHS (HAC01-02 to HAC01-06) affected the activity of these derivatives, although were all inhibitory (Appendix Table 8.1). Similar to the HAC01 series, the structural correlates of inhibition for HAC02 were examined using the enzyme and the replicon models. The removal of the furan ring on the RHS was critical for the enzymatic activity of HAC02, as HAC02-09, which retained this group, displayed potent inhibition of the RdRp in vitro. However, the RHS of the molecule containing the furan group was not sufficient for the inhibitory activity (HAC02-13). Neither HAC02-01 nor HAC02-09, however, inhibited the HCV replicon replication in culture. Conversely, the imidazole containing HAC02-05 and the amides were potent inhibitors of the HCV replicon but had modest RdRp inhibition in vitro. It is not uncommon for small molecule inhibitors to display variability in inhibitory effect between in vitro enzyme and cell culture models.127, 130, 295-297 Firstly, cellular uptake and serum stability properties often lead to differing activities between enzyme assays and cell culture models. The compound absorption, distribution, 82

metabolism and excretion (ADME) properties were not assessed in this study. However, the correlation between the calculated compound lipophilicity and replicon inhibition is indicative that ADME properties may play a role the cell culture, but not enzyme activity of the identified molecules. Conversely, cellular activation of inactive RdRp inhibitors upon internalisation has also been reported in the literature.44 Furthermore, the association of the RdRp with other viral and cellular proteins in the replicon model has been suggested to play a role in similar discrepancies.127, 296 Lastly, non-specific binding of inhibitor compounds to other proteins has also been shown to reduce their activity in cell culture models297, and might reduce the antiviral activity of the HAC molecules in cell culture. Further studies will need to elucidate the mechanism of replicon inhibition by the RdRp-inactive molecules like HAC02-05 and HAC01-03.

4.4.5 Binding site of the HAC molecules

Although HAC01 and HAC02 derivatives displayed inhibitory activity against the HCV replicon, attempts to identify key resistance mutations in the RdRp were unsuccessful. The binding site of the HAC molecules therefore remains to be resolved. Quinoxaline-containing compounds have been reported in a HTS for HCV RdRp inhibitors.298 While X-ray structures of these benzimidazole mimics complexed with the RdRp are not available, in silico docking models predict the binding to the Thumb I pocket.298 This is consistent with the cross-genotypic activity of HAC01-01, demonstrated by the G3a RdRp and the G1b replicon activity; benzimidazole molecules that bind to this site similarly display inhibitory activity against multiple HCV genotypes (chapter 5). Heterocyclic compounds containing a quinolinone moiety fused to a benzothiadiazine group were also described131, and extensive SAR studies led to the development of the Palm I inhibitor Setrobuvir.126 However, the benzothiadiazine core was essential for the antiviral activity of these molecules126, which is not present in the molecules identified in this study. Crystallographic studies of the HAC compounds in complex with the polymerase will provide valuable insights into the site and key amino acids involved in HAC binding to the RdRp. In summary, this study describes the development of a HTS format which utilises a fluorescent dye, PicoGreen, to identify inhibitors of the HCV G3a RdRp. The assay was robust, cost-effective and capable of identifying inhibitors of HCV G3a RdRp activity with 83

IC50s in the low micromolar range. Hits identified in the present study and chemical derivatives also displayed inhibition in the HCV replicon model. However, the mechanism of action for these compounds remains to be resolved, and future SAR analyses are required in order to improve potency, specificity and ADME profiles for these molecules. The molecules identified in this study represent the first NNIs specifically targeting G3a, and provide potential antiviral scaffolds where IFN-based therapy remains as the standard of intervention for this genotype.

5 Mechanistic examination of HCV polymerase inhibitors reveals a novel mechanism for thumb binders.

Acknowledgements:

Dr. Enoch Tay (Storr Liver Unit, Westmead Millennium Institute, University of Sydney at Westmead Hospital, NSW, Australia) performed clonal selection for HCV G2a replicon (section 3.3.1).

The following work has been submitted for publication:

Introduction

Direct acting antivirals which target the HCV RdRp have been classified into nucleoside inhibitors (NI) and non-nucleoside inhibitors (NNI) (section 1.4.2.3). Two NNI binding sites have been characterised within the thumb subdomain; T1 and T2, and two have been identified within the palm subdomain, P1 and P2. A fifth, less characterised site, has been described in palm domain of the polymerase which also involves an interaction with the β-hairpin from the thumb domain (site P-β), reviewed in section 1.4.2.3. In the absence of culture models for HCV genotypes other than G1 and G2299, studies to determine the efficacy of NNIs which bind to these sites on G3-6 viruses have largely involved recombinant enzymes made in Escherichia coli280, 287, or used chimeric sub-genomic replicons containing the NS5B encoding region on a G1b/G2a backbone.119, 169, 300, 301 However chimeric replicons are often prone to replication fitness losses, and the contribution of the backbone RNA/proteins to the differential antiviral effects is poorly characterised.17 Recently, replicons of HCV G3a and G4a have been described71, 72, 167 therefore allowing a closer examination of the efficacies of HCV DAAs against these genotypes, which represent between 40% and 90% of all HCV infections in some countries (section 1.3.2). The RdRp of HCV has been shown to catalyse the RNA transcription through primed elongation as well as primer-independent (de novo) mechanism. These two modes of activity are believed to correspond to different conformations of the enzyme; the closed conformation facilitates the de novo formation of a dinucleotide for replication initiation60, whereas the open conformation is thought to be responsible for the primer-extension activity of the RdRp (section 1.3.3). Studies involving recombinant HCV RdRps and NNIs have focussed primarily on the ability of the NNIs to inhibit the primer-dependent activity of the HCV RdRp, and little is currently known on what effect they have on the de novo RdRp activity (section 1.3.3). Yi et al.274 have recently demonstrated that T2 binding HCV NNIs inhibit the primed activity of the HCV G1b RdRp, but had little effect on the de novo activity of the RdRp. Conversely, compounds which bind to the P1 and P2 sites inhibited both modes of RdRp activity.274, 302 These compounds, however, were only examined at a narrow range of concentrations (0 to 200 nM), and the effect of NNIs binding to T1 and P- sites remains to be fully 86

understood.274 Furthermore, it remains unclear how NNIs which bind to different allosteric sites affect the two modes activity, de novo and primed elongation, across RdRp of different HCV genotypes. In this study, RdRp inhibitors representing molecules that bind to all known allosteric sites on HCV polymerase were screened for their ability to inhibit the G1b, G2a and G3a replicons, and G1b and G3a recombinant enzymes. Furthermore, the inhibitory profiles of HCV NNIs against de novo and primed RdRp activity was analysed, revealing a novel mechanism of action for T2 and P-β binders.

Materials and Methods

5.2.1 Compounds

All compounds were purchased from commercial vendors, dissolved in 100% DMSO and freshly diluted to the desired concentration on the day of the experiment. These compounds, shown with the commercial supplier, included 2’-C-Methylcytidine (Sigma-Aldrich, St. Louis, USA), JTK-109 (Dalton Pharma Services, Toronto, Canada), Tegobuvir (GS-9190) and Nesbuvir (HCV-796, Haoyuan Chemexpress, Shanghai, China), Filibuvir (PF-00868554) and Setrobuvir (ANA-598, Acme Biosciences, Palo Alto, USA), and Lomibuvir (VX-222, Selleckchem, Houston, USA). The structures of molecules examined in this study are shown in Figure 5.1.

Setrobuvir (P1) Nesbuvir (P2) JTK-109 (T1)

Lomibuvir (T2) Filibuvir (T2) Tegobuvir (P-β)

Figure 5.1. Chemical structures of HCV NNIs examined in this study. Compounds were selected which bind to the palm (P1 and P2), thumb (T1 and T2), or palm-β-hairpin interaction site (P-β). The binding site for each NNI is indicated in brackets.

5.2.2 Recombinant RdRps and quantitative assays

Recombinant RdRps were expressed in E. coli and purified by affinity chromatography (section 3.2.3). RdRp sequences with the following database accession numbers were used in this study: HCV G1b strain Con1 (GenBank accession number AJ238799) and G3a strain VRL69b (accession number EF189901). The de novo activity of RdRps was measured by monitoring the formation of double-stranded RNA from a single stranded homopolymeric template, poly(C), using the fluorescent dye PicoGreen, or by using the radioactive-nucleotide incorporation assays described in section 3.2.5.

Results

5.3.1 Inhibitory activity of NNIs against HCV replicons

In order to examine the cross-genotype activity of HCV NNIs, replicons from HCV G1b, G2a and G3a were used (section 3.3.1). NNIs were selected that bind to each of the five allosteric sites and their ability to inhibit HCV replicon replication was assessed at increasing concentrations. As a positive control, an NI known to be cross-genotypic, 2CM, was also used. All three replicons were susceptible to inhibition by 2CM.

Specifically, EC50 value for the G1b replicon was 767.1 nM; however, a 5-fold (3.8 µM) and 3-fold (2.2 µM) increase in EC50 was observed with the G2a and G3a replicons, respectively (Figure 5.2, Table 5.1). In contrast, the five NNIs demonstrated differential inhibitory efficacy across replicons from genotypes 1b, 2a and 3a. Firstly, in comparison to a potent EC50 value of 8.1 nM for the G1b replicon, the P1 inhibitor Setrobuvir demonstrated significant loss of inhibitory activity against HCV G2a and G3a replicons. Only 44% ± 14.6% inhibition of the G2a replicon replication was observed with the maximal concentration of 100 µM (>12,000-fold increase in EC50). Similarly, when examined using the G3a replicon the EC50 value was 26.1 µM, >3,200-fold higher when compared to G1b (Figure 5.2, Table 5.1). The P2 inhibitor Nesbuvir had an EC50 value of 16.6 µM when examined using the G1b replicon, and showed comparable activity against all three genotypes with only a 3-fold and 2-fold reduction in potency against

G2a (EC50 43.3 µM) and G3a (EC50 39.9 µM) replicons, respectively compared to the G1b replicon (Figure 5.2, Table 5.1). Overall these results show that P2 binding HCV Nesbuvir is a better cross-genotype inhibitor than the NNI Setrobuvir which binds to the P1 site. For the thumb binders, JTK-109, which binds to the T1 site, was a potent inhibitor of G1b replicon replication (EC50 0.26 µM), whilst a 6-fold increase in EC50 was observed for the G3a replicon (EC50 1.5 µM), and a 41-fold increase in EC50 for the G2a replicon was also observed (EC50 10.6 µM, Figure 5.2, Table 5.1). Both T2 binders, Lomibuvir and

Filibuvir, inhibited the G1b replicon with EC50 values 5.9 nM and 79.2 nM, respectively. The two T2 inhibitors appeared to be specific G1b inhibitors, with a significant loss of inhibitory activity observed with Lomibuvir against the G2a and G3a replicons (678-fold and >2,000-fold increase in EC50s, respectively), whilst Filibuvir demonstrated a 40-fold and 59-fold increase in EC50 for G2a and G3a, respectively (Table 5.1). Lastly, Tegobuvir, 90

a P-β binder, inhibited the replication of the G1b replicon with an EC50 value of 3.2 nM, but was its inhibitory activity was significantly reduced against G2a (>3,400-fold) and G3a replicons (822-fold) compared to G1b replicon (Figure 5.2, Table 5.1). .

Figure 5.2. Inhibitory activity of HCV NNIs against sub-genomic replicons. The activity of HCV NNIs was examined using sub-genomic replicons of HCV G1b (blue bars), G2a (red bars) or G3a (green bars). Cells harbouring the HCV replicon were treated with the compounds for 72 h, and luciferase activity was measured. Results are shown as a percentage of the mock (DMSO)

92 treatment. All results are the average of triplicate experiments shown with standard deviations.

Table 5.1 Cross-genotype activity of HCV inhibitors against sub-genomic replicons

G1b G2a G3a Compound Binding site a b a b EC50 [nM] 95% CI EC50 [nM] 95% CI Fold change EC50 [nM] 95% CI Fold change

2CM NI 767.1 520.5 - 1,130 3,765 1,548 - 9,156 5 2,249 1,437 - 3,517 3

Setrobuvir P1 8.1 6.1 - 10.6 >100,000 >12,000 26,060 17,690 - 38,390 3,217

Nesbuvir P2 16.6 10.9 - 25.4 43.3 27.3 - 68.8 3 39.9 27.4 - 58.1 2

JTK-109 T1 257.1 153.6 - 430.2 10,620 6,434 - 17,520 41 1,508 944 - 2,409 6

Lomibuvir T2 5.9 4.4 - 7.9 3,998 2,406 - 6,643 678 12,030 8,744 - 1,654 2,039

Filibuvir T2 79.2 55.8 - 112.6 3,176 1,904 - 5,296 40 4,698 3,448 - 6,401 59

Tegobuvir P-β 3.2 2.3 - 4.3 10,960 3,207 - 37,440 3,425 2,629 1,845 - 3,746 822 a CI, confidence interval b Fold change in EC50 value when compared to those obtained using the G1b replicon.

5.3.2 Inhibitory activity of HCV NNIs against recombinant RdRps

The effect of HCV NNIs on the de novo activity of G1b and G3a RdRps was analysed. Inhibition of recombinant RdRp activity was measured by a reduction of dsRNA produced from a polyC template, as determined by PicoGreen fluorescence, and compared to control reactions (section 3.2.5). The P1 binder Setrobuvir inhibited the

G1a RdRp with an IC50 value of 157.5 nM, and was 17-fold less effective (IC50 2.6 µM) when examined using the G3a RdRp (Figure 5.3, Table 5.2). In contrast, the P2 RdRp inhibitor Nesbuvir demonstrated similar efficacy against both G1b and G3a RdRps, with

IC50 values of 76.7 nM and 75.0 nM, respectively (Figure 5.3, Table 5.2). In a similar pattern, JTK-109 (T1 binder), showed comparable inhibitory activities using both RdRps, with IC50 values of 35.4 nM (G3a) and 107.6 nM (G1b, Table 5.2). A surprising observations was made when the T2 binders, which were potent inhibitors of the G1 replicon, were examined using the recombinant RdRp in vitro. Remarkably, an increase in the de novo activity of the HCV G1b RdRp was observed upon the addition of more than 100 nM of Lomibuvir and Filibuvir, as measured by increased dsRNA formation (Figure 5.3). With both these compounds, the G1b RdRp de novo activity appeared to be induced by 50% at 100 nM, and > 100% at 1 µM (Figure 5.3). This effect was not observed for the G3a RdRp, i.e. the de novo activity for the G3a enzyme was not induced by Lomibuvir or Filibuvir (Figure 5.3). In fact Lomibuvir inhibited the G3a de novo RdRp activity, but only at high concentrations (45.5% ± 6.6% inhibition at 100 µM), whereas Filibuvir did not inhibit the G3a RdRp at any of the examined concentrations (Figure 5.3). The induction of the de novo activity of the G1b RdRp was not limited to T2 binders as Tegobuvir, which binds to the P-β site, also specifically enhanced G1b RdRp activity, although this effect was not as potent as that of Lomibuvir and Filibuvir, with a 75.5% ± 7.5% increase in G1b RdRp activity at 100 µM (Figure 5.3). Like the T2 binders, Tegobuvir had no observable effect on the activity of the G3a RdRp (Figure 5.3). In order to eliminate the possibility that the enhancement of G1b de novo RdRp activity by T2 and P- binding NNIs was an artefact of the assay system, an alternative radioactive-nucleotide incorporation assay was used (section 3.2.5), and Lomibuvir was examined as a representative of de novo enhancer NNIs. A marked increase in de novo activity was again seen with the radioactive assay; Lomibuvir enhanced the de novo 94

activity of the G1b enzyme by 39.1% ± 10.6% at 100 nM, and 115% ± 29.7% at 10 µM, but did not enhance the de novo activity of the G3a RdRp (Appendix Figure 8.3). Lomibuvir also inhibited both RdRps at higher concentrations, although was more potent against G3a (81% ± 1.5% inhibition) compared to G1b (41% ± 3.3% inhibition) at 100 µM (Appendix Figure 8.3).

Figure 5.3. Effect of HCV NNIs on the de novo activity of recombinant RdRps. The inhibitory activity of HCV NNIs was examined using recombinant RdRps of HCV G1b (blue bars) and G3a (green bars). Assays were performed using a homopolymeric poly(C) RNA template and RdRp activity was measured via the quantitation of dsRNA formation using PicoGreen. RdRp activity is shown as a percentage of control reactions with the compound vehicle DMSO. Results are the average of triplicate experiments shown with standard 96 deviations.

Table 5.2 Inhibitory activity of HCV NNIs against recombinant RdRp

G1b G3a Compound Site Fold IC50 [nM] 95% CI IC50 [nM] 95% CI changea Setrobuvir P1 157.5 82.8 - 299.5 2,605.0 1,392 - 4,873 17 Nesbuvir P2 76.7 21.6 - 271.9 75.0 57.6 - 97.7 1 JTK-109 T1 107.6 45.6 - 253.8 35.4 15.0 - 83.6 0.3 Lomibuvir T2 Enhanced >100,000 Filibuvir T2 Enhanced >100,000 Tegobuvir P-β Enhanced >100,000 a Fold change in IC50 when compared to those obtained with the G1b RdRp

5.3.3 Gel Based examination of RdRp inhibition

The mechanism of G1b RdRp inhibition by the different NNIs was further analysed using two gel-based assays (section 3.2.6) developed by Yi et al.274 Only the G1b RdRp was used for this analysis as it is the target enzyme for all six NNIs used in this study. One template (PE46) was designed to form a hairpin to initiate primed transcription, whilst the other template (LE19p) had a puromycin modification of the 3’ terminus, allowing only de novo dsRNA synthesis (section 3.2.6). NNIs that bind to the palm and thumb sites (T1, T2, P1 and P2) inhibited the primer-dependent activity of the G1b, as assessed using the PE46 template (Figure 5.4A). These inhibitors exerted their inhibitory activity at concentrations between 10 nM and 1 µM with the exception of JTK- 109, which required a concentration between 1 µM and 10 µM (Figure 5.4A). The NNI Tegobuvir however, had no observable effect on primer-dependent activity of the G1b RdRp with a concentration up to 100 µM (Figure 5.4A). Gel-based analysis of the de novo G1b RdRp activity was performed to further confirm observations of enhanced activity using the quantitative fluorescent and radioactive assays by T2 and P- binding NNIs (section 5.3.2). Nesbuvir (P2) and Lomibuvir (T2) were examined to represent NNIs which inhibited and enhanced the RdRp activity, respectively (Figure 5.3). In the gel-based assay, Nesbuvir abolished the de novo activity at concentrations > 10 nM (Figure 5.4B). In contrast, an increase in RdRp activity was again observed with the T2 inhibitor Lomibuvir. A 35% increase in de novo activity was observed at 100 nM, and 144% increase at a concentration of 10 µM

(Figure 5.4B). At higher concentrations (100 µM) a 9% reduction in the activity was observed (Figure 5.4B). Overall, these results were consistent with the pattern observed using the florescent and radioactive RdRp assays, where T2 binders enhance the de novo activity, but inhibit primed elongation of the HCV G1b RdRp.

Figure 5.4. Analysis of the effect of HCV NNIs on different modes of RdRp activity. The effect HCV NNIs on primed and de novo activities of HCV G1b RdRp was analysed. Reactions were run in the absence of compound (negative control), in the absence of RdRp (positive control) or in the presence of increasing compound concentration. (A) The primer-dependent activity was examined using the PE46 RNA template and reaction products were run on a 15% denaturing gel stained with SYBR Green II. (B) de novo RdRp activity was analysed using LE46 and products were run under non-denaturing conditions and stained with SYBR Green II. The RNA templates PE46 and LE19p are observed as the bottom band in both panels, whereas the RdRp product is observed as the top band in the same lane. 98

5.3.4 Analysis of resistant mutations in HCV RdRp sequences

In order to gain insights into the differential efficacy of HCV inhibitors in this study, the amino acid sequences for the G1, G2 and G3 RdRps was analysed for known mutations conferring resistance to all compounds tested in this study (reviewed in 303). A summary of amino acid substitutions associated with G1b resistance to NNIs in this study is shown in Figure 5.5. Residues are also shown for the RdRps from the replicon strains G2a (JFH-1) and G3a (S52), and for the G3a strain VRL69b which was used for recombinant enzyme experiments in this study (Figure 5.5). A complete alignment of the amino acid sequences of HCV RdRps in this study is shown in Appendix Figure 8.2. The examination of HCV G2a and G3a RdRp sequences revealed multiple naturally occurring residues in both G2a and G3a RdRps, which may confer resistance to HCV NNIs that bind to the site P1, T2 and P-β, but not to the NNIs which bind to P2 or T1. Specifically, residues G556 and F445, implicated in G1 RdRp resistance to Setrobuvir (P1) 304, 305 and Tegobuvir (P-)134, respectively, were found in both G2a and G3a RdRp sequences (Figure 5.5). In contrast, none of the substitutions previously known to confer resistance to JTK-109 (T1) and Nesbuvir (P2) were present in G2a or G3a RdRps119 (Figure 5.5) Both G2a and G3a RdRps contained residues I419 and L482119, 287 (Figure 5.5), which are associated with resistance to the T2 binders Lomibuvir and Filibuvir.21,46,45 Furthermore, the amino acid residue A494, also associated with T2 NNI resistance, was present in the G2a RdRp sequence, but not in G3a (Figure 5.5). The amino acid substitution V494A has been selected upon treating G1 HCV patients with Lomibuvir285, and using the G1b replicon in this study (section 4.3.10).

Known RdRp resistance mutation

P495S/L/A/T

L419 M/V/I

M423T/I/V

I482L/V/T

M426T/V

M414T/L

C316Y/N

Y448C/H

S365T/A

V494I/A

G554D D559G

A486V

Y452H

S556G

C445F

S282T RdRp Compound site

NI 2CM P1 Setrobuvir P2 Nesbuvir P-β Tegobuvir T1 JTK-109 Filibuvir T2 Lomibuvir

HCV G2a (JFH-1) Q I F L A G HCV G3a (S52) I F L C G HCV G3a (VRL69b) I F L C G

Figure 5.5. Amino acid substitutions conferring resistance to HCV inhibitors in this study. Previously characterised mutations in the HCV G1 RdRp which confer resistance to compounds analysed in this study are shown. The corresponding amino acid residues for non-G1 RdRps analysed in this study are shown at the bottom, and those that may confer resistance to HCV inhibitors are highlighted for G2a and G3a RdRps.

100

Discussion

The treatment standard for HCV is currently undergoing significant changes from interferon-based regimens to combination DAAs for shorter duration and in the absence of interferon (reviewed in section 1.4.2). A few dozen DAAs are in different stages of clinical trials121, and four have so far been approved for use in combination with PEG- IFN/RBV. With an all-oral interferon-free combination therapy now on the horizon, a better understanding of the cross-genotypic specificity and mode of action for the different NNIs is warranted. In this study we examined six NNIs, which encompass all known RdRp binding pockets, for their ability to inhibit G1, G2 and G3 HCV replicons. Furthermore, the ability of the NNIs to inhibit recombinant G1b and G3a RdRps in their two modes of transcription (de novo and primed elongation), was also assessed. Of the NNIs selected in this study, Lomibuvir, Setrobuvir and Tegobuvir are currently in phase II clinical trials, whereas the clinical development has been halted for JTK-109, Nesbuvir, and more recently for Filibuvir.306 The examination of the cross-genotypic efficacy of the NNI Setrobuvir revealed it had little effect of the replication of the G2a replicon (EC50 >100 µM) and G3a replicon

(EC50 = 26 µM) compared to G1b (EC50 = 8.1 nM, Table 5.1). Similarly, the G3a RdRp was far less susceptible to inhibition by Setrobuvir (IC50 2.6 µM) compared to the G1b RdRp

(IC50 157.5 nM, Table 5.2). The loss of activity of benzothiadiazines against G2a and G3a viruses is consistent with previous reports using chimeric replicons119, 169, 301, and recombinant G2a and G3a RdRps.280, 287 Resistance to benzothiadiazines in G2a and G3a appears to be due to the glycine residue at position 556304, 305, a mutation which was detected in benzothiadiazine-resistant replicons307, and upon treating G1 HCV patients.308 Surprisingly, however, the mutagenesis of G556 in G3a enzyme to the G1b serine residue does not confer susceptibility to benzothiadiazines, at least in recombinant RdRp studies287, and the G3a resistance to this class of molecules remains to be resolved. Of the six NNIs examined, only Nesbuvir (site P2) demonstrated cross-genotypic inhibitory activity against G1b, G2a and G3a replicons, with EC50 values of between 16.6 nM and 43.3 nM (Table 5.1). Nesbuvir also inhibited the de novo activity of the G3a

RdRp, with an IC50 of 75 nM compared to 76.7 nM for the G1b RdRp (Table 5.2). These 101

findings are also in agreement with previous HCV replicon and RdRp studies, where Nesbuvir demonstrated equivalent potency against all HCV genotypes119, 167, 169, 280, 301, which corresponds with the absence of naturally occurring mutations in the P2 binding region of G2a and G3a RdRps.

As well as inhibiting the G1b replicon (EC50 257.1 nM), the benzimidazole JTK-109

(site T1) also demonstrated similar inhibitory activity against the G3a replicon (EC50 1.5

µM) and enzyme (IC50 35.4 nM). However, JTK-109 was 41-fold less active when tested against the G2a replicon (Figure 5.2, Table 5.1). Reasons for reduced efficacy of T1 binders, like JTK-109, against G2a viruses are not fully understood.119 For instance, no substitution is observed at the P495 residue of the G2a RdRp (Figure 5.5), which is known to confer resistance to JTK-109, both in replicon and recombinant enzyme models.284 However, a V494A mutation has been associated with resistance of recombinant RdRp to other thumb I binders, such as indole-N-acetamides.309 The naturally occurring alanine at this position for G2a, but not G3a might result in reduced inhibitory activity against the G2 replicon (Figure 5.5), although further work is needed to confirm this. Resistance to the T2 inhibitors Lomibuvir and Filibuvir has been attributed to L419I and I482L substitutions21, 46,45 both of which are present in G2a and G3a viruses.119, 287 This is consistent with our findings of a lack of activity for these drugs against G2a and G3a replicons and the G3a RdRp (Figure 5.5), where T2 binders were 678 to 2,039-fold (Lomibuvir) and 40 to 59-fold (Filibuvir) less potent compared to G1b. Similarly resistance to Tegobuvir (P-) is likely to be due to the C445F substitution in G2a and G3a RdRps (Figure 5.5), a mutation in the β-hairpin that has been shown to confer resistance to this class of compounds in replicon studies.134 Tegobuvir was less potent against the G2a (>3,400-fold) and G3a (822-fold) replicons, when compared to G1b (Table 5.1). It is worth noting that the reduced potency of Lomibuvir, Filibuvir and Tegobuvir against HCV G3a is higher than those recently reported using a different G3a replicon based on the same HCV strain S52 (63-fold, 13-fold and 22-fold, respectively)167, but result in this thesis are more consistent with levels reported previously using chimeric HCV replicons.119, 169, 301 To our surprise, de novo, but not primed, RdRp activity of the recombinant G1b RdRp was enhanced around two-fold with 1 µM of the T2 binders Lomibuvir and

102

Filibuvir, and to a lesser degree with the P-β binder Tegobuvir (Figure 5.3 and Figure 5.4). The increase in the RdRp activity was confirmed using de novo RdRp assays with both fluorescent and radioactive output (section 5.3.2), and was further visualised by gel- based assays (Figure 5.4), indicating that it was a true effect and not an artefact of the assay used. Furthermore, no increase in de novo activity was detected when these compounds were examined using the G3a RdRp. Most studies thus far that analyse NNI RdRp inhibition have used primer-dependent assays and therefore it is likely the enhancement of de novo RdRp activity has gone unnoticed. In fact, both thiophene-2- carboxylic acids127 and dihydropyranones128, 310, scaffolds from which Lomibuvir and Filibuvir were developed, were identified from HTS studies using primed assays followed by replicon evaluation of the hits. In a recent study, Yi et al. reported no observable effect on de novo activity of the G1b RdRp by Lomibuvir and Filibuvir.274 However, Lomibuvir and Filibuvir were only examined at a relatively narrow range of concentrations (50-200 nM), and a small increase in RdRp activity can be observed in figure 3.274 In contrast to Lomibuvir and Filibuvir, Tegobuvir was identified using HCV replicon and HCVcc (JFH-1) systems.133 Interestingly, in another recent study, Tegobuvir was shown to increase the G1b RdRp primed activity by 50% at a concentration of 3.7 µM, but inhibited the RdRp by 40% at 100 µM, an effect that was also unexplained by the authors.311 In summary, de novo enhancement by T2 and P- NNIs can be identified in two previous reports, and this is clearly demonstrated in the current study. The mechanism of action for Tegobuvir and T2 inhibitors has been poorly understood. Tegobuvir requires intracellular activation to form a covalent inhibitor of the HCV RdRp295, and was not thought to interact with the enzyme before activation.311 In contrast, recent evidence suggest that Filibuvir and Lomibuvir reduce RdRp binding to RNA, but do not block the interaction completely.311 Structural analysis of the RdRp in complex with thiophene-2-carboxylic acids indicate that these molecules only bind to the closed conformation of the enzyme312, and that binding induces conformational changes that may interfere with enzymatic activity.312, 313 Although the authors of this study suggested this resulted in an initiation-incompetent enzyme, our results indicate that Filibuvir and Lomibuvir in fact increase the initiation (de novo), but inhibit the elongation activity of the G1 RdRp. Recently, recombinant HCV RdRp has been proposed

103

to exist as a mixture of conformations which are in dynamic equilibrium, and stabilisation of one conformation occurs at the expense of the other in solution.61 Given that the formation first few phosphodiester bonds, and the transition-to-elongation, are known rate-limiting steps of the HCV RdRp reaction60, 314, 315, it is likely that T2 and P-β enhance the initiation efficiency (de novo) of the HCV RdRp by stabilising the RdRp conformations required for these rate-limiting steps. However, further studies are needed to validate such effects and to detail the mechanism of action for the thumb- interacting subset of HCV NNIs. In summary we have analysed the inhibitory activity of six representative HCV NNIs that bind to the five known allosteric sites across three genotypes, G1b, G2a and G3a. Our data indicate that only the P2 inhibitor Nesbuvir is cross-genotypic, whereas naturally occurring amino acid substitutions largely confer resistance to non-G1 RdRps. We also report a previously uncharacterised enhancement effect on de novo RdRp activity for T2 binders Lomibuvir and Filibuvir, as well as the imidazopyridine Tegobuvir, which provides a better understanding of the mechanism by which these compounds possess antiviral activity against HCV.

104

6 Non-nucleoside inhibitors of the norovirus RNA polymerase

Acknowledgements: Kun Lee Lim (School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW, Australia) performed the antiviral experiments using the infectious MNV model (section 6.3.5).

The following work has been published in: Eltahla, A. A., Lim, K. L., Eden, J. S., Kelly, A. G., Mackenzie, J. M., & White, P. A. (2014). Non-nucleoside inhibitors of the norovirus RNA polymerase; scaffolds for rational drug design. Antimicrobial Agents and Chemotherapy, 58(6), 3115-3123.

105

Introduction

Noroviruses cause around 50% of all gastroenteritis cases worldwide316, and are associated with the deaths of more than 200,000 people per year, mainly in developing countries.183 Of particular importance are NoVs that belong to Genogroup II, genotype 4 (GII.4) which have been associated with all six major NoV pandemics of acute gastroenteritis in the last two decades and account for 80% of all human NoV infections. 192 In addition, NoV is increasingly recognised as an important cause of chronic gastroenteritis in immunocompromised patients.198, 200 The highly infectious nature of NoV, and its association with outbreaks in hospitals, age-care facilities and cruise ships, highlights the need for specific approaches to control NoV infections either through vaccines or antivirals. This is of particular importance for individuals with a high risk of exposure, for immunocompromised patients with chronic NoV infections, and for those who are susceptible to complications and dehydration including young children and the elderly. The NoV infectious cycle offers a number of potential targets for the development of direct-acting antivirals (DAA). One key target for NoV antivirals is the viral polymerase (RdRp) because of its essential role in viral replication and the lack of homologous human enzymes. A number of studies have used recombinant NoV RdRp to characterise its biochemical properties in vitro, and the X-ray crystal structures of RdRps from human GI, GII and mouse GV NoVs have been solved.217-219 There are only a handful of publications reporting NoV DAA development, reviewed in section 2.4.2. The most advanced pre-clinical studies have so far focussed on protease inhibitors260, 261 or have repurposed available drugs as NoV RdRp inhibitors which were originally developed to treat other viral262-264 and non-viral infections.297 However, thus far no novel molecules or scaffolds have been identified as specific inhibitors of the NoV RdRp. In this study, we conducted a high throughput screen to identify small molecule inhibitors of GII.4 NoV RdRp transcription that may provide a platform for the development of antivirals against this important clinical pathogen. Four scaffolds were identified and their mode of RdRp inhibition was characterised. We further examined specificity of these compounds across a range of calicivirus RdRps, and

106

the antiviral activity was assessed using the human Norwalk GI.1 sub-genomic replicon, and the MNV infectious cell culture model.

107

Materials and Methods

6.2.1 Recombinant RdRp expression, purification and comparison

Recombinant RdRps with a with a C-terminal hexahistidine tag, were expressed in E. coli and purified by nickel affinity chromatography (section 3.2.3) The RdRp of the following caliciviruses (shown along with their corresponding GenBank accession numbers) were used in this study: NoV GII.4 Den Haag 2006b variant (EF684915), NoV GII.4 New Orleans 2009 variant (JQ613573), NoV GI.1 Norwalk virus (NC_001959), NoV GV.1 (MNV, DQ285629) NoV GII.7 (GQ849131) and Sapovirus (SaV) GII (AY237420). Amino acid sequence analysis was performed using the MEGA5 software package44, and a phylogenetic tree of protein sequences was produced using the Neighbour-Joining method.

6.2.2 Biochemical RdRp assays

Polymerase activity was measured by monitoring the formation of double- stranded RNA from a single stranded homopolymeric template, poly(C), using the fluorescent dye PicoGreen in 384-well plates (section 3.2.5). Each reaction contained 20 ng enzyme (13.3 nM), 5 µM GTP, 6 µg/mL poly(C) RNA, 2.5 mM MnCl2, 5 mM DTT, 0.01% BSA and 0.005% Tween-20 in 20 mM Tris-HCl pH 7.5 with a final volume of 25 μL. Reactions were run for 10 min at 23°C and terminated with 10 mM EDTA followed by PicoGreen staining and dsRNA quantitation. Alternatively, radioactive GTP incorporation was measured on a scintillation counter (section 3.2.5).

6.2.3 High-throughput screening

A high-throughput screen was carried out to identify inhibitors of the NoV using the RdRp of a representative GII.4 variant Den Haag 2006b which was associated with a global pandemic, and was the predominant NoV in circulation between 2006 and 2008.317 A random selection of 19,956 compounds from the Walter and Eliza lead-like compound library (The Walter and Eliza Hall Institute, Parkville, Australia) were screened at a final concentration of 10 µM, as outlined in section 3.2.7. Hits from the HTS were subjected to a confirmatory counter-assay at 10 µM using radioactive-nucleotide incorporation to further exclude false positives that could have affected the fluorescence signal in the primary assay. 108

6.2.4 Mode of RdRp inhibition

To examine the mode of enzyme inhibition by the lead hits, the kinetics of substrate (GTP) incorporation was examined in the presence or absence of inhibitor. Reactions were performed with increasing concentrations of GTP (from 0.2 to 66 µM) and 5, 10, 15, or 20 µM of inhibitor. Kinetic parameters for each compound were determined by non-linear regression and used to generate Lineweaver–Burk double reciprocal plots.

6.2.5 Inhibition of murine norovirus replication

Inhibition of MNV replication in RAW 264.7 cells was determined by plaque reduction assays, as described in reference 318 with modifications. Briefly, 6-well plates were seeded with 1 × 106 RAW 264.7 cells/well and incubated overnight at 37°C. Monolayers were then inoculated with 80 plaque-forming units (PFUs) of MNV in DMEM. After a 1h adsorption at 37°C, the medium was removed and wells were overlaid with 0.75% (w/v) low melting agarose in minimum essential media (MEM, Life Technologies) containing test compounds. Cells were incubated for 48 hours at 37°C then fixed with formaldehyde (4% v/v) and stained with crystal violet (0.2% w/v). Inhibition of MNV in was measured by quantitation of total plaque surface area using the image processing program ImageJ.319 To measure MNV RNA replication, RAW 264.7 cells were seeded in 96-well plates at a density of 2.0 × 104 cells/well. Test compounds were added on the following day and cells were incubated for 1 h at 37°C. Cells were then infected with MNV (MOI = 0.1) and incubated for 48 h. Total RNA was extracted for quantitation using QIAamp Viral RNA Mini kit (Qiagen).

6.2.6 Quantitative reverse transcriptase polymerase chain reaction

Viral RNA was quantitated from either HG23 cells or MNV-infected RAW 264.7 cells by qRT-PCR.192 In brief, cDNA was synthesised using a SuperScript VILO cDNA Synthesis Kit (Life Technologies). Replicon (GI.1) and MNV (GV.1) RNA was measured using an iTaq Universal SYBR Green Supermix (Bio-Rad, California) following the manufacturer’s instructions. NS7-specific primers were used for both GI-based replicon and MNV quantitation and included Replicon Fwd (5´-CCAACTGAAACCCTTTGCGG-3´),

109

Replicon Rev (5´-AGGCATCAGCGTAAGACCAC-3´), MNV Fwd (5´- TGGACGTCGGCGACTATAAG-3´) and MNV Rev (5´-ACCACCTCGTCATCACCATA-3´).

6.2.7 Structure-activity relationship analysis

To identify molecules from the remaining 90,000 compounds in the library with structural similarity to leading HTS hits, all functionality was removed from the hit structures (Appendix Figure 8.5) and the library was screened using ActivityBase SARview software (IDBS, Guilford, UK).320 All identified molecules were screened in triplicate at 10 µM for their ability to inhibit the NoV GII.4 RdRp, and results were compared to the primary HTS hits.

110

Results

6.3.1 Identification of RdRp inhibitors by high-throughput screening (HTS)

Using an in vitro HTS, NNIs of the NoV GII.4 RdRp (Den Haag 2006b variant) were identified from 19,956 compounds randomly selected from a larger 110,000 compound library. Figure 6.1 shows a summary of the pathway to the identification of the four most potent RdRp inhibitors in this study.

Primary HTS: 20,000 compounds

Compounds screened at 10 µM Hit: > 15.5 % inhibitory activity Hit rate: 0.18 %

Secondary screen: 35 compounds

Compounds counter-screened with a radioactive based assay Hit: > 40 % inhibitory activity

Dose-response curves: 12 compounds

Half-maximal inhibitory concentrations (IC50) calculated Hit: IC50 < 10 µM

Characterisation of top inhibitors: 4 compounds

Figure 6.1. Outline of the pathway to identify NoV RdRp inhibitors in this study

Overall, test plates demonstrated suitable measures for HTS quality275, as quantitated by Z´ and Z factors (Figure 6.2A). Both measures were above the acceptable limit of 0.5 (average Z´ = 0.77 ± 0.06, Z = 0.72 ± 0.07) with the exception of two out of 57 plates which had Z factor scores of -0.64 and 0.31. The apparent drop in quality was attributed to two highly fluorescent compounds, which were subsequently omitted

111

from the analysis. Of the 19,956 compounds screened, 35 hits demonstrated greater than 3 × standard deviation (SD) of the average inhibition relative to control reactions, or > 15.5% inhibition of RdRp activity (Figure 6.2B).

Figure 6.2. HTS for inhibitors of the NoV GII.4 RdRp. Compounds were screened for inhibitory activity against the NoV RdRp. (A) Control measurement for test plates in chronological order. The Z′ factor (blue spheres) and Z factor (red spheres) were determined for each plate and a value of > 0.5 was considered acceptable, which is highlighted with a dashed red line. (B) Inhibition results of the HTS. RdRp inhibition was calculated as a percentage of internal plate controls. The hit selection cut-off, which represented inhibition exceeding 3 × SD of the mean distribution, is shown as a dashed red line. 112

All 35 hits were subsequently counter-screened using a radioactive-nucleotide incorporation RdRp assay and 12 compounds demonstrated > 40% inhibition of RdRp activity, while 14 had no inhibitory activity and were most likely fluorescent-quenchers. Four other compounds which appeared to increase the activity of NoV RdRp by ~2-fold (Figure 6.2B) also had no effect when retested at 10 µM using the fluorescent and radioactive assays (data not shown). Dose-response curves for RdRp inhibition were generated for the 12 most potent hits and half-maximal inhibitory concentrations (IC50) ranged from 5 to 32 µM (Figure 6.3). The most potent of these compounds, a phenylthiazole-carboxamide (NIC02) and a pyrazole-acetamide (NIC04) inhibited the

GII.4 NoV RdRp with IC50 values of 5.0 and 5.5 µM, respectively (Figure 6.3). NIC10, a triazole and NIC12, a pyrazolidinedione demonstrated slightly higher IC50 values of 9.2 and 9.8 µM, respectively (Figure 6.3).

113

NIC01 NIC02 NIC03

IC50 = 20 (14.7-27.1) IC50 = 5 (3.6-6.9) IC50 = 11.3 (8.8-14.3)

NIC04 NIC05 NIC06

IC50 = 5.5 (4.5-6.7) IC50 = 19.1 (14.9-24.5) IC50 = 16 (10.1-25.3)

NIC07 NIC08 NIC09

IC50 = 32.4 (18.7-56.3) IC50 = 10.1 (8.4-12.0) IC50 = 13 (10.4-16.3)

NIC10 NIC11 NIC12

IC50 = 9.2 (7.4-11.3) IC50 = 14.8 (10.7-20.5) IC50 = 9.8 (7.4-13.0)

Figure 6.3. Chemical structures and inhibitory activity of NoV inhibitors in this study. Dose-response curves were generated for the twelve most potent compounds identified from the primary HTS, using the radioactive nucleotide incorporation assay. IC50 values were calculated using non-linear regression, and are shown for each compound together with the 95% confidence interval in brackets.

114

6.3.2 Mode of Inhibition

To characterise the mode of NoV GII.4 RdRp inhibition by each of the four lead hits, the kinetics of GTP incorporation was examined in the absence or presence of increasing inhibitor concentrations (Appendix Figure 8.4). Double reciprocal Lineweaver–Burk plots indicated a mixed mode of inhibition for NIC02 and NIC04, where both the substrate affinity and the reaction velocity decreased with higher inhibitor concentrations (Figure 6.4A and B). In contrast, the Lineweaver–Burk plots for NIC10 and NIC12 were representative of an uncompetitive mechanism of RdRp inhibition. For these two compounds, the apparent substrate affinity increased, as indicated by a decrease in Km values, while the reaction velocity decreased at higher compound concentrations (Figure 6.4C and D).

115

Figure 6.4. Differential mechanism of RdRp inhibition by the four NIC compounds In order to determine the mode of inhibition, RdRp activity was measured at different substrate concentrations (0.2 to 66 µM) in the presence of 0 to 20 µM of inhibitors. Double-reciprocal plots were generated for NIC02 (A), NIC04 (B), NIC10 (C) and NIC12 (D).

116

6.3.3 Activity of identified NNIs across related calicivirus RdRps

To determine the breadth of the inhibitory activity for the four identified compounds, purified recombinant RdRps were used from five caliciviruses of varying relatedness to the target GII.4 Den Haag 2006b (Figure 6.5). These represented a different variant within GII.4 (GII.4, New Orleans 2009 variant), a different genotype within the same genogroup (GII.7) and different genogroups within the genus (GI.1 Norwalk and GV.1 MNV). Additionally, an RdRp from Sapovirus (GII), which represents another genus within the Caliciviridae family, was examined. A phylogenetic analysis of the protein sequences reveals the relationship of the enzymes examined in this study (Figure 6.5), with amino acid identity ranging from 32.6 % (Sapovirus) to 97.1% (New Orleans 2009), when compared to the target GII.4 Den Haag 2006b RdRp sequence (Table 6.1).

Figure 6.5 Amino acid sequence analysis of RdRps from representative viruses. Unrooted Neighbour-Joining tree of RdRp amino acid sequences used in this study. The evolutionary distances were computed using the number of differences method and are in the units of the number of amino acid differences per sequence, indicated by the scale bar.

Table 6.1 Amino acid identity between the RdRp enzymes in this study. Amino acid identity [%] SaV RdRp strain GII.4 2006b GII.4 2009 GII.7 GI.1 GV.1 GII GII.4 2006b 100.0 GII.4 2009 97.1 100.0 GII.7 80.0 80.6 100.0 GI.1 65.3 65.6 66.4 100.0 GV.1 59.9 59.5 60.3 59.9 100.0 SaV GII 32.6 32.4 31.3 32.4 30.5 100.0

117

All four lead hits demonstrated inhibitory activity against both GII.4 enzymes

(New Orleans 2009 and Den Haag 2006b variants, 97% identity) with similar IC50 values for each NIC (Figure 6.6), as assessed by in vitro RdRp assays. Of the four lead hits, NIC02 showed the broadest inhibitory activity, with similar levels of inhibition across RdRps from all six caliciviruses (±0.19-fold change in IC50, Figure 6.6). Given its inhibitory activity against distantly related RdRps, we examined the inhibitory activity of NIC02 against different classes of polymerases. NIC02 did not demonstrate any inhibition of enzyme activity, even at concentrations up to 100 µM, against an RNA dependent DNA polymerase (Avian Myeloblastosis Virus RdDp) or a DNA-dependent DNA polymerase (Taq DdDp, data not shown). NIC10 demonstrated a narrower spectrum of inhibitory activity, with an 11-fold and 56-fold reduction in inhibitory activity against SaV and GI.1 RdRps, respectively when compared to the GII.4 RdRp, and a 1.9-fold reduction against MNV, but had comparable activity against the GII.7 RdRp (Figure 6.6). NIC12 showed a modest loss of potency against the GI.1 (2.3-fold) and GII.7 (4.6-fold) RdRps, compared to GII.4, and had little inhibitory activity against the SaV RdRp, with 33.9% ± 14.1 inhibition of RdRp activity at 100 µM (Figure 6.6). Interestingly, however, NIC12 was 4-fold more active against the MNV RdRp, compared to the GII.4 enzymes. Finally, NIC04 appeared to be the most specific scaffold for the GII.4 enzymes with significant losses of inhibitory activity against GII.7 (4.6-fold), GI.1 (3.1-fold) and SaV RdRp (8.7-fold), while no inhibition was observed against the MNV RdRp (Figure 6.6).

118

Figure 6.6. Inhibitory profiles of lead NoV inhibitors against RdRps from related caliciviruses. The inhibitory activity of the identified compounds against enzymes from related viruses. Compounds were assayed in triplicate at increasing concentrations and average changes in IC50 values are shown with standard deviations for each enzyme relative to the target GII.4 Den Haag 2006b RdRp.

6.3.4 Inhibition of the NoV GI Replicon

The antiviral activity of the identified compounds was assessed by monitoring the replication of the GI.1 Norwalk virus replicon in Huh-7 cells.242 Cells were treated with increasing concentrations of each compound (1 to 100 µM) and replicon RNA levels were quantitated 72 h later by qRT-PCR. NIC02 and NIC04 inhibited replicon replication in a dose-dependent manner, with EC50 values of 30.1 µM (95% CI 19.6 to 45.9 µM) and 71.1 µM (95% CI 56.7 to 89.1 µM), respectively (Figure 6.7A and Table 6.2). NIC10 and NIC12 had no effect on the replication of the GI.1 replicon when tested at concentrations up to 100 µM. Compound cytotoxicity was assessed simultaneously and compared to vehicle (0.5% DMSO) treated cells. NIC02 was toxic to the Huh-7 cells (Figure 6.7B and Table 6.2) at concentrations greater than 10 µM, with a half-maximal cytotoxic concentration (CC50) of 134 µM. In contrast, NIC04, NIC10 and NIC12 had no cytotoxic effects at concentrations up to 100 µM. The nucleoside analogue 2CM was used as a positive control and at 10 µM reduced RNA replicon levels by 84.9% (± 1.4) relative to untreated cells, with no observable cytotoxicity (data not shown).

119

Figure 6.7. Inhibition of the Norwalk sub-genomic replicon by lead norovirus NNIs (A) The inhibitory activity of the top four compounds was assessed using Huh-7 cells harbouring the Norwalk replicon (HG23 cells). NIC02 and NIC04 inhibited the replicon with EC50 values of 30.1 µM and 71.1 µM, respectively. NIC10 and NIC12 had limited effect on the replication of the replicon. (B) Cytotoxicity of the NIC compounds at concentrations ranging from 1 µM to 100 µM. Only NIC02 demonstrated cytotoxic effect at high concentrations (CC50 = 134 µM). All results are the average of triplicate experiments plotted with standard deviations.

Table 6.2 Summary of the inhibitory activity of top NoV inhibitors in this study GII.4 RdRp GI.1 replicon GV.1 MNV Compound CLogP IC50 (µM) EC50 (µM) EC50 (µM) NIC02 3.6 5.0 (3.6-6.9) 30.1 (19.6-45.9) 4.8 (1.7-13.3) NIC04 3.5 5.5 (4.5-6.7) 71.1 (56.7-89.1) 32.8 (22.0-48.9) NIC10 0.8 9.2 (7.4-11.3) >100 34.5 (22.6-52.5) NIC12 0.4 9.8 (7.4-13.0) >100 38.1 (17.7-82.4)

IC50 values were determined by in vitro radioactive GTP incorporation RdRp assays, shown with 95% CI EC50 values were determined using cell-based replicon and infectious NoV models, shown with 95% CI

120

6.3.5 Inhibition of infectious murine norovirus GV.1

To examine the antiviral activity of the four lead hits (NIC02, NIC04, NIC10 and NIC12) in an infectious norovirus cell culture system, the GV.1 murine norovirus (MNV strain CW1) was used. Monolayers of RAW 264.7 cells were treated with test compounds and infected with MNV for 48 h. MNV replication was assessed either by plaque reduction assays or by viral RNA genome quantitation by qRT-PCR (NIC02 only). Inhibitory activity was demonstrated by a reduction in both plaque size and numbers with all four compounds compared to DMSO vehicle-treated MNV infected cells (Figure 6.8). Quantitation of inhibitory activity, based on reduction in plaque area, revealed an EC50 of 4.8 μM for NIC02 (95% CI 1.7 to 13.3), the most potent of the four inhibitors of MNV replication (Figure 6.9A). The quantitation of MNV RNA genomes from infected cells also revealed a potent inhibitory activity of NIC02, with an EC50 of 2.3 µM (Figure 6.9C). Higher concentrations were required to inhibit the replication of MNV with NIC04, NIC10 and NIC12, with EC50 values of 32.8 (22.0-48.9), 34.5 (22.6-52.5) and 38.1 µM (17.7-82.4), respectively (Table 6.2).

Figure 6.8. MNV plaque reduction by lead NoV NNIs. Plaque formation was visualised 48 h after treatment with different inhibitor and infection with MNV. Control wells for each experiment, treated with DMSO (vehicle), are shown in the top panel. Inhibitor treatment is shown at a non-toxic concentration to RAW 264.7 cells in the bottom panel

121

The reduction in plaque size for NIC04, NIC10 and NIC12 was not due to cytotoxic effects of the test compounds as no cell death was observed with concentrations up to 100 μM (Figure 6.9B). However, toxicity to RAW 264.7 cells was observed with NIC02, with a CC50 of 57.1 μM, which was more than 10-fold higher than the EC50 of NIC02. The nucleoside analogue 2CM was cytotoxic to RAW 264.7 cells at concentrations above

1 µM and demonstrated a CC50 value of 12.2 µM (data not shown); however, at 1 µM MNV plaque area was reduced by 93.5% (± 0.3) relative to untreated cells (data not shown).

122

Figure 6.9. Effect of NIC compounds on the replication of murine norovirus. The antiviral activity of the NIC compounds was examined using the infections MNV. (A) The mean plaque area for the different treatments shown as percentage of mock (DMSO) treated cells. NIC02 demonstrated the highest inhibition of MNV replication (EC50 = 4.8 μM). NIC04,

NIC10 and NIC12 had similar efficacies with EC50 values of 32.8, 34.5 and 38.1 μM, respectively. (B) Cytotoxicity profiles of the NIC compounds with uninfected RAW 264.7 cells, shown as percentage of viable cells compared to untreated wells. (C) Inhibition of MNV by NIC02 was assessed by quantitation of viral RNA levels where the antiviral activity was similar to that observed with the plaque reduction assay, with EC50 2.3 µM. Results are the average of triplicate experiments plotted with standard deviations.

123

6.3.6 Preliminary structure-activity analysis

In order to gain initial insights into the structure-activity relationships for the four molecules, the compound library used in the primary HTS was searched to identify relevant analogues to NIC02, NIC04, NIC10 and NIC12. A total of 182 molecules were identified which were examined for inhibitory activity (10µM) against the NoV GII.4 RdRp (Appendix Table 8.2). Only three analogues of NIC02 were found, and one retained moderate inhibitory activity at 10 µM; 17% compared to 63% enzyme inhibition for the original hit (Appendix Table 8.2). None of the analogues for NIC04 (112 compounds) or NIC10 (33 compounds) demonstrated increased activity when compared to the original hit molecules (Appendix Table 8.2). Of 34 NIC12 analogues identified, three were more potent than the original hit (48% inhibition at 10 µM) with 55%, 62% and 76% inhibition of RdRp activity at 10 µM, respectively (Appendix Table 8.2).

124

Discussion

In the absence of protective vaccines, there is an unmet need for safe, effective antiviral therapies to combat norovirus infections, both for prophylactic use to prevent NoV transmission in an outbreak setting, and to treat immunocompromised patients with chronic NoV infections. Four small “drug-like” molecules were identified as inhibitors of NoV RdRp activity and represent new scaffolds for the development of therapeutic molecules (Figure 6.3). The compounds demonstrated inhibitory activity in the low micromolar range and included; a phenylthiazole-5-carboxamide (NIC02), a pyrazole-4-acetamide (NIC04), a triazole (NIC10) and a pyrazolidine-3,5-dione (NIC12). Three of these compounds were novel scaffolds that have not been previously reported as viral NNIs, however, NIC12 has been reported in a separate HTS as a “weak” inhibitor of the poliovirus RdRp activity, with approximately 22% inhibition when tested at 86 µM321, suggesting NIC12 is more potent against NoV than poliovirus RdRp. The mechanism by which each of these compounds inhibited the NoV RdRp was examined using double reciprocal Lineweaver–Burk plots (Figure 6.4). Interestingly, the two larger compounds, NIC02 and NIC04 (365 and 347 daltons, respectively), inhibited the NoV RdRp by a mixed mechanism, whereas NIC10 and NIC12 (189 and 233 daltons, respectively) had a mode of action that represented uncompetitive inhibition. This suggests that NIC02 and NIC04 bind to both free enzyme and enzyme-substrate complexes, while NIC10 and NIC12 only bind to the enzyme-substrate complex and therefore likely bind to an allosteric pocket that is distinct from the substrate-binding site. Taken together these findings indicate that NIC02 and NIC04 occupy a different binding pocket/s of the NoV RdRp compared to NIC10 and NIC12. The four compounds demonstrated variable inhibition profiles when examined across a panel of related RdRps in vitro. A broad-spectrum of inhibitory activity was observed for NIC02, with similar IC50 values across RdRps from different species within the Caliciviridae family (Figure 6.6). These results suggest that the NIC02 could inhibit the enzymes in a non-specific manner, e.g. protein reactivity.293 However the lack of NIC02 inhibitory activity against an RdDp and a DdDp, as well as our subsequent validation of its inhibitory activity in available cell culture models of NoV, demonstrated

125

this was an inhibitor of the viral RdRp. An explanation for its broad inhibitory activity therefore could be that NIC02 binds to a highly conserved RdRp motif. Using the infectious GV.1 MNV system, the antiviral activity of NIC02 was demonstrated both by reduction of plaque size (EC50 = 4.8 µM, Figure 6.9), and by a reduction in viral genome replication in treated cells (EC50 = 2.3 µM, Table 6.2, Figure 6.9). NIC02 also inhibited the replication of the GI.1 NoV replicon, although it was less potent compared to MNV, with an EC50 of 30.1 µM (Figure 6.9). It should be noted, however, that NIC02 was the only compound of the four lead hits to display cytotoxicity against Huh-7 cells (CC50 = 134

µM) and RAW 264.7 cells (CC50 = 57.1 μM) The inhibitory profile of the remaining three hits was more restricted; NIC10 was significantly less potent against the RdRps of NoV GI.1 and SaV (56 and 11-fold, respectively), compared to GII and MNV RdRps. These findings were consistent with the cell culture observations; NIC10 did not inhibit the replication of the GI.1 replicon in

Huh-7 cells, but did inhibit MNV in cell culture with an EC50 of 34.5 µM (Table 6.2). Interestingly, in the RdRp assays NIC12 was more potent against GV.1 MNV when compared to all three GII enzymes, however, it demonstrated only modest activity against MNV in cell culture (EC50 = 38.1 µM, Table 6.2, Figure 6.9). When examined for inhibitory activity against NoV GI.1, NIC12 was only 2.3-fold less potent compared to the GII RdRps (Figure 6.6), but had no effect on the replication of GI.1 replicon. The lack of activity of NIC12 in the viral culture systems could be explained by a lack of cell internalisation or metabolic instability; these parameters were not measured in the current study. This is further supported by the large difference in the lipophilicity between NIC12 (CLogP = 0.4) when compared to NIC02 and NIC04 (CLogP 3.6 and 3.5, respectively), which were both active in the viral cell culture models. Finally, NIC04 demonstrated the highest specificity towards GII enzymes, which is consistent with the high EC50 observed with the GI.1 replicon (71.1 µM). However, NIC04 was not active against GV.1 MNV RdRp in the in vitro assays but weakly inhibited MNV replication (EC50 = 32.8 µM). Only a handful of studies have reported the development of small compound RdRp inhibitors as potential NoV DAAs (section 2.4.2) These studies, however, have so far been limited to the use of known viral polymerase inhibitors repositioned against a new viral target – the NoV RdRp, or alternative uses for existing

126

drugs. Therefore, no new compounds have yet been explored as potential inhibitors of the NoV RdRp. The identification of the new scaffolds in this study provides a platform for NoV-specific antiviral developments. However, current limitations for NoV drug development associated with the lack of a human GII.4 NoV culture system, indicate a more difficult pathway to clinical use than that of other viruses that can be easily cultured in vitro. Further screening of the compound library revealed three NIC12–like molecules with increased potency against the NoV GII.4 RdRp compared to the original hit (Appendix Table 8.2). This indicates that NIC12 could represent an attractive scaffold for medicinal chemistry optimisation in the search for viral polymerases inhibitors. The examination of the three remaining scaffolds, in contrast, did not reveal any compounds with increased RdRp inhibitory activity. However, the analysis was limited by either large structural modifications of the hit molecules (NIC04 and NIC10) or by the limited number of chemical analogues in the library (Appendix Table 8.2), and further SAR would be useful to identify more potent derivatives. In summary, through a HTS approach we have identified four inhibitors of the NoV polymerase with low micromolar activity. These can be explored for further design efforts to yield potent NNIs with minimal undesirable biological effects. We next intend to determine the RdRp binding sites through mutational and crystallography studies, in order to guide structure activity relationship analyses for the development of effective NoV therapeutics.

127

7 General discussion

Infections with HCV and NoV pose a significant health and economic burden in developing and developed countries. Every year, over 360,000 deaths are attributed to HCV-related illness such as cirrhosis, liver failure and hepatocellular carcinoma.3 Similarly, NoV is the cause of over 200,000 deaths every year, the majority of which are children in developing countries.183 Preventative vaccines are not available for either HCV or NoV, and their development is faced by a number of challenges for both pathogens.235, 258, 322 There are no antivirals for the treatment of chronic NoV infections, or for use as a prophylactic measures in an outbreak setting, which typically affects hospitals, nursing homes and other enclosed environments. Conversely, interferon- based therapies for HCV are limited by poor tolerability and variable response rates among patients. First generation HCV DAAs have so far shown limited coverage against non-G1 genotypes, particularly against HCV G3, which infects over 33 million people worldwide. Two aims of this study were to; (i) characterise the cross-genotypic inhibitory activity and mechanism of action for all 5 classes of HCV NNI in clinical development, and (ii) to identify new small molecules inhibitors which target the RdRp of the HCV G3a. Other aims of this study were to identify and characterise novel RdRp inhibitors of GII.4 NoVs, which represents 80% of all NoV infections, and are associated with all six major pandemics of acute gastroenteritis in the last two decades.

Findings and implications

7.1.1 Quinoxalines and quinolinones as HCV G3a inhibitors

In order to identify small molecule inhibitors of viral RdRps in this study, an HTS approach was employed. In the absence of a permissive cell culture system for HCV G3a and NoV GII.4, an enzyme-based HTS had to be used. Indeed, a simple, inexpensive, non- radioactive and HTS-suitable assay was required for screening of thousands of molecules from lead-like chemical libraries. In chapter 4, the development of a HTS-amenable assay is described. The assay, which quantitates the formation of dsRNA as a measure of RdRp activity, could easily be adapted for any positive sense RNA viruses that cannot be easily cultured.323 The assay was used for the identification of inhibitors of the G3a

128

HCV RdRp (chapter 4). To our knowledge, this is the first screen in which this HCV genotype was the main target for antiviral development. By screening 10,208 molecules with “drug-like” characteristics, four novel compounds were identified that had micromolar activity against the G3a HCV RdRp (Figure 4.10). Exploring the two leading scaffolds, the quinoxalines (HAC01) and quinolinones (HAC02) resulted in the identification of analogue molecules with antiviral activity as demonstrated by enzyme and cell culture models (Supplementary Table 8.1). The differential efficacy of HAC01 and HAC02 derivatives provides preliminary insights into the structural determinants of their activities to guide further SAR efforts (section 4.2.7). As the standard-of-care (SOC) for the treatment of HCV moves past IFN-based therapies, antiviral development for G3 infections lags far behind. Full genotype coverage of HCV DAAs has long been a challenge in antiviral development (chapter 5). Results from clinical trials which examined new HCV DAAs in G3 patients provide evidence that G3a is far less susceptible to the new DAAs than the G1, which is not surprising given that the drugs were developed for G1 in the first place (Table 1.2). Even amongst the traditionally “easy-to-treat” with the old SOC, genotypes 2 and 3, G3 patients were consistently less responsive to new DAA based antiviral therapy than those infected with G2 HCV.161 Further in vitro studies of all classes of DAAs are also indicative of a particular need for G3a-specific molecules83, 84, 280, 287 and the prospects of a pan-genotype SOC therefore remains in doubt. The molecules identified in this study are first-in class HCV G3a RdRp NNIs, which alone infects around as many people as HIV globally. The work in this chapter provides two potential new antiviral scaffolds for the development of G3a specific NNIs, where IFN-based therapy remains as the standard of intervention.

7.1.2 Only palm II HCV NNIs are cross-genotypic

The current interferon-based antiviral treatments for HCV are long, associated with numerous side effects, and sustained virological response (SVR) rates vary between 40% and 80%.91, 92 DAAs targeting the viral protease, NS5A encoded phosphoprotein, and the RdRp show great potential for the treatment of G1 HCV patents (section 1.4.2). However, the efficacy of G1 RdRp inhibitors against non-G1 viruses has not been fully analysed, primarily due to the lack of a cell culture and replicons models for genotypes 129

other than G1 and G2.299 Very recently (2012-2013), and while this study was underway, replicon models became available for G3a HCV.71, 72, 167 Although this was more than a decade after the development of the first HCV replicon62, it represented a significant advancement for the study of G3a HCV. In chapter 5, a comprehensive examination of the efficacy of all five known classes of HCV NNIs was performed. This study utilised HCV G1b, G2a and G3a replicons and G1b and G3a recombinant enzymes. Of all the NNIs which bound the 5 described allosteric pockets324, only benzofurans, which bind to RdRp site palm II (Nesbuvir), were cross-genotypic, as demonstrated by enzyme and replicon models (Figure 5.2 and Figure 5.3). The broad activity of Nesbuvir could be explained by the absence of naturally occurring resistance mutations in non-G1 viruses (Figure 5.5). Thumb I inhibitors also demonstrated inhibitory activity against G3a HCV, but were > 40- fold less effective against the G2a replicon (Figure 5.2). Conversely, NNIs which bound to sites P1, T2 and P- were all significantly less potent against non-G1 viruses, which all contained naturally occurring resistance amino acid residues in the RdRp sequence (Figure 5.5). Benzofurans, including Nesbuvir, remain the only class of molecules which have been reported to bind the palm II site of the HCV RdRp.29, 95 Furthermore, despite the potential of these inhibitors, very few reports described further development of compounds from the same scaffold, and almost all studies examining the P2 RdRp site focused on the compound Nesbuvir.126 Given that NNIs which bind to the same site share similar resistance profiles325, and palm II resistance is rarely observed in HCV isolates326 (section 5.3.4), results in this study highlight the significance of RdRp sites to be targeted in future development of HCV NNIs. Molecules which bind to the palm II site could, for example, be developed by specific design efforts using in silico based approaches, to develop highly potent, pan-genotypic NNIs. The six compounds examined in this study were at different phases in the clinical trial pathway; JTK-109 (thumb I) and Nesbuvir (palm II) were halted after reaching phase II (Figure 1.4). During the course of this study, in 2013, the development of Filibuvir (thumb II) was also discontinued. Discouragingly, none of the three NNIs in this study which are currently still in clinical trials demonstrated cross-genotypic activity. ABT-333 is a benzothiadiazine compound, similar to Setrobuvir (site P1) which was examined in

130

this study, has progressed to phase III clinical trials while this study was underway. However, like Setrobuvir, ABT-333 also has limited genotype coverage, and is >400-fold less potent against all non-G1 HCV RdRps.327 Similarly, Deleobuvir (BI 207127, Boehringer-Ingelheim) is an indole derivative, similar to JTK-109, which has also progressed recently to phase III for the treatment of G1 HCV.328 There is limited data on the efficacy of BI 207127 across HCV genotypes; however, G1a patients are less responsive to Deleobuvir when compared to patients infected with G1b.329 Furthermore, other indole derivatives like TMC647055 (Janssen) and BMS791325 (Bristol-Myers Squibb) share a similar inhibitory profile with JTK-109, with reduced potency against the G2 RdRp330 (Figure 5.2). Overall, all NNIs of the HCV RdRps in current development have limited cross-genotypic activity, and further development is required for specific genotype targeted NNIs, such as the two initial scaffolds described in this chapter, for future IFN-free regiments for G2a and G3a HCV infections.

7.1.3 Thumb II NNIs enhance the de novo RdRp activity.

Antiviral inhibitors, in addition to their significance for therapy, are vital tools for studying biochemical processes which occur during viral infection and replication, and for studying the timing of these processes as well as dissecting the functions of the proteins involved.331 In chapter 5 of this thesis, the effect of NNIs on the biochemical activity of the HCV RdRp was investigated using polymerase reactions. With the exception of Tegobuvir, which is thought to require cellular activation, all HCV NNIs inhibited the primer-dependent activity of the RdRp (Figure 5.4). However, only compounds which bind to three of the five NNI pockets , P1, P2 and T1, inhibited de novo activity of the HCV RdRp. Molecules which bound to the T2 and P-β sites were serendipitously found to enhance the de novo RdRp activity, an unexpected observation given that these molecules were developed as inhibitors of the HCV RdRp (Figure 5.3, Figure 5.4). These molecules included T2 binders Lomibuvir and Filibuvir, as well as Tegobuvir, which is unique in its binding to a palm-β-hairpin interaction domain. These findings, although preliminary, emphasise the importance of different RdRp conformations on the biochemical activity of the enzyme, and add to the current understanding of the initiation of genome replication by the HCV polymerase, and its importance for viral fitness. For instance, the capability of the G2a JFH-1 strain to 131

replicate in cell culture has been partly attributed to the higher de novo transcription efficiency of the JFH-1 RdRp (~10-fold), even when compared to other G2a isolates.332, 333 The findings in this thesis also add to the understanding of the mechanism by which these molecules exert their activity on the replication of HCV, which remains poorly understood. De novo enhancing molecules have been very promising for the treatment of HCV G1 infected patients; both Lomibuvir and Tegobuvir are in phase II clinical trials, while the development of Filibuvir was halted for strategic reasons despite its potent clinical activity.334 Tegobuvir has also very recently shown promising SVR rates when combined with NS5A and Ribavirin in an IFN-free combination therapy.335 Findings in this study, therefore, also have implication for the development of new NNIs, particularly for genotypes 2-6, where RdRp enhancers of de novo transcription could be specifically targeted to identify T2 or P- binders for these genotypes, none of which have so far been described.

7.1.4 NoV NNIs: scaffolds for rational drug design

In chapter 6 of this thesis, the discovery of the first NoV-specific NNIs was described. Using high-throughput screening, nearly 20,000 “lead-like” compounds were tested for inhibitory activity against the NoV GII.4 RNA polymerase. The four most potent hits demonstrated IC50 values between 5.0 µM and 9.8 µM. Compounds NIC02 and NIC04 demonstrated a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious MNV cell culture system. NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with EC50 values of 30.1µM and 71.1 µM, respectively, while NIC10 and NIC12 had no observable effect on NoV GI.1 replicon replication in the cells. In the MNV model, NIC02 reduced plaque numbers, size and MNV

RNA levels, in a dose-dependent manner (EC50 values 2.3 µM to 4.8 µM). The remaining three compounds also reduced MNV replication, although with higher EC50 values, (32 µM to 38 µM). In a preliminary SAR analysis of the NIC molecules identified in this study (Section 6.3.6), NIC12 was shown to be an attractive scaffold for medicinal chemistry optimisation in the search for NoV polymerases inhibitors, and at least three molecules 132

were identified with improved potency over the initial HTS hit. In summary, we have identified novel NoV RdRp NNI scaffolds which will provide a starting framework for the development and future optimisation of targeted antivirals against NoV.

Limitations and future directions

7.2.1 Binding sites of the identified NNIs

Although this study was successful in the identification of novel inhibitors for both HCV and NoV, further development of the identified NNIs is currently limited by the lack of a characterised RdRp binding pocket for these molecules. Attempts to identify the mode of RdRp binding were carried out either by means of resistance selection or using molecular docking approaches, however no clear pocket has been identified despite rigorous efforts. Not only could the identification of resistance mutations help in mapping the site of inhibitor-enzyme interaction, they could also aid in understanding drug selectivity, the mechanism of action, as well as the mutational threshold for these inhibitors.331, 336 Selection of HCV resistant replicons was attempted for the HAC molecules, although this was not successful and no mutations were isolated (section 4.3.10). Conversely, for the NoV NNIs, the low antiviral activity of the identified molecules in cell culture, particularly using the replicon model (Table 6.2), made it difficult to treat the cells with compound concentrations that are typically required to generate resistant replicons. For the most potent inhibitor in the study, NIC02, the observed cytotoxicity for both examined cell lines (CC50 between 57 µM and 134 µM) also limited the prospects of such experiments. Resistance selection for both HAC and NIC molecules would be possible once more potent derivatives are identified from SAR studies, which are currently underway. Nevertheless, alternative pathways to the one applied in this study could be used for the identification of resistance mutations. A recent analysis of different pathways towards HCV NNI resistance revealed that resistance to some NNIs could only be isolated following a lengthy selection procedure, in which the concentration of the compound is increased in a stepwise manner307, instead of the single-concentration protocol applied in this study. Another approach would be to engineer known amino

133

acid mutations known to confer resistance to different classes of NNIs to generate a panel of mutant enzymes or replicons which could be used to test for cross-resistance to molecules identified in this study.287 However, this remains challenging for the NIC molecules as the NNI-resistance mutations are yet to be identified. Molecular docking experiments were also conducted for both the HAC and NIC compounds identified in this study in order to generate binding models which could then be biologically tested. Standard software packages such as GOLD337 and Glide338 were used for such experiments. However, as these analyses relied heavily on prior knowledge at least with regards to the binding site to generate a binding model, the results were often inconclusive, and had no biological evidence to support any of the docked models for either virus. These models, therefore, were not reported in this study. In silico docking using the available crystal structures of HCV and NoV RdRps was problematic for two main reasons; firstly, there are at least five pockets for the HCV RdRp, to which small-molecule inhibitors can bind. In the absence of supporting biological data, we could not convincingly draw conclusions on the binding mode of the HAC molecules to the RdRp. Secondly, as yet, no allosteric pockets have been identified for the NoV RdRp. One study reports the crystallographic binding of a class of NNIs to the NoV RdRp. 297 However, this particular study specifically targeted the active site of the enzyme using in silico approaches. 297 The lack of knowledge about NoV allosteric pockets meant that additional software had to be used for “pocket prediction” such as SiteMap software339, after which further prediction programs (GOLD337 and Glide338) were used to predict the binding mode. This added complexity to the analysis, and the models generated were not consistent with the biological data in chapter 6. Crystallographic identification of the binding site for both HAC and NIC molecules are therefore needed, and should be the focus of future efforts in order to guide further SAR analyses, to aid discovery efforts of NNIs which bind to the same pocket, and to further understand the detailed mechanism of action for these inhibitors.

7.2.2 Model systems used in the study

While model systems used in this study have been instrumental for antiviral development and the study of the replication of both HCV and NoV, they suffer from a number of limitations. Recombinant enzymes for both viruses are assayed in vitro in 134

isolation from all other viral and cellular factors which make up the replication complex within infected cells.28, 216 In the case of HCV, this was only possible with the deletion of a transmembrane anchor domain which would otherwise render the enzyme insoluble.47, 48 In contrast, sub-genomic replicon models were not available at the beginning of this study for the viruses targeted for antiviral development (HCV G3a and NoV GII.4). For NoV we had to rely on a GI.1 replicon which represents a diverse and minor virus compared to GII NoVs. However, replicons for HCV G3a were developed during the course of this study.72, 167 Replicon models are often highly adapted to cell culture (Section 1.3.4) and, therefore, results should always be interpreted with care, and validated using other methods of assessing HCV replication which may provide a more complete picture of drug efficacy. For instance, Sofosbuvir demonstrates equivalent potency against of G3a and G1b replicons.167 Despite this, however, G3a patients were 30% to 40% less responsive to Sofosbuvir compared to all other genotypes.98, 170 Ideally, in order to fully characterise the antiviral activity of identified molecules in this study, infectious cell culture and animal model systems are needed. Currently, infectious models are only available for G1 and G2 HCV, and GV.1 for NoV. These viruses are between 20% to 40% divergent from the target G3a HCV and GII.4 NoV RdRps at the amino acid level. It is therefore essential to continue efforts to establish cell culture systems for both human NoVs, and G3-6 HCV, which would greatly facilitate antiviral discovery and development, as well as addressing fundamental questions about the replication and pathogenesis of these viral genotypes.

7.2.3 Past hit identification

Nearly 28% of all drugs approved today are enzyme-targeting molecules.340 Viral polymerases have long been an attractive target for antiviral development, given their importance for viral replication, and in most cases, the absence of human homologues, which minimises the possibility of undesirable side-effects. As a result of extensive research both from academic and industrial bodies, numerous polymerase inhibitors have been approved for the treatment of different viral infections, both NIs and NNIs, and for DNA and RNA viruses. A repertoire of DdDp, RdDp NIs and NNIs are now available for the treatment of HIV, HSV and HBV infections, reviewed in 331, and more recently the first RdRp NI Sofosbuvir was also approved for the treatment of HCV infections. 135

Although only available for two decades, HTS has been an extremely fruitful approach for the discovery of drugs to treat various diseases, including viral infections.341 HTS in this study led to the identification of chemical hits for the treatment and prevention of HCV and NoV. These molecules represent a good starting point towards a successful pre- clinical candidate (Figure 7.1). Hit-to-lead optimisation is a phase which typically follows hit identification, and could be carried out by two different strategies, each involving several key considerations. The process could be performed by synthesis or screening of analogues of the original hits to create “focus libraries”, an approach that was applied for HAC01 molecules, and the four NIC scaffolds. Alternatively, hit fragmentation could be applied to isolate the chemical moieties important for biological activity, which was briefly applied to characterise HAC02 scaffold in this study. Information obtained from these analyses can used to synthesise compounds with improved potency and selectivity (Figure 7.1).342 However, while compound potency is critical for hit selection in HTS, other factors arguably play a more important role in the process of hit-to-lead optimisation. For hits generated from HTS similar to those described in this thesis, these factors include binding mechanism, ease of chemical manipulation (i.e. synthetic accessibility), selectivity and ADME properties.342, 343 The awareness of these factors early on during drug development is crucial to avoid undesirable effects, and the significant costs associated with later stages of clinical trials.340 However, industrial factors such as patentability and market potential are of equal importance as the biological activity, and are key factors in the prospects of any developed drug, including antivirals.342, 343

Final remarks

In conclusion, this study describes the discovery and characterisation of the first non-nucleoside inhibitors specifically targeted against the RdRp of HCV G3a, a neglected but increasingly important pathogen. The efficacy of previously identified inhibitors against this RdRp was examined, and a novel mechanism of action for a subclass of these antivirals was described. This study also describes the identification of the first NoV-directed NNIs to form the basis of future rational drug design for antivirals against another pathogen with thus far limited control measures.

136

Figure 7.1. Pathways towards pre-clinical development of antivirals. The figure shows possible routes for the identification of antiviral molecules for any RNA virus. In the absence of cell culture models for viruses in this study, and the availability of a robust RdRp assay, HTS was conducted by in vitro screening for inhibitors of the HCV and NoV replication. The current stage achieved in this study within the overall path towards a pre-clinical candidate is highlighted in red.

137

138

8 Appendix

139

140

Figure 8.1 Nanospray mass spectrometry analysis of HAC molecules Mass spectra are shown for lots 1 and 2 of HAC01-01 (A), HAC02-01 (B), HAC03-01 (C) and HAC04-01 (D). Protonated and sodiated ions are detected for each molecule. However, an additional ion (m/z = 407) was detected for lot 1 but not lot 2 of HAC02-01. 141

Table 8.1 Inhibitory activity of HAC01 and HAC02 derivatives in the recombinant enzyme and replicon models*

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM] HAC01 compounds

HAC01-01 60.2 ± 5.6 60.7 ± 5.3 103.8 ± 4.32 26.30 19.5

HAC01-02 15.5 ± 2.9 23.0 ± 1.4 94.3 ± 4.4

HAC01-03 2.3 ± 7.0 77.3 ± 3.3 99.8 ± 2.0 22.3

HAC01-04 0.7 ± 0.2 15.0 ± 3.8 105.4 ± 6.9

HAC01-05 -2.0 ± 11.4 55.8 ± 7.9 97.2 ± 1.3

HAC01-06 -14.1 ± 7.7 39.7 ± 5.7 95.2 ± 3.0

142

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM]

HAC01-07 12.7 ± 5.7 -2.2 ± 2.1 104.2 ± 5.0

HAC01-08 15.7 ± 7.4 -13.7 ± 13.8 104.2 ± 3.6

HAC01-09 0.0 ± 5.1 -40.6 ± 3.6 101.3 ± 4.4

HAC01-10 -3.5 ± 6.7 -35.7 ± 3.0 105.5 ± 2.6

HAC01-11 9.9 ± 7.7 -23.6 ± 11.3 104.6 ± 1.3

HAC01-12 11.1 ± 8.4 -23.3 ± 12.8 106.0 ± 2.9

HAC01-13 16.3 ± 2.6 -22.8 ± 5.1 106.9 ± 0.7

HAC01-14 14.1 ± 5.8 -20.4 ± 14.3 104.6 ± 2.2

143

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM]

HAC01-15 26.1 ± 3.1 7.0 ± 9.5 105.9 ± 2.7

HAC01-16 -30.3 ± 3.4 -12.8 ± 16.4 106.2 ± 0.7

HAC01-17 0.0 ± 7.1 -13.2 ± 8.8 104.0 ± 1.3

HAC01-18 -6.3 ± 3.4 -18.1 ± 39.17 106.8 ± 1.8

HAC01-19 5.6 ± 5.6 -37.5 ± 12.16 104.2 ± 1.42

HAC01-20 -0.6 ± 4.3 -37.3 ± 8.66 103.6 ± 2.0

HAC01-21 2.3 ± 7.9 -16.1 ± 5.52 107.2 ± 3.1

HAC01-22 8.53 ± 2.8 -6.87 ± 9.74 107.4 ± 1.5

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM]

HAC01-23 16.62 ± 7.9 -18 ± 9.3 104.8 ± 0.5

HAC01-24 7.73 ± 2.0 -6.55 ± 1.91 106.4 ± 1.6

HAC02 compounds

HAC02-01 84.5 ± 2.6 12.7 ± 9.3 89.8 ± 0.3 12.8

HAC02-02 2.9 ± 3.7 -2.9 ± 8.7 82.9 ± 8.5

HAC02-03 12.3 ± 4.8 -13.1 ± 3.5 87.3 ± 9.6

HAC02-04 9.5 ± 11.2 2.5 ± 2.7 78.5 ± 17.5

HAC02-05 -0.2 ± 6.5 85.6 ± 3.7 96.2 ± 4.4 2.6

145

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM]

HAC02-06 11.0 ± 3.2 -18.2 ± 8.9 91.8 ± 4.1

HAC02-07 2.8 ± 9.2 -13.2 ± 15.9 77.8 ± 1.3

HAC02-08 13.6 ± 20 -17.9 ± 17.3 90.4 ± 2.7

HAC02-09 45.0 ± 5 -93.5 ± 8.1 121.6 ± 17 30.1

HAC02-10 3.9 ± 17.4 -72.3 ± 2.4 123.1 ± 10.5

HAC02-11 -3.2 ± 7.4 16.8 ± 3.8 96.6 ± 2.8

146

Enzyme Replicon Cell Viability Enzyme Replicon Compound Structure a b c Inhibition [%] Inhibition [%] [%] IC50 [µM] EC50 [µM]

HAC02-12 -23 ± 4.7 2.3 ± 11.2 99.4 ± 2.7

HAC02-13 -3.7 ± 5.1 -11.4 ± 23.0 98.4 ± 3.3

HAC02-14 16.7 ± 3.2 68.0 ± 12.3 78.7 ± 5.5

HAC02-15 21.4 ± 1.6 86.5 ± 7.3 63.9 ± 13.4

HAC02-16 -1.1 ± 0.8 72.2 ± 2.9 99.9 ± 3.4

HAC02-17 7.5 ± 15.9 -17.1 ± 2.7 95 ± 3.4

a Values obtained from an in vitro HCV G3a polymerase assay with 10µM compound b Values obtained from an HCV G1b replicon assay in Huh-7 cells with 20µM compound c Viability of Huh-7 cells after a 72 h treatment with 20µM compound compared to mock-treated cells * All results are shown as the average of triplicate experiments with standard deviation

147

Figure 8.2 Alignment of the RdRp amino acid sequences for HCV genotypes 1b, 2a and 3a Amino acid sequences were aligned using ClustalW and visualised in Geneious 344. The RdRp fingers, palm and thumb subdomains are indicated together with the C-terminal linker and the 21 amino acid transmembrane anchor, which is deleted for the expression of recombinant RdRp. Sequence similarity

148 between HCV genotype is indicated by yellow shading (60-80% similarity) whilst black shades indicates < 60% similarity. Numbers indicate amino acid residue position.

Figure 8.3. Effect of the HCV NNI Lomibuvir on the de novo RdRp activity. The de novo RdRp activity of HCV G1b and G3a RdRp was examined at increasing concentration of the T2 binder Lomibuvir, using the radioactive nucleotide incorporation assay. Lomibuvir enhanced the de novo activity of the G1b enzyme by > 100% at 10 µM, but not the G3a RdRp. At 100 µM, however, Lomibuvir inhibited the de novo activities of G1b and G3a RdRps by 41% and 81%, respectively, compared to control reactions. Results are shown as the average of triplicate experiments plotted with standard deviations.

149

Figure 8.4 Kinetics of NoV GII.4 RdRp activity in the presence of NIC 2 inhibitors. The activity of the NoV RdRp was tested at various concentrations of GTP (substrate) and inhibitor. For each of the substrate concentrations (0.2 to 66 μM), the inhibitor concentration was also varied from 0 to 20 μM. RdRp assays were performed in triplicate and presented as the average with standard deviations. The data was used to generate double reciprocal Lineweaver–Burk plots (Figure 6.4).

150

Figure 8.5 Core structures used for SAR analysis of the NoV RdRp inhibitors. Structures of the four leading compounds, without their functional groups, were used to identify analogue molecules from the compound library. These core structures are shown for NIC02 (C1), NIC04 (C2), NIC10 (C3) and NIC12 (C4).

151

Table 8.2 Inhibitory activity of NoV NNI analogues Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

NIC02 64.1 ± 1.0

WECC-0147676 NIC02 17.1 ± 0.4

WECC-0124682 NIC02 -1.6 ± 1.8

WECC-0000826 NIC02 -3.5 ± 4.5

NIC04 30.4 ± 7.5

WECC-0121840 NIC04 10.8 ± 16

WECC-0119984 NIC04 9.8 ± 9.7

WECC-0155998 NIC04 9.3 ± 0.8

WECC-0155058 NIC04 8.0 ± 2.8

WECC-0129793 NIC04 7.8 ± 9.2

WECC-0131157 NIC04 7.5 ± 10.2

WECC-0130545 NIC04 7.4 ± 1.0

152

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0159854 NIC04 7.1 ± 4.0

WECC-0131506 NIC04 6.8 ± 5.1

WECC-0133030 NIC04 6.1 ± 8.6

WECC-0157421 NIC04 5.8 ± 5.4

WECC-0155007 NIC04 5.3 ± 3.4

WECC-0124111 NIC04 5.2 ± 10.2

WECC-0151645 NIC04 5.2 ± 4.2

WECC-0141628 NIC04 5.0 ± 7.6

WECC-0135652 NIC04 4.7 ± 13

WECC-0153396 NIC04 4.6 ± 2.3

WECC-0132604 NIC04 4.3 ± 6.1

153

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0136981 NIC04 4.2 ± 6.7

WECC-0127545 NIC04 4.0 ± 6.4

WECC-0127616 NIC04 3.9 ± 9.0

WECC-0129123 NIC04 3.8 ± 4.6

WECC-0145342 NIC04 3.8 ± 4.6

WECC-0157113 NIC04 3.6 ± 2.7

WECC-0153684 NIC04 3.6 ± 5.3

WECC-0154797 NIC04 3.5 ± 1.9

WECC-0088299 NIC04 3.5 ± 9.5

WECC-0126967 NIC04 3.3 ± 6.3

WECC-0084573 NIC04 3.1 ± 7.4

154

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0138117 NIC04 3.0 ± 7.3

WECC-0124815 NIC04 2.9 ± 7.9

WECC-0138218 NIC04 2.7 ± 16.3

WECC-0141598 NIC04 2.6 ± 2.4

WECC-0158058 NIC04 2.5 ± 7.8

WECC-0155892 NIC04 2.1 ± 2.5

WECC-0124388 NIC04 2.0 ± 2.8

WECC-0141334 NIC04 2.0 ± 3.7

WECC-0154307 NIC04 1.9 ± 2.5

WECC-0118923 NIC04 1.7 ± 6.4

WECC-0087233 NIC04 1.5 ± 12.0

WECC-0087236 NIC04 1.1 ± 16.4

155

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0152270 NIC04 0.9 ± 10.8

WECC-0139047 NIC04 0.7 ± 11.8

WECC-0158431 NIC04 0.7 ± 3.5

WECC-0114628 NIC04 0.6 ± 7.3

WECC-0130765 NIC04 0.5 ± 11.7

WECC-0129673 NIC04 0.2 ± 7.1

WECC-0137813 NIC04 0 ± 3.3

WECC-0148245 NIC04 0 ± 0.8

WECC-0127985 NIC04 -0.3 ± 5.7

WECC-0157037 NIC04 -0.3 ± 5.3

WECC-0154971 NIC04 -0.3 ± 4.2

156

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0138748 NIC04 -0.4 ± 2.0

WECC-0159453 NIC04 -0.5 ± 5.1

WECC-0127208 NIC04 -0.7 ± 6.4

WECC-0137534 NIC04 -0.7 ± 7.2

WECC-0087169 NIC04 -0.7 ± 5.5

WECC-0133646 NIC04 -0.8 ± 5.9

WECC-0004464 NIC04 -0.8 ± 5.9

WECC-0122129 NIC04 -0.9 ± 2.5

WECC-0140492 NIC04 -0.9 ± 2.7

WECC-0153988 NIC04 -1.0 ± 9.3

WECC-0087065 NIC04 -1.1 ± 3.5

157

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0136288 NIC04 -1.2 ± 4.4

WECC-0153838 NIC04 -1.2 ± 11.8

WECC-0121462 NIC04 -1.2 ± 6.4

WECC-0084669 NIC04 -1.7 ± 8.0

WECC-0138256 NIC04 -1.8 ± 1.3

WECC-0137579 NIC04 -2.0 ± 9.1

WECC-0130415 NIC04 -2.4 ± 5.4

WECC-0087067 NIC04 -2.5 ± 1.6

WECC-0143711 NIC04 -2.7 ± 5.0

WECC-0122543 NIC04 -2.7 ± 5.8

WECC-0152365 NIC04 -2.9 ± 4.3

WECC-0151606 NIC04 -3.0 ± 3.3

158

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0159049 NIC04 -3.1 ± 3.1

WECC-0154458 NIC04 -3.1 ± 3.2

WECC-0123651 NIC04 -3.4 ± 9.5

WECC-0149289 NIC04 -3.4 ± 4.0

WECC-0125513 NIC04 -3.4 ± 2.6

WECC-0084725 NIC04 -3.6 ± 7.9

WECC-0156093 NIC04 -3.9 ± 4.9

WECC-0147229 NIC04 -4.1 ± 5.1

WECC-0133556 NIC04 -4.2 ± 5.1

WECC-0086989 NIC04 -4.3 ± 5.9

WECC-0148560 NIC04 -4.3 ± 1.6

WECC-0158829 NIC04 -4.6 ± 4.7

WECC-0142703 NIC04 -4.7 ± 5.1

159

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0142013 NIC04 -5.1 ± 4.3

WECC-0003117 NIC04 -5.2 ± 3.1

WECC-0123846 NIC04 -5.2 ± 3.7

WECC-0151472 NIC04 -5.6 ± 3.0

WECC-0121988 NIC04 -5.6 ± 3.2

WECC-0149206 NIC04 -5.7 ± 6.0

WECC-0131954 NIC04 -6.3 ± 2.4

WECC-0086990 NIC04 -6.4 ± 2.0

WECC-0143716 NIC04 -6.4 ± 10.5

WECC-0087232 NIC04 -7.1 ± 8.2

WECC-0086991 NIC04 -7.3 ± 2.1

WECC-0147453 NIC04 -7.6 ± 0.8

WECC-0149778 NIC04 -8.5 ± 12.1

160

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0145176 NIC04 -8.5 ± 9.7

WECC-0150495 NIC04 -8.5 ± 1.1

WECC-0150513 NIC04 -8.5 ± 5.8

WECC-0149135 NIC04 -8.9 ± 4.0

WECC-0120518 NIC04 -9.1 ± 0.8

WECC-0087380 NIC04 -9.4 ± 8.3

WECC-0146702 NIC04 -10.4 ± 7.8

WECC-0143988 NIC04 -11.1 ± 5.8

WECC-0157612 NIC04 -11.4 ± 2.0

WECC-0144033 NIC04 -11.5 ± 2.4

WECC-0151320 NIC04 -13.5 ± 4.4

161

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

NIC10 41.2 ± 0.5

WECC-0029330 NIC10 22.1 ± 13.2

WECC-0141606 NIC10 15.5 ± 14.8

WECC-0159361 NIC10 15.2 ± 18.2

WECC-0029329 NIC10 12.5 ± 6.1

WECC-0029328 NIC10 12.1 ± 16.4

WECC-0131788 NIC10 11.7 ± 6.4

WECC-0022218 NIC10 8.0 ± 4.5

WECC-0111306 NIC10 7.4 ± 4.2

162

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0029327 NIC10 6.7 ± 11

WECC-0007325 NIC10 5.8 ± 10.9

WECC-0155904 NIC10 5.2 ± 7.1

WECC-0007329 NIC10 3.8 ± 2.8

WECC-0020694 NIC10 3.5 ± 2.5

WECC-0000368 NIC10 1.8 ± 7.9

WECC-0150391 NIC10 1.8 ± 9.2

WECC-0140134 NIC10 1.7 ± 3.2

WECC-0018094 NIC10 1.6 ± 13.0

WECC-0115190 NIC10 0.9 ± 1.2

WECC-0110971 NIC10 -0.2 ± 2.7

WECC-0017416 NIC10 -0.2 ± 5.4

163

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0158809 NIC10 -0.7 ± 7.0

WECC-0015626 NIC10 -1.0 ± 2.3

WECC-0020931 NIC10 -2.2 ± 5.2

WECC-0009462 NIC10 -3.0 ± 6.8

WECC-0007328 NIC10 -3.7 ± 5.3

WECC-0007327 NIC10 -4.4 ± 3.0

WECC-0007320 NIC10 -4.7 ± 3.0

WECC-0056532 NIC10 -7.0 ± 4.9

WECC-0019921 NIC10 -9.1 ± 1.2

WECC-0018026 NIC10 -10.5 ± 18

WECC-0144671 NIC10 -10.6 ± 5.4

WECC-0015501 NIC10 -14.2 ± 16

WECC-0003882 NIC10 -22.1 ± 13.7

164

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

NIC12 47.8 ± 4.6

WECC-0056153 NIC12 75.7 ± 1.2

WECC-0056152 NIC12 61.8 ± 6.6

WECC-0041278 NIC12 54.5 ± 5.0

WECC-0055263 NIC12 35.2 ± 8.6

WECC-0037505 NIC12 32.7 ± 11.6

WECC-0041308 NIC12 32.0 ± 20.6

WECC-0041226 NIC12 31.4 ± 6.1

WECC-0041162 NIC12 30.3 ± 9.5

165

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0041342 NIC12 30.1 ± 7.0

WECC-0038991 NIC12 28.7 ± 6.2

WECC-0036746 NIC12 25.0 ± 2.5

WECC-0056747 NIC12 24.2 ± 4.7

WECC-0038666 NIC12 23.1 ± 4.8

WECC-0037742 NIC12 22.8 ± 5.4

WECC-0041320 NIC12 19.4 ± 9.5

WECC-0037399 NIC12 18.4 ± 4.6

166

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0041293 NIC12 17.9 ± 0.0

WECC-0037895 NIC12 17.7 ± 1.1

WECC-0041194 NIC12 16.2 ± 7.8

WECC-0036860 NIC12 15.9 ± 4.9

WECC-0038046 NIC12 15.6 ± 5.5

WECC-0038578 NIC12 13.4 ± 7.0

WECC-0056154 NIC12 13.1 ± 3.3

WECC-0038695 NIC12 12.4 ± 7.1

167

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0038714 NIC12 12.3 ± 0.4

WECC-0028330 NIC12 12.2 ± 4.9

WECC-0036826 NIC12 11.7 ± 1.1

WECC-0038507 NIC12 9.9 ± 9.1

WECC-0037083 NIC12 8.5 ± 2.7

WECC-0038688 NIC12 6.8 ± 4.3

WECC-0037573 NIC12 4.7 ± 1.2

168

Parent NoV RdRp Inhibition Entry number Structure compound [% of control]*

WECC-0046425 NIC12 3.2 ± 8.8

WECC-0036872 NIC12 2.8 ± 4.4

WECC-0045626 NIC12 -1.7 ± 11.6

* Inhibition of the NoV GII.4 RdRp at a compound concentration of 10 µM ± SD

169

170

9 References

1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-62. 2. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. New England Journal of Medicine 1989;321:1494-500. 3. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. Journal of Hepatology 2006;45:529-38. 4. Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013;57:1333-42. 5. Dore GJ, Law M, MacDonald M, Kaldor JM. Epidemiology of hepatitis C virus infection in Australia. Journal of Clinical Virology 2003;26:171-184. 6. Razali K, Thein HH, Bell J, Cooper-Stanbury M, Dolan K, Dore G, George J, Kaldor J, Karvelas M, Li J. Modelling the hepatitis C virus epidemic in Australia. Drug and Alcohol Dependence 2007;91:228-235. 7. Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur RR, Magder LS, El Khoby T, Abdel-Wahab Y, Aly Ohn ES, Anwar W, Sallam I. The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. The Lancet 2000;355:887-91. 8. Ray SC, Arthur RR, Carella A, Bukh J, Thomas DL. Genetic epidemiology of hepatitis C virus throughout Egypt. Journal of Infectious Diseases 2000;182:698- 707. 9. Zoulim F, Chevallier M, Maynard M, Trepo C. Clinical consequences of hepatitis C virus infection. Reviews in Medical Virology 2003;13:57-68. 10. Alter MJ, Hadler SC, Judson FN, Mares A, Alexander WJ, Hu PY, Miller JK, Moyer LA, Fields HA, Bradley DW, et al. Risk factors for acute non-A, non-B hepatitis in the United States and association with hepatitis C virus infection. Journal of the American Medical Association 1990;264:2231-5. 11. Alter MJ. Prevention of spread of hepatitis C. Hepatology 2002;36:S93-8. 12. Simonsen L, Kane A, Lloyd J, Zaffran M, Kane M. Unsafe injections in the developing world and transmission of bloodborne pathogens: a review. Bull World Health Organ 1999;77:789-800. 13. Mosley JW, Operskalski EA, Tobler LH, Andrews WW, Phelps B, Dockter J, Giachetti C, Busch MP. Viral and host factors in early hepatitis C virus infection. Hepatology 2005;42:86-92. 14. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology 1997;26:15S-20S. 15. Hoofnagle JH. Course and outcome of hepatitis C. Hepatology 2002;36:S21-9. 16. Lavanchy D. Evolving epidemiology of hepatitis C virus. Clinical Microbiology and Infection 2011;17:107-15. 17. Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002;36:S35-S46. 171

18. Merican I, Sherlock S, McIntyre N, Dusheiko GM. Clinical, biochemical and histological features in patients with chronic hepatitis C virus infection. Quarterly Journal of Medicine 1993;86:119-25. 19. Stapleton JT, Foung S, Muerhoff AS, Bukh J, Simmonds P. The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae. Journal of General Virology 2011;92:233- 246. 20. Kapoor A, Simmonds P, Gerold G, Qaisar N, Jain K, Henriquez JA, Firth C, Hirschberg DL, Rice CM, Shields S. Characterization of a canine homolog of hepatitis C virus. Proceedings of the National Academy of Sciences 2011;108:11608-11613. 21. Lyons S, Kapoor A, Sharp C, Schneider BS, Wolfe ND, Culshaw G, Corcoran B, McGorum BC, Simmonds P. Nonprimate hepaciviruses in domestic horses, United Kingdom. Emerging Infectious Diseases 2012;18:1976. 22. Kapoor A, Simmonds P, Scheel TK, Hjelle B, Cullen JM, Burbelo PD, Chauhan LV, Duraisamy R, Leon MS, Jain K. Identification of Rodent Homologs of Hepatitis C Virus and Pegiviruses. MBio 2013;4. 23. Quan P-L, Firth C, Conte JM, Williams SH, Zambrana-Torrelio CM, Anthony SJ, Ellison JA, Gilbert AT, Kuzmin IV, Niezgoda M. Bats are a major natural reservoir for hepaciviruses and pegiviruses. Proceedings of the National Academy of Sciences 2013;110:8194-8199. 24. Simmonds P. The origin of hepatitis C virus. Hepatitis C Virus: From Molecular Virology to Antiviral Therapy: Springer, 2013:1-15. 25. Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. Journal of Virology 1992;66:1476-1483. 26. Wang C, Sarnow P, Siddiqui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. Journal of Virology 1993;67:3338-3344. 27. Lindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function. Nature 2005;436:933-8. 28. Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, Moradpour D. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. Journal of Virology 2003;77:5487-5492. 29. Bartenschlager R. Hepatitis c virus: from molecular virology to antiviral therapy: Springer, 2013. 30. Cuevas JM, Gonzalez-Candelas F, Moya A, Sanjuan R. Effect of ribavirin on the mutation rate and spectrum of hepatitis C virus in vivo. Journal of Virology 2009;83:5760-4. 31. Sanjuan R, Nebot MR, Chirico N, Mansky LM, Belshaw R. Viral mutation rates. Journal of Virology 2010;84:9733-48. 32. Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, Perelson AS. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-α therapy. Science 1998;282:103-107. 33. Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, Feinstone S, Halfon P, Inchauspe G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin IT, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ,

172

Widell A. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005;42:962-73. 34. Simmonds P. Genetic diversity and evolution of hepatitis C virus--15 years on. Journal of General Virology 2004;85:3173-88. 35. Martell M, Esteban J, Quer J, Genesca J, Weiner A, Esteban R, Guardia J, Gomez J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. Journal of Virology 1992;66:3225-3229. 36. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 2005;439:344-348. 37. Zein NN, Rakela J, Krawitt EL, Reddy KR, Tominaga T, Persing DH. Hepatitis C virus genotypes in the United States: epidemiology, pathogenicity, and response to interferon therapy. Annals of Internal Medicine 1996;125:634-639. 38. Yusim K, Fischer W, Yoon H, Thurmond J, Fenimore PW, Lauer G, Korber B, Kuiken C. Genotype 1 and global hepatitis C T-cell vaccines designed to optimize coverage of genetic diversity. Journal of General Virology 2010;91:1194-1206. 39. McOmish F, Yap PL, Dow BC, Follett EA, Seed C, Keller AJ, Cobain TJ, Krusius T, Kolho E, Naukkarinen R, et al. Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative survey. Journal of Clinical Microbiology 1994;32:884-92. 40. Nguyen MH, Keeffe EB. Prevalence and treatment of hepatitis C virus genotypes 4, 5, and 6. Clinical Gastroenterology and Hepatology 2005;3:S97-S101. 41. Chowdhury A, Santra A, Chaudhuri S, Dhali GK, Maity SG, Naik TN, Bhattacharya SK, Mazumder DN. Hepatitis C virus infection in the general population: a community-based study in West Bengal, India. Hepatology 2003;37:802-9. 42. Bassit L, Van Heuverswyn H, De Bosschere K, Nishiya AS, Carrilho FJ, Moraes CR, Sablon E. Comparative study of two anti-HCV screening tests in a large genotyped population of Brazilian dialysis patients. European Journal of Clinical Microbiology & Infectious Diseases 2002;21:404-6. 43. White PA, Zhai X, Carter I, Zhao Y, Rawlinson WD. Simplified hepatitis C virus genotyping by heteroduplex mobility analysis. Journal of Clinical Microbiology 2000;38:477-82. 44. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 2011;28:2731-2739. 45. Miller RH, Purcell RH. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proceedings of the National Academy of Sciences 1990;87:2057-2061. 46. Hwang SB, Park K-J, Kim Y-S, Sung YC, Lai M. Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology 1997;227:439-446. 47. Ferrari E, Wright-Minogue J, Fang JW, Baroudy BM, Lau JY, Hong Z. Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. Journal of Virology 1999;73:1649-54.

173

48. Yamashita T, Kaneko S, Shirota Y, Qin W, Nomura T, Kobayashi K, Murakami S. RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. Journal of Biological Chemistry 1998;273:15479-86. 49. Bressanelli S, Tomei L, Roussel A, Incitti I, Vitale RL, Mathieu M, De Francesco R, Rey FA. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proceedings of the National Academy of Sciences 1999;96:13034-9. 50. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Structural Biology 1999;6:937-43. 51. Ago H, Adachi T, Yoshida A, Yamamoto M, Habuka N, Yatsunami K, Miyano M. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 1999;7:1417-26. 52. Butcher SJ, Grimes JM, Makeyev EV, Bamford DH, Stuart DI. A mechanism for initiating RNA-dependent RNA polymerization. Nature 2001;410:235-240. 53. Bressanelli S, Tomei L, Rey FA, De Francesco R. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. Journal of Virology 2002;76:3482-3492. 54. Dutartre H, Boretto J, Guillemot JC, Canard B. A relaxed discrimination of 2′-O- methyl-GTP relative to GTP between de novo and elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B. Journal of Biological Chemistry 2005;280:6359-6368. 55. Behrens SE, Tomei L, De Francesco R. Identification and properties of the RNA- dependent RNA polymerase of hepatitis C virus. EMBO Journal 1996;15:12-22. 56. Lohmann V, Körner F, Herian U, Bartenschlager R. Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. Journal of Virology 1997;71:8416-8428. 57. Luo G, Hamatake RK, Mathis DM, Racela J, Rigat KL, Lemm J, Colonno RJ. De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. Journal of Virology 2000;74:851-63. 58. Zhong W, Uss AS, Ferrari E, Lau JY, Hong Z. De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. Journal of Virology 2000;74:2017-22. 59. Kao CC, Singh P, Ecker DJ. De novo initiation of viral RNA-dependent RNA synthesis. Virology 2001;287:251-60. 60. Harrus D, Ahmed-El-Sayed N, Simister PC, Miller S, Triconnet M, Hagedorn CH, Mahias K, Rey FA, Astier-Gin T, Bressanelli S. Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis. Journal of Biological Chemistry 2010;285:32906-32918. 61. Scrima N, Caillet-Saguy C, Ventura M, Harrus D, Astier-Gin T, Bressanelli S. Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405. Journal of Virology 2012;86:7107-17. 62. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999;285:110-3.

174

63. Guo J-T, Bichko VV, Seeger C. Effect of alpha interferon on the hepatitis C virus replicon. Journal of Virology 2001;75:8516-8523. 64. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science 2000;290:1972-1974. 65. Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. Journal of Virology 2003;77:3181-3190. 66. Krieger N, Lohmann V, Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. Journal of Virology 2001;75:4614- 4624. 67. Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. Journal of Virology 2002;76:2997-3006. 68. Yi M, Lemon SM. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells. Journal of Virology 2004;78:7904- 7915. 69. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 2003;125:1808-1817. 70. Targett-Adams P, McLauchlan J. Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. Journal of General Virology 2005;86:3075-3080. 71. Saeed M, Gondeau C, Hmwe S, Yokokawa H, Date T, Suzuki T, Kato T, Maurel P, Wakita T. Replication of hepatitis C virus genotype 3a in cultured cells. Gastroenterology 2013;144:56-58 e7. 72. Saeed M, Scheel TK, Gottwein JM, Marukian S, Dustin LB, Bukh J, Rice CM. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrobial Agents and Chemotherapy 2012;56:5365-73. 73. Lindenbach BD, Evans MJ, Syder AJ, Wölk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating Ja, Rice CM. Complete replication of hepatitis C virus in cell culture. Science 2005;309:623-6. 74. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wieland SF, Uprichard SL, Wakita T, Chisari FV. Robust hepatitis C virus infection in vitro. Proceedings of the National Academy of Sciences 2005;102:9294-9. 75. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Kräusslich H-G, Mizokami M, Bartenschlager R, Liang TJ. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature medicine 2005;11:791-6. 76. Ramirez S, Li YP, Jensen SB, Pedersen J, Gottwein JM, Bukh J. Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors. Hepatology 2013. 77. Date T, Kato T, Kato J, Takahashi H, Morikawa K, Akazawa D, Murayama A, Tanaka-Kaneko K, Sata T, Tanaka Y. Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone. Journal of Virology 2012;86:10805-10820.

175

78. Li Y-P, Ramirez S, Jensen SB, Purcell RH, Gottwein JM, Bukh J. Highly efficient full- length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proceedings of the National Academy of Sciences 2012;109:19757-19762. 79. Morikawa K, Tanaka Y, Tanaka‐Kaneko K, Sata T, Mizokami M, Wakita T. Replication and infectivity of a novel genotype 1b hepatitis C virus clone. Microbiology and Immunology 2012;56:308-317. 80. Pietschmann T, Kaul A, Koutsoudakis G, Shavinskaya A, Kallis S, Steinmann E, Abid K, Negro F, Dreux M, Cosset F-L. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proceedings of the National Academy of Sciences 2006;103:7408-7413. 81. Gottwein JM, Scheel TK, Hoegh AM, Lademann JB, Eugen–Olsen J, Lisby G, Bukh J. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 2007;133:1614-1626. 82. Yi M, Ma Y, Yates J, Lemon SM. Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. Journal of Virology 2007;81:629-638. 83. Gottwein JM, Scheel TK, Jensen TB, Ghanem L, Bukh J. Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses. Gastroenterology 2011;141:1067-79. 84. Scheel TK, Gottwein JM, Mikkelsen LS, Jensen TB, Bukh J. Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-alpha. Gastroenterology 2011;140:1032-42. 85. Jensen TB, Gottwein JM, Scheel TK, Hoegh AM, Eugen-Olsen J, Bukh J. Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. Journal of Infectious Diseases 2008;198:1756-1765. 86. Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009;49:1335-1374. 87. Craxì A. EASL Clinical Practice Guidelines: Management of hepatitis C virus infection. Journal of Hepatology 2011. 88. Mangia A, Santoro R, Minerva N, Ricci GL, Carretta V, Persico M, Vinelli F, Scotto G, Bacca D, Annese M. Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. New England Journal of Medicine 2005;352:2609-2617. 89. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. The Lancet 2001;358:958-65. 90. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Gonçales Jr FL, Häussinger D, Diago M, Carosi G, Dhumeaux D. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. New England Journal of Medicine 2002;347:975-982. 91. Manns MP, Wedemeyer H, Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications. Gut 2006;55:1350-9. 92. Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005;436:967-72. 93. Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology 2002;36:S237-S244. 176

94. Jacobson IM, McHutchison JG, Dusheiko G, Di Bisceglie AM, Reddy KR, Bzowej NH, Marcellin P, Muir AJ, Ferenci P, Flisiak R. Telaprevir for previously untreated chronic hepatitis C virus infection. New England Journal of Medicine 2011;364:2405-2416. 95. Sarrazin C, Hezode C, Zeuzem S, Pawlotsky JM. Antiviral strategies in hepatitis C virus infection. Journal of Hepatology 2012;56 Suppl 1:S88-100. 96. Poordad F, McCone Jr J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, Jacobson IM, Reddy KR, Goodman ZD, Boparai N. Boceprevir for untreated chronic HCV genotype 1 infection. New England Journal of Medicine 2011;364:1195-1206. 97. Gane EJ, Stedman CA, Hyland RH, Ding X, Svarovskaia E, Symonds WT, Hindes RG, Berrey MM. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. New England Journal of Medicine 2013;368:34-44. 98. Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, Schultz M, Davis MN, Kayali Z, Reddy KR. Sofosbuvir for previously untreated chronic hepatitis C infection. New England Journal of Medicine 2013. 99. Lawitz E, Lalezari JP, Hassanein T, Kowdley KV, Poordad FF, Sheikh AM, Afdhal NH, Bernstein DE, DeJesus E, Freilich B. Sofosbuvir in combination with peginterferon alfa-2a and ribavirin for non-cirrhotic, treatment-naive patients with genotypes 1, 2, and 3 hepatitis C infection: a randomised, double-blind, phase 2 trial. The Lancet Infectious Diseases 2013. 100. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Bös M, Cameron DR, Cartier M, Cordingley MG. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 2003;426:186-189. 101. Reiser M, Hinrichsen H, Benhamou Y, Reesink HW, Wedemeyer H, Avendano C, Riba N, Yong CL, Nehmiz G, Steinmann GG. Antiviral efficacy of NS3‐serine protease inhibitor BILN‐2061 in patients with chronic genotype 2 and 3 hepatitis C. Hepatology 2005;41:832-835. 102. Reesink HW, Zeuzem S, Weegink CJ, Forestier N, Van Vliet A, Van De Wetering De Rooij J, McNair L, Purdy S, Kauffman R, Alam J. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology 2006;131:997-1002. 103. Aghemo A, De Francesco R. New horizons in Hepatitis C antiviral therapy with direct-acting antivirals. Hepatology 2013. 104. Summa V, Ludmerer SW, McCauley JA, Fandozzi C, Burlein C, Claudio G, Coleman PJ, DiMuzio JM, Ferrara M, Di Filippo M. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrobial Agents and Chemotherapy 2012;56:4161-4167. 105. Petry A, Brainard DM, Van Dyck K, Nachbar RB, De Lepeleire I, Caro L, Stone JA, Sun P, Uhle M, Wagner F. Safety and antiviral activity of MK-5172, a novel HCV NS3/4a protease inhibitor with potent activity against known resistance mutants, in genotype 1 and 3 HCV-infected patients, In 61st Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), 2010. 106. Clark VC, Peter JA, Nelson DR. New therapeutic strategies in HCV: second‐ generation protease inhibitors. Liver International 2013;33:80-84. 107. Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T, Kobayashi K, Murakami S. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP)

177

NS5B and modulates RNA-dependent RNA polymerase activity. Journal of Biological Chemistry 2002;277:11149-11155. 108. Tellinghuisen TL, Foss KL, Treadaway JC, Rice CM. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. Journal of Virology 2008;82:1073-1083. 109. Tellinghuisen TL, Foss KL, Treadaway J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathogens 2008;4:e1000032. 110. Appel N, Zayas M, Miller S, Krijnse-Locker J, Schaller T, Friebe P, Kallis S, Engel U, Bartenschlager R. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathogens 2008;4:e1000035. 111. Macdonald A, Harris M. Hepatitis C virus NS5A: tales of a promiscuous protein. Journal of General Virology 2004;85:2485-2502. 112. Lan K-H, Sheu M-L, Hwang S-J, Yen S-H, Chen S-Y, Wu J-C, Wang Y-J, Kato N, Omata M, Chang F-Y. HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis. Oncogene 2002;21:4801. 113. Ghosh AK, Steele R, Meyer K, Ray R, Ray RB. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. Journal of General Virology 1999;80:1179-1183. 114. Lemm JA, O'Boyle D, Liu M, Nower PT, Colonno R, Deshpande MS, Snyder LB, Martin SW, Laurent DRS, Serrano-Wu MH. Identification of hepatitis C virus NS5A inhibitors. Journal of Virology 2010;84:482-491. 115. Conte I, Giuliano C, Ercolani C, Narjes F, Koch U, Rowley M, Altamura S, Francesco RD, Neddermann P, Migliaccio G. Synthesis and SAR of piperazinyl-< i> N- phenylbenzamides as inhibitors of hepatitis C virus RNA replication in cell culture. Bioorganic & Medicinal Chemistry Letters 2009;19:1779-1783. 116. Gao M. Antiviral activity and resistance of HCV NS5A replication complex inhibitors. Current Opinion in Virology 2013;3:514-520. 117. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun J-H, O’Boyle II DR. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010;465:96-100. 118. Fridell RA, Wang C, Sun JH, O'Boyle DR, Nower P, Valera L, Qiu D, Roberts S, Huang X, Kienzle B. Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS‐ 790052 in Humans: In Vitro and In Vivo Correlations. Hepatology 2011;54:1924- 1935. 119. Herlihy KJ, Graham JP, Kumpf R, Patick AK, Duggal R, Shi ST. Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors. Antimicrobial Agents and Chemotherapy 2008;52:3523-31. 120. Pawlotsky J-M, Najera I, Jacobson I. Resistance to mericitabine, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase. Antiviral Therapy 2012;17:411-423. 121. Scheel TK, Rice CM. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nature Medicine 2013;19:837-849. 122. Lawitz E, Nguyen T, Younes Z, Santoro J, Gitlin N, McEniry D, Chasen R, Goff J, Knox S, Kleber K. Valopicitabine (NM283) plus PEG-interferon in treatment-naive 178

hepatitis C patients with HCV genotype-1 infection: HCV RNA clearance during 24 weeks of treatment. Hepatology 2006;44:223A-223A. 123. Pockros P, Jensen D, Tsai N, Taylor R, Ramji A, Cooper C, Dickson R, Tice A, Stande S, Ipe D. First SVR data with the nucleoside analogue polymerase inhibitor Mericitabine (RG7128) combined with Peginterferon/Ribavirin in treatment- naive HCV g1/4 patients: interim analysis from the JUMP-C trial. Journal of Hepatology 2011;54:S538. 124. Gane E, Pockros P, Zeuzem S, Marcellin P, Shikhman A, Bernaards C, Yetzer E, Shulman N, Tong X, Najera I. Interferon-free treatment with a combination of Mericitabine and Danoprevir with or without Ribavirin in treatment-naive HCV genotype 1-infected patients. Journal of Hepatology 2012;56:S555-S556. 125. Caillet-Saguy C, Simister PC, Bressanelli S. An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non- nucleoside inhibitors. Journal of Molecular Biology 2011;414:370-384. 126. Haudecoeur R, Peuchmaur M, Ahmed-Belkacem A, Pawlotsky JM, Boumendjel A. Structure-activity relationships in the development of allosteric hepatitis C virus RNA-dependent RNA polymerase inhibitors: ten years of research. Medicinal Research Reviews 2013;33:934-84. 127. Chan L, Das SK, Reddy TJ, Poisson C, Proulx M, Pereira O, Courchesne M, Roy C, Wang W, Siddiqui A. Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides. Bioorganic & Medicinal Chemistry Letters 2004;14:793-796. 128. Li H, Tatlock J, Linton A, Gonzalez J, Jewell T, Patel L, Ludlum S, Drowns M, Rahavendran SV, Skor H. Discovery of (R)-6-cyclopentyl-6-(2-(2, 6-diethylpyridin- 4-yl) ethyl)-3-((5, 7-dimethyl-[1, 2, 4] triazolo [1, 5-a] pyrimidin-2-yl) methyl)-4- hydroxy-5, 6-dihydropyran-2-one (PF-00868554) as a potent and orally available hepatitis C virus polymerase inhibitor. Journal of Medicinal Chemistry 2009;52:1255-1258. 129. Gopalsamy A, Chopra R, Lim K, Ciszewski G, Shi M, Curran KJ, Sukits SF, Svenson K, Bard J, Ellingboe JW. Discovery of proline sulfonamides as potent and selective hepatitis C virus NS5b polymerase inhibitors. Evidence for a new NS5b polymerase binding site. Journal of Medicinal Chemistry 2006;49:3052-3055. 130. Nyanguile O, Pauwels F, Van den Broeck W, Boutton CW, Quirynen L, Ivens T, van der Helm L, Vandercruyssen G, Mostmans W, Delouvroy F, Dehertogh P, Cummings MD, Bonfanti JF, Simmen KA, Raboisson P. 1,5-benzodiazepines, a novel class of hepatitis C virus polymerase nonnucleoside inhibitors. Antimicrobial Agents and Chemotherapy 2008;52:4420-31. 131. Dhanak D, Duffy KJ, Johnston VK, Lin-Goerke J, Darcy M, Shaw AN, Gu B, Silverman C, Gates AT, Nonnemacher MR, Earnshaw DL, Casper DJ, Kaura A, Baker A, Greenwood C, Gutshall LL, Maley D, DelVecchio A, Macarron R, Hofmann GA, Alnoah Z, Cheng HY, Chan G, Khandekar S, Keenan RM, Sarisky RT. Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase. Journal of Biological Chemistry 2002;277:38322-7. 132. Kneteman NM, Howe AY, Gao T, Lewis J, Pevear D, Lund G, Douglas D, Mercer DF, Tyrrell DLJ, Immermann F. HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti‐hepatitis C virus activity In Vitro, in mice 179

with chimeric human livers, and in humans infected with hepatitis C virus. Hepatology 2009;49:745-752. 133. Vliegen I, Paeshuyse J, De Burghgraeve T, Lehman LS, Paulson M, Shih IH, Mabery E, Boddeker N, De Clercq E, Reiser H, Oare D, Lee WA, Zhong W, Bondy S, Purstinger G, Neyts J. Substituted imidazopyridines as potent inhibitors of HCV replication. Journal of Hepatology 2009;50:999-1009. 134. Shih IH, Vliegen I, Peng B, Yang H, Hebner C, Paeshuyse J, Purstinger G, Fenaux M, Tian Y, Mabery E, Qi X, Bahador G, Paulson M, Lehman LS, Bondy S, Tse W, Reiser H, Lee WA, Schmitz U, Neyts J, Zhong W. Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase. Antimicrobial Agents and Chemotherapy 2011;55:4196-203. 135. Vermehren J, Sarrazin C. New HCV therapies on the horizon. Clinical Microbiology and Infection 2011;17:122-34. 136. Chatterji U, Bobardt M, Selvarajah S, Yang F, Tang H, Sakamoto N, Vuagniaux G, Parkinson T, Gallay P. The isomerase active site of cyclophilin A is critical for hepatitis C virus replication. Journal of Biological Chemistry 2009;284:16998- 17005. 137. Kaul A, Stauffer S, Berger C, Pertel T, Schmitt J, Kallis S, Lopez MZ, Lohmann V, Luban J, Bartenschlager R. Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. PLoS Pathogens 2009;5:e1000546. 138. Yang F, Robotham JM, Nelson HB, Irsigler A, Kenworthy R, Tang H. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro. Journal of Virology 2008;82:5269-5278. 139. Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 2003;38:1282-1288. 140. Coelmont L, Hanoulle X, Chatterji U, Berger C, Snoeck J, Bobardt M, Lim P, Vliegen I, Paeshuyse J, Vuagniaux G. DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A. PLoS One 2010;5:e13687. 141. Paeshuyse J, Kaul A, De Clercq E, Rosenwirth B, Dumont JM, Scalfaro P, Bartenschlager R, Neyts J. The non‐immunosuppressive cyclosporin DEBIO‐025 is a potent inhibitor of hepatitis C virus replication in vitro. Hepatology 2006;43:761-770. 142. Flisiak R, Feinman SV, Jablkowski M, Horban A, Kryczka W, Pawlowska M, Heathcote JE, Mazzella G, Vandelli C, Nicolas‐Métral V. The cyclophilin inhibitor Debio 025 combined with PEG IFNα2a significantly reduces viral load in treatment‐naïve hepatitis C patients. Hepatology 2009;49:1460-1468. 143. Pawlotsky J-M, Sarin S, Foster G, Peng C-Y, Rasenack J, Flisiak R, Piratvisuth T, Wedemeyer H, Chuang W-L, Zhang W. Alisporivir plus Ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated HCV-gt2 or gt3 patients: SVR12 results from vital-1 phase 2b study. Journal of Hepatology 2012;56:S553. 144. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Watts L, Booten SL, Graham M, McKay R, Subramaniam A, Propp S, Lollo BA, Freier S, Bennett CF, Bhanot S,

180

Monia BP. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 2006;3:87-98. 145. Larsen L, Rosenstierne MW, Gaarn LW, Bagge A, Pedersen L, Dahmcke CM, Nielsen JH, Dalgaard LT. Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism. PLoS One 2011;6:e25997. 146. Jopling CL, Schütz S, Sarnow P. Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host & Microbe 2008;4:77-85. 147. Jangra RK, Yi M, Lemon SM. Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122. Journal of Virology 2010;84:6615-6625. 148. Jopling C. Regulation of hepatitis C virus by microRNA-122. Biochemical Society Transactions 2008;36:1220. 149. Shimakami T, Yamane D, Jangra RK, Kempf BJ, Spaniel C, Barton DJ, Lemon SM. Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex. Proceedings of the National Academy of Sciences 2012;109:941-946. 150. Li Y-P, Gottwein JM, Scheel TK, Jensen TB, Bukh J. MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR. Proceedings of the National Academy of Sciences 2011;108:4991-4996. 151. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, Kauppinen S, Ørum H. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010;327:198-201. 152. Janssen HL, Reesink HW, Zeuzem S, Lawitz E, Rodriguez-Torres M, Chen A, Davis C, King B, Levin AA, Hodges MR. A randomized, double-blind, placebo (plb) controlled safety and anti-viral proof of concept study of miravirsen (MIR), an oligonucleotide targeting miR-122, in treatment naïve patients with genotype 1 (gt1) chronic HCV infection. Hepatology 2011;54:1430A-1430A. 153. Zeisel MB, Lupberger J, Fofana I, Baumert TF. Host-targeting agents for prevention and treatment of viral hepatitis C-perspectives and challenges. Journal of Hepatology 2012. 154. Gerold G, Pietschmann T. Opportunities and Risks of Host-targeting Antiviral Strategies for Hepatitis C. Current Hepatitis Reports 2013;12:200-213. 155. Schinazi R, Halfon P, Marcellin P, Asselah T. HCV direct‐acting antiviral agents: the best interferon‐free combinations. Liver International 2014;34:69-78. 156. Rubbia-Brandt L, Quadri R, Abid K, Giostra E, Malé P-J, Mentha G, Spahr L, Zarski J-P, Borisch B, Hadengue A. Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3. Journal of Hepatology 2000;33:106-115. 157. Hui JM, Kench J, Farrell GC, Lin R, Samarasinghe D, Liddle C, Byth K, George J. Genotype‐specific mechanisms for hepatic steatosis in chronic hepatitis C infection. Journal of Gastroenterology and Hepatology 2002;17:873-881. 158. Adinolfi LE, Gambardella M, Andreana A, Tripodi Mf, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001;33:1358-1364.

181

159. Westin J, Nordlinder H, Lagging M, Norkrans G, Wejstål R. Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients. Journal of Hepatology 2002;37:837-842. 160. Bochud P-Y, Cai T, Overbeck K, Bochud M, Dufour J-F, Müllhaupt B, Borovicka J, Heim M, Moradpour D, Cerny A. Genotype 3 is associated with accelerated fibrosis progression in chronic hepatitis C. Journal of Hepatology 2009;51:655- 666. 161. Goossens N, Negro F. Is the genotype 3 of the hepatitis C virus the new villain? Hepatology 2013. 162. Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura Y, Eder G, Schaff Z, Chapman M, Miyamura T. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proceedings of the National Academy of Sciences 1997;94:1200-1205. 163. Hourioux C, Patient R, Morin A, Blanchard E, Moreau A, Trassard S, Giraudeau B, Roingeard P. The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model. Gut 2007;56:1302-1308. 164. Zein NN. Clinical significance of hepatitis C virus genotypes. Clinical Microbiology Reviews 2000;13:223-235. 165. Imhof I, Simmonds P. Genotype differences in susceptibility and resistance development of hepatitis C virus to protease inhibitors telaprevir (VX‐950) and danoprevir (ITMN‐191). Hepatology 2011;53:1090-1099. 166. Foster GR, Hezode C, Bronowicki JP, Carosi G, Weiland O, Verlinden L, van Heeswijk R, van Baelen B, Picchio G, Beumont M. Telaprevir alone or with peginterferon and ribavirin reduces HCV RNA in patients with chronic genotype 2 but not genotype 3 infections. Gastroenterology 2011;141:881-889 e1. 167. Yu M, Corsa AC, Xu S, Peng B, Gong R, Lee Y-J, Chan K, Mo H, Delaney IV W, Cheng G. In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons. Antiviral Research 2013;100:439-445. 168. Dore G, Lawitz E, Hezode C, Shafran S, Ramji A, Tatum H, Taliani G, Tran A, Brunetto M, Zaltron S. Daclatasvir combined with peginterferon alfa-2a and ribavirin for 12 or 16 weeks in patients with HCV genotype 2 or 3 infection: COMMAND GT2/3 study. The 48th Annual Meeting of the European Association for the Study of the Liver 2013. 169. Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Niu C, Zennou V, Keilman M, Zhu Y, Lan S, Otto MJ, Furman PA. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrobial Agents and Chemotherapy 2012;56:3359-68. 170. Jacobson IM, Gordon SC, Kowdley KV, Yoshida EM, Rodriguez-Torres M, Sulkowski MS, Shiffman ML, Lawitz E, Everson G, Bennett M. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. New England Journal of Medicine 2013. 171. Jones LA. Aptamers to the hepatitis C virus. Volume PhD. Sydney: University of New South Wales, 2006. 172. Mathers CD, Fat DM, Boerma J. The global burden of disease: 2004 update: World Health Organization, 2008. 182

173. Bryce J, Boschi-Pinto C, Shibuya K, Black RE. WHO estimates of the causes of death in children. The Lancet 2005;365:1147-1152. 174. Hellard ME, Sinclair M, Harris AH, Kirk M, Fairley CK. Cost of community gastroenteritis. Journal of Gastroenterology and Hepatology 2003;18:322-328. 175. Majowicz S, McNab W, Sockett P, Henson S, Dore K, Edge V, Buffett M, Fazil A, Read S, McEwen S. Burden and cost of gastroenteritis in a Canadian community. Journal of Food Protection 2006;69:651-659. 176. Hoffmann S, Batz MB, Morris Jr JG. Annual cost of illness and quality-adjusted life year losses in the United States due to 14 foodborne pathogens. Journal of Food Protection 2012;75:1292-1302. 177. Scharff RL. Economic burden from health losses due to foodborne illness in the United States. Journal of Food Protection 2012;75:123-131. 178. Elliott EJ. Acute gastroenteritis in children. British Medical Journal 2007;334:35. 179. Dey A, Wang H, Menzies R, Macartney K. Changes in hospitalisations for acute gastroenteritis in Australia after the national rotavirus vaccination program. Medical Journal of Australia 2012;197:453. 180. Macartney KK, Porwal M, Dalton D, Cripps T, Maldigri T, Isaacs D, Kesson A. Decline in rotavirus hospitalisations following introduction of Australia's national rotavirus immunisation programme. Journal of Paediatrics and Child Health 2011;47:266-270. 181. Curns AT, Steiner CA, Barrett M, Hunter K, Wilson E, Parashar UD. Reduction in acute gastroenteritis hospitalizations among US children after introduction of rotavirus vaccine: analysis of hospital discharge data from 18 US states. Journal of Infectious Diseases 2010;201:1617-1624. 182. Estes MK, Prasad BV, Atmar RL. Noroviruses everywhere: has something changed? Current Opinion in Infectious Diseases 2006;19:467-474. 183. Patel MM, Widdowson M-A, Glass RI, Akazawa K, Vinjé J, Parashar UD. Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerging Infectious Diseases 2008;14:1224. 184. Gordon I, Ingraham HS, Korns RF. Transmission of epidemic gastroenteritis to human volunteers by oral administration of fecal filtrates. The Journal of Experimental Medicine 1947;86:409-422. 185. Dolin R, Blacklow NR, DuPont H, Formal S, Buscho RF, Kasel JA, Chames RP, Hornick R, Chanock RM. Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. Journal of Infectious Diseases 1971;123:307-312. 186. Blacklow NR, Dolin R, Fedson DS, Dupont H, Northrup RS, Hornick RB, Chanock RM. Acute infectious nonbacterial gastroenteritis: etiology and pathogenesis. Annals of Internal Medicine 1972;76:993-1008. 187. Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, Chanock RM. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. Journal of Virology 1972;10:1075-1081. 188. Hall AJ, Vinjé J, Lopman B, Park GW, Yen C, Gregoricus N, Parashar U. Updated norovirus outbreak management and disease prevention guidelines: US Department of Health and Human Services, Centers for Disease Control and Prevention, 2011.

183

189. Control CfD, Prevention. Surveillance for foodborne disease outbreaks—United States, 2009-2010. Annals of Emergency Medicine 2013;62:91-93. 190. Teunis PF, Moe CL, Liu P, Miller SE, Lindesmith L, Baric RS, Le Pendu J, Calderon RL. Norwalk virus: how infectious is it? J Med Virol 2008;80:1468-76. 191. Atmar RL, Opekun AR, Gilger MA, Estes MK, Crawford SE, Neill FH, Ramani S, Hill H, Ferreira J, Graham DY. Determination of the Human Infectious Dose-50% for Norwalk Virus. Journal of Infectious Diseases 2013:jit620. 192. Tu ET-V, Bull RA, Greening GE, Hewitt J, Lyon MJ, Marshall JA, McIver CJ, Rawlinson WD, White PA. Epidemics of gastroenteritis during 2006 were associated with the spread of norovirus GII. 4 variants 2006a and 2006b. Clinical Infectious Diseases 2008;46:413-420. 193. Tu ET, Bull RA, Kim MJ, McIver CJ, Heron L, Rawlinson WD, White PA. Norovirus excretion in an aged-care setting. Journal of Clinical Microbiology 2008;46:2119- 21. 194. Atmar RL, Opekun AR, Gilger MA, Estes MK, Crawford SE, Neill FH, Graham DY. Norwalk virus shedding after experimental human infection. Emerging Infectious Diseases 2008;14:1553. 195. Gallimore CI, Cubitt D, du Plessis N, Gray JJ. Asymptomatic and symptomatic excretion of noroviruses during a hospital outbreak of gastroenteritis. Journal of Clinical Microbiology 2004;42:2271-2274. 196. Isakbaeva ET, Widdowson M-A, Beard RS, Bulens SN, Mullins J, Monroe SS, Bresee J, Sassano P, Cramer EH, Glass RI. Norovirus transmission on cruise ship. Emerging Infectious Diseases 2005;11:154. 197. Green KY. Caliciviridae: the noroviruses. Fields Virology 2007;5:949-79. 198. Bok K, Green KY. Norovirus gastroenteritis in immunocompromised patients. New England Journal of Medicine 2012;367:2126-2132. 199. Krones E, Högenauer C. Diarrhea in the immunocompromised patient. Gastroenterology Clinics of North America 2012;41:677. 200. Bull RA, Eden JS, Luciani F, McElroy K, Rawlinson WD, White PA. Contribution of intra- and interhost dynamics to norovirus evolution. Journal of Virology 2012;86:3219-29. 201. Xi J, Graham DY, Wang K, Estes MK. Norwalk virus genome cloning and characterization. Science 1990;250:1580-1583. 202. Jiang X, Wang M, Wang K, Estes MK. Sequence and genomic organization of Norwalk virus. Virology 1993;195:51-61. 203. Scheffler U, Rudolph W, Gebhardt J, Rohayem J. Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease. Journal of General Virology 2007;88:2013-2018. 204. Goodfellow I, Chaudhry Y, Gioldasi I, Gerondopoulos A, Natoni A, Labrie L, Laliberté J-F, Roberts L. Calicivirus translation initiation requires an interaction between VPg and eIF4E. EMBO Reports 2005;6:968-972. 205. Daughenbaugh KF, Fraser CS, Hershey JW, Hardy ME. The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment. EMBO Journal 2003;22:2852-2859. 206. Morales M, Bárcena J, Ramírez MA, Boga JA, Parra F, Torres JM. Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (-

184

)sense genomic RNA: mapping of a subgenomic promoter. Journal of Biological Chemistry 2004;279:17013-17018. 207. Vinjé J, Hamidjaja RA, Sobsey MD. Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. Journal of Virological Methods 2004;116:109-117. 208. Zheng D-P, Ando T, Fankhauser RL, Beard RS, Glass RI, Monroe SS. Norovirus classification and proposed strain nomenclature. Virology 2006;346:312-323. 209. Kroneman A, Vega E, Vennema H, Vinjé J, White PA, Hansman G, Green K, Martella V, Katayama K, Koopmans M. Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology 2013:1-10. 210. Mattison K, Shukla A, Cook A, Pollari F, Friendship R, Kelton D, Bidawid S, Farber JM. Human noroviruses in swine and cattle. Emerging Infectious Diseases 2007;13:1184. 211. Wolf S, Williamson W, Hewitt J, Lin S, Rivera-Aban M, Ball A, Scholes P, Savill M, Greening GE. Molecular detection of norovirus in sheep and pigs in New Zealand farms. Veterinary Microbiology 2009;133:184-189. 212. Karst SM, Wobus CE, Lay M, Davidson J, Virgin HW. STAT1-dependent innate immunity to a Norwalk-like virus. Science 2003;299:1575-1578. 213. Martella V, Lorusso E, Decaro N, Elia G, Radogna A, D’Abramo M, Desario C, Cavalli A, Corrente M, Camero M. Detection and molecular characterization of a canine norovirus. Emerging Infectious Diseases 2008;14:1306. 214. Wang Q-H, Han MG, Cheetham S, Souza M, Funk JA, Saif LJ. Porcine noroviruses related to human noroviruses. Emerging Infectious Diseases 2005;11:1874. 215. Martella V, Campolo M, Lorusso E, Cavicchio P, Camero M, Bellacicco AL, Decaro N, Elia G, Greco G, Corrente M. Norovirus in captive lion cub (Panthera leo). Emerging Infectious Diseases 2007;13:1071. 216. Hyde JL, Sosnovtsev SV, Green KY, Wobus C, Virgin HW, Mackenzie JM. Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway. Journal of Virology 2009;83:9709-9719. 217. Lee J-H, Alam I, Han KR, Cho S, Shin S, Kang S, Yang JM, Kim KH. Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase. Journal of General Virology 2011;92:1607-1616. 218. Ng KK-S, Pendás-Franco N, Rojo J, Boga JA, Machín À, Alonso JMM, Parra F. Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft. Journal of Biological Chemistry 2004;279:16638-16645. 219. Högbom M, Jäger K, Robel I, Unge T, Rohayem J. The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity. Journal of General Virology 2009;90:281-91. 220. Rohayem J, Robel I, Jäger K, Scheffler U, Rudolph W. Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. Journal of Virology 2006;80:7060- 9. 221. Zamyatkin DF, Parra F, Alonso JMM, Harki DA, Peterson BR, Grochulski P, Ng KK- S. Structural insights into mechanisms of catalysis and inhibition in Norwalk virus polymerase. Journal of Biological Chemistry 2008;283:7705-7712.

185

222. Bull RA, Hyde J, Mackenzie JM, Hansman GS, Oka T, Takeda N, White PA. Comparison of the replication properties of murine and human calicivirus RNA- dependent RNA polymerases. Virus Genes 2011;42:16-27. 223. Eden J-S, Sharpe LJ, White Pa, Brown AJ. Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt. Journal of Virology 2011;85:10894-8. 224. Eden J-S, Hewitt J, Lim KL, Boni MF, Merif J, Greening G, Ratcliff RM, Holmes EC, Tanaka MM, Rawlinson WD. The emergence and evolution of the novel epidemic norovirus GII. 4 variant Sydney 2012. Virology 2014;450:106-113. 225. Zheng D-P, Widdowson M-A, Glass RI, Vinjé J. Molecular epidemiology of genogroup II-genotype 4 noroviruses in the United States between 1994 and 2006. Journal of Clinical Microbiology 2010;48:168-177. 226. Bok K, Abente EJ, Realpe-Quintero M, Mitra T, Sosnovtsev SV, Kapikian AZ, Green KY. Evolutionary dynamics of GII. 4 noroviruses over a 34-year period. Journal of Virology 2009;83:11890-11901. 227. Bull RA, White PA. Mechanisms of GII. 4 norovirus evolution. Trends in Microbiology 2011;19:233-240. 228. Noel J, Fankhauser R, Ando T, Monroe S, Glass R. Identification of a distinct common strain of “Norwalk-like viruses” having a global distribution. Journal of Infectious Diseases 1999;179:1334-1344. 229. Widdowson M-A, Cramer EH, Hadley L, Bresee JS, Beard RS, Bulens SN, Charles M, Chege W, Isakbaeva E, Wright JG. Outbreaks of acute gastroenteritis on cruise ships and on land: identification of a predominant circulating strain of norovirus—United States, 2002. Journal of Infectious Diseases 2004;190:27-36. 230. Bull RA, Tu ET, McIver CJ, Rawlinson WD, White PA. Emergence of a new norovirus genotype II.4 variant associated with global outbreaks of gastroenteritis. Journal of Clinical Microbiology 2006;44:327-33. 231. Yen C, Wikswo ME, Lopman BA, Vinje J, Parashar UD, Hall AJ. Impact of an emergent norovirus variant in 2009 on norovirus outbreak activity in the United States. Clinical Infectious Diseases 2011;53:568-571. 232. van Beek J, Ambert-Balay K, Botteldoorn N, Eden J, Fonager J, Hewitt J, Iritani N, Kroneman A, Vennema H, Vinje J. Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II. 4, late 2012. Euro Surveillance 2013;18:8-9. 233. Eden J-S, Tanaka MM, Boni MF, Rawlinson WD, White PA. Recombination within the pandemic norovirus GII. 4 lineage. Journal of Virology 2013;87:6270-6282. 234. Lindesmith LC, Donaldson EF, LoBue AD, Cannon JL, Zheng D-P, Vinje J, Baric RS. Mechanisms of GII. 4 norovirus persistence in human populations. PLoS Medicine 2008;5:e31. 235. Lindesmith LC, Donaldson EF, Baric RS. Norovirus GII. 4 strain antigenic variation. Journal of Virology 2011;85:231-242. 236. Bull Ra, Eden J-S, Rawlinson WD, White Pa. Rapid evolution of pandemic noroviruses of the GII.4 lineage. PLoS Pathogens 2010;6:e1000831. 237. Lindesmith LC, Beltramello M, Donaldson EF, Corti D, Swanstrom J, Debbink K, Lanzavecchia A, Baric RS. Immunogenetic mechanisms driving norovirus GII. 4 antigenic variation. PLoS Pathogens 2012;8:e1002705.

186

238. Bull RA, Hansman GS, Clancy LE, Tanaka MM, Rawlinson WD, White PA. Norovirus recombination in ORF1/ORF2 overlap. Emerging Infectious Diseases 2005;11:1079. 239. Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MP, Estes MK. Laboratory efforts to cultivate noroviruses. Journal of General Virology 2004;85:79-87. 240. Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, Krug A, Mackenzie JM, Green KY, Virgin HW. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biology 2004;2:e432. 241. Taube S, Kolawole AO, Hohne M, Wilkinson JE, Handley SA, Perry JW, Thackray LB, Akkina R, Wobus CE. A mouse model for human norovirus. MBio 2013;4. 242. Chang K-O, Sosnovtsev SV, Belliot G, King AD, Green KY. Stable expression of a Norwalk virus RNA replicon in a human hepatoma cell line. Virology 2006;353:463-73. 243. Guix S, Asanaka M, Katayama K, Crawford SE, Neill FH, Atmar RL, Estes MK. Norwalk virus RNA is infectious in mammalian cells. Journal of Virology 2007;81:12238-12248. 244. Kingsley DH, Vincent EM, Meade GK, Watson CL, Fan X. Inactivation of human norovirus using chemical sanitizers. International Journal of Food Microbiology 2014;171:94-99. 245. Barker J, Vipond I, Bloomfield S. Effects of cleaning and disinfection in reducing the spread of Norovirus contamination via environmental surfaces. Journal of Hospital Infection 2004;58:42-49. 246. O’Ryan M, Prado V, Pickering LK. A millennium update on pediatric diarrheal illness in the developing world, In Seminars in Pediatric Infectious Diseases, Elsevier, 2005. 247. Jiang X, Wang M, Graham D, Estes M. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. Journal of Virology 1992;66:6527-6532. 248. Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Estes MK. Humoral, mucosal, and cellular immune responses to oral Norwalk virus-like particles in volunteers. Clinical Immunology 2003;108:241-247. 249. Hutson AM, Atmar RL, Marcus DM, Estes MK. Norwalk virus-like particle hemagglutination by binding to H histo-blood group antigens. Journal of Virology 2003;77:405-415. 250. Marionneau S, Ruvoën N, Le Moullac–Vaidye B, Clement M, Cailleau–Thomas A, Ruiz–Palacois G, Huang P, Jiang X, Le Pendu J. Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals. Gastroenterology 2002;122:1967-1977. 251. Harrington PR, Lindesmith L, Yount B, Moe CL, Baric RS. Binding of Norwalk virus- like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice. Journal of Virology 2002;76:12335-12343. 252. Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, Lindblad L, Stewart P, LePendu J, Baric R. Human susceptibility and resistance to Norwalk virus infection. Nature Medicine 2003;9:548-553. 253. Bok K, Parra GI, Mitra T, Abente E, Shaver CK, Boon D, Engle R, Yu C, Kapikian AZ, Sosnovtsev SV. Chimpanzees as an animal model for human norovirus infection

187

and vaccine development. Proceedings of the National Academy of Sciences 2011;108:325-330. 254. Atmar RL, Bernstein DI, Harro CD, Al-Ibrahim MS, Chen WH, Ferreira J, Estes MK, Graham DY, Opekun AR, Richardson C. Norovirus vaccine against experimental human Norwalk Virus illness. New England Journal of Medicine 2011;365:2178- 2187. 255. Bernstein DI, Atmar RL, Lyon M, Treanor JJ, Chen WH, Frenck R, Jiang X, Vinje J, Al-Ibrahim MS, ChB JB, Graham DY, Richardson C, Goodwin R, Borkowski A, Clemens R, Mendelman PM. An Intramuscular (IM) Bivalent Norovirus GI.1/GII.4 Virus Like Particle (VLP) Vaccine Protects Against Vomiting and Diarrhea in an Experimental Human GII.4 Oral Challenge Study, In IDWeek 2013, San Francisco, CA, Oct. 2-6, 2013. 256. Tan M, Huang P, Xia M, Fang P-A, Zhong W, McNeal M, Wei C, Jiang W, Jiang X. Norovirus P particle, a novel platform for vaccine development and antibody production. Journal of Virology 2011;85:753-764. 257. Fang H, Tan M, Xia M, Wang L, Jiang X. Norovirus P Particle Efficiently Elicits Innate, Humoral and Cellular Immunity. PloS One 2013;8:e63269. 258. Parra GI, Bok K, Taylor R, Haynes JR, Sosnovtsev SV, Richardson C, Green KY. Immunogenicity and specificity of norovirus Consensus GII. 4 virus-like particles in monovalent and bivalent vaccine formulations. Vaccine 2012;30:3580-3586. 259. Rohayem J, Bergmann M, Gebhardt J, Gould E, Tucker P, Mattevi A, Unge T, Hilgenfeld R, Neyts J. Antiviral strategies to control calicivirus infections. Antiviral Research 2010;87:162-178. 260. Kim Y, Lovell S, Tiew K-C, Mandadapu SR, Alliston KR, Battaile KP, Groutas WC, Chang K-O. Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses. Journal of Virology 2012;86:11754-62. 261. Tiew K-C, He G, Aravapalli S, Mandadapu SR, Gunnam MR, Alliston KR, Lushington GH, Kim Y, Chang K-O, Groutas WC. Design, synthesis, and evaluation of inhibitors of Norwalk virus 3C protease. Bioorganic & Medicinal Chemistry Letters 2011;21:5315-9. 262. Rocha-Pereira J, Jochmans D, Dallmeier K, Leyssen P, Nascimento MSJ, Neyts J. Favipiravir (T-705) inhibits in vitro norovirus replication. Biochemical and Biophysical Research Communications 2012:5-8. 263. Costantini VP, Whitaker T, Barclay L, Lee D, McBrayer TR, Schinazi RF, Vinje J. Antiviral activity of nucleoside analogues against norovirus. Antiviral Therapy 2012;17:981-91. 264. Rocha-Pereira J, Jochmans D, Dallmeier K, Leyssen P, Cunha R, Costa I, Nascimento MSJ, Neyts J. Inhibition of norovirus replication by the nucleoside analogue 2'-C-methylcytidine. Biochemical and Biophysical Research Communications 2012:1-5. 265. Rocha-Pereira J, Jochmans D, Debing Y, Verbeken E, Nascimento MS, Neyts J. The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model. Journal of Virology 2013. 266. Furuta Y, Takahashi K, Fukuda Y, Kuno M, Kamiyama T, Kozaki K, Nomura N, Egawa H, Minami S, Watanabe Y, Narita H, Shiraki K. In vitro and in vivo activities 188

of anti-influenza virus compound T-705. Antimicrobial Agents and Chemotherapy 2002;46:977-81. 267. Arias A, Emmott E, Vashist S, Goodfellow I. Progress towards the prevention and treatment of norovirus infections. Future Microbiology 2013;8:1475-1487. 268. Hansman GS, Shahzad-ul-Hussan S, McLellan JS, Chuang G-Y, Georgiev I, Shimoike T, Katayama K, Bewley CA, Kwong PD. Structural basis for norovirus inhibition and fucose mimicry by citrate. Journal of Virology 2012;86:284-292. 269. Rademacher C, Guiard J, Kitov PI, Fiege B, Dalton KP, Parra F, Bundle DR, Peters T. Targeting norovirus infection—multivalent entry inhibitor design based on NMR experiments. Chemistry 2011;17:7442-7453. 270. Feng X, Jiang X. Library screen for inhibitors targeting norovirus binding to histo- blood group antigen receptors. Antimicrobial Agents and Chemotherapy 2007;51:324-331. 271. Perry JW, Ahmed M, Chang K-O, Donato NJ, Showalter HD, Wobus CE. Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response. PLoS Pathogens 2012;8:e1002783. 272. Kaufman SS, Green KY, Korba BE. Treatment of norovirus infections: Moving antivirals from the bench to the bedside. Antiviral Research 2014. 273. Jones LA, Clancy LE, Rawlinson WD, White PA. High-affinity aptamers to subtype 3a hepatitis C virus polymerase display genotypic specificity. Antimicrobial Agents and Chemotherapy 2006;50:3019-27. 274. Yi G, Deval J, Fan B, Cai H, Soulard C, Ranjith-Kumar CT, Smith DB, Blatt L, Beigelman L, Kao CC. Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrobial Agents and Chemotherapy 2012;56:830-7. 275. Zhang JH, Chung TDY, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening 1999;4:67-73. 276. Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High- throughput screening assays for the identification of chemical probes. Nature Chemical Biology 2007;3:466-79. 277. Vrolijk JM, Kaul A, Hansen BE, Lohmann V, Haagmans BL, Schalm SW, Bartenschlager R. A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C. Journal of Virological Methods 2003;110:201-9. 278. Jones DM, Domingues P, Targett-Adams P, McLauchlan J. Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication. Journal of General Virology 2010;91:2238-48. 279. Beaulieu PL, Bos M, Bousquet Y, Fazal G, Gauthier J, Gillard J, Goulet S, LaPlante S, Poupart MA, Lefebvre S, McKercher G, Pellerin C, Austel V, Kukolj G. Non- nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery and preliminary SAR of benzimidazole derivatives. Bioorganic & Medicinal Chemistry Letters 2004;14:119-24. 280. May MM, Lorengel H, Kreuter J, Zimmermann H, Ruebsamen-Schaeff H, Urban A. RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities. Biochimica et Biophysica Acta 2011;1814:1325-32. 189

281. Williams KP, Scott JE. Enzyme assay design for high-throughput screening. Methods in Molecular Biology 2009;565:107-26. 282. Canvas, version 1.8, Schrödinger, LLC, New York, NY, 2013. 283. Sastry M, Lowrie JF, Dixon SL, Sherman W. Large-Scale Systematic Analysis of 2D Fingerprint Methods and Parameters to Improve Virtual Screening Enrichments. Journal of Chemical Information and Modeling 2010;50:771-784. 284. Tomei L, Altamura S, Bartholomew L, Biroccio A, Ceccacci A, Pacini L, Narjes F, Gennari N, Bisbocci M, Incitti I, Orsatti L, Harper S, Stansfield I, Rowley M, De Francesco R, Migliaccio G. Mechanism of action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis C virus RNA-dependent RNA polymerase. Journal of Virology 2003;77:13225-31. 285. Bartels D, Jiang M, Zhang E, Tigges A, Sullivan J, Dorrian J, Spanks J, Ardzinski A, Nicolas O, Bedard J. Characterization of HCV variants in genotype 1 patients administered VX-222, a non-nucleoside polymerase inhibitor, In 5th International Workshop of Hepatitis C Resistance and New Compounds, Boston, MA, 2010. 286. Kim SS, Peng LF, Lin W, Choe WH, Sakamoto N, Kato N, Ikeda M, Schreiber SL, Chung RT. A cell-based, high-throughput screen for small molecule regulators of hepatitis C virus replication. Gastroenterology 2007;132:311-20. 287. Pauwels F, Mostmans W, Quirynen LM, van der Helm L, Boutton CW, Rueff AS, Cleiren E, Raboisson P, Surleraux D, Nyanguile O, Simmen KA. Binding-site identification and genotypic profiling of hepatitis C virus polymerase inhibitors. Journal of Virology 2007;81:6909-19. 288. Mestas SP, Sholders AJ, Peersen OB. A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity. Analytical Biochemistry 2007;365:194-200. 289. Tang G-Q, Anand VS, Patel SS. Fluorescence-based assay to measure the real- time kinetics of nucleotide incorporation during transcription elongation. Journal of Molecular Biology 2011;405:666-78. 290. Niyomrattanakit P, Abas SN, Lim CC, Beer D, Shi PY, Chen YL. A fluorescence- based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase. Journal of Biomolecular Screening 2011;16:201-10. 291. Jones LJ, Yue ST, Cheung C-Y, Singer VL. RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Analytical Biochemistry 1998;265:368-374. 292. Clemente-Casares P, Lopez-Jimenez AJ, Bellon-Echeverria I, Encinar JA, Martinez- Alfaro E, Perez-Flores R, Mas A. De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5. PLoS One 2011;6:e18515. 293. Baell JB, Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. Journal of Medicinal Chemistry 2010;53:2719-40. 294. Auld DS, Thorne N, Nguyen D-T, Inglese J. A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chemical Biology 2008;3:463-470. 295. Hebner CM, Han B, Brendza KM, Nash M, Sulfab M, Tian Y, Hung M, Fung W, Vivian RW, Trenkle J, Taylor J, Bjornson K, Bondy S, Liu X, Link J, Neyts J, Sakowicz 190

R, Zhong W, Tang H, Schmitz U. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS One 2012;7:e39163. 296. Chan L, Pereira O, Reddy TJ, Das SK, Poisson C, Courchesne M, Proulx M, Siddiqui A, Yannopoulos CG, Nguyen-Ba N. Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 2: tertiary amides. Bioorganic & Medicinal Chemistry Letters 2004;14:797- 800. 297. Hirashima S, Suzuki T, Ishida T, Noji S, Yata S, Ando I, Komatsu M, Ikeda S, Hashimoto H. Benzimidazole derivatives bearing substituted biphenyls as hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors: structure- activity relationship studies and identification of a potent and highly selective inhibitor JTK-109. Journal of Medicinal Chemistry 2006;49:4721-4736. 298. Rong F, Chow S, Yan S, Larson G, Hong Z, Wu J. Structure–activity relationship (SAR) studies of quinoxalines as novel HCV NS5B RNA-dependent RNA polymerase inhibitors. Bioorganic & Medicinal Chemistry Letters 2007;17:1663- 1666. 299. Lohmann V, Bartenschlager R. On the history of hepatitis C virus cell culture systems. J Med Chem 2014;57:1627-42. 300. Fenaux M, Eng S, Leavitt SA, Lee Y-J, Mabery EM, Tian Y, Byun D, Canales E, Clarke MO, Doerffler E. Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase. Antimicrobial Agents and Chemotherapy 2013;57:804-810. 301. Wong KA, Xu S, Martin R, Miller MD, Mo H. Tegobuvir (GS-9190) potency against HCV chimeric replicons derived from consensus NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a. Virology 2012;429:57-62. 302. Tomei L, Altamura S, Bartholomew L, Bisbocci M, Bailey C, Bosserman M, Cellucci A, Forte E, Incitti I, Orsatti L. Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides. Journal of Virology 2004;78:938-946. 303. Legrand-Abravanel F, Nicot F, Izopet J. New NS5B polymerase inhibitors for hepatitis C. Expert Opinion on Investigational Drugs 2010;19:963-75. 304. Thompson PA, Patel R, Showalter RE, Li C, Applemon JR, Steffy K. In vitro studies demonstrate that combinations of antiviral agents that include HCV polymerase inhibitor ANA598 have the potential to overcome viral resistance. Hepatology 2008;48:1164A. 305. Le Pogam S, Seshaadri A, Kosaka A, Chiu S, Kang H, Hu S, Rajyaguru S, Symons J, Cammack N, Nájera I. Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients. Journal of Antimicrobial Chemotherapy 2008;61:1205-1216. 306. Ahn J, Flamm SL. Frontiers in the Treatment of Hepatitis C Virus Infection. Gastroenterology & Hepatology 2014;10:91. 307. Delang L, Vliegen I, Froeyen M, Neyts J. Comparative study of the genetic barriers and pathways towards resistance of selective inhibitors of hepatitis C virus replication. Antimicrobial Agents and Chemotherapy 2011;55:4103-4113. 308. Rodriguez-Torres M, Lawitz E, Cohen D, Larsen LM, Menon R, Collins C, Marsh T, Gibbs S, Bernstein B. Treatment-naïve, HCV genotype 1-infected subjects show significantly greater HCV RNA decreases when treated with 28 days of ABT-333 191

plus peginterferon and ribavirin compared to peginterferon and ribavirin alone. Hepatology 2009;50:5A-5A. 309. Rydberg EH, Cellucci A, Bartholomew L, Mattu M, Barbato G, Ludmerer SW, Graham DJ, Altamura S, Paonessa G, De Francesco R. Structural basis for resistance of the genotype 2b hepatitis C virus NS5B polymerase to site A non- nucleoside inhibitors. Journal of Molecular Biology 2009;390:1048-1059. 310. Love RA, Parge HE, Yu X, Hickey MJ, Diehl W, Gao J, Wriggers H, Ekker A, Wang L, Thomson JA. Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme. Journal of Virology 2003;77:7575-7581. 311. Winquist J, Abdurakhmanov E, Baraznenok V, Henderson I, Vrang L, Danielson UH. Resolution of the interaction mechanisms and characteristics of non- nucleoside inhibitors of hepatitis C virus polymerase. Antiviral Res 2013;97:356- 68. 312. Biswal BK, Cherney MM, Wang M, Chan L, Yannopoulos CG, Bilimoria D, Nicolas O, Bedard J, James MNG. Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. Journal of Biological Chemistry 2005;280:18202-10. 313. Biswal BK, Wang M, Cherney MM, Chan L, Yannopoulos CG, Bilimoria D, Bedard J, James MN. Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition. Journal of Molecular Biology 2006;361:33-45. 314. Shim JH, Larson G, Wu JZ, Hong Z. Selection of 3′-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase. Journal of Virology 2002;76:7030-7039. 315. Ferrari E, He Z, Palermo RE, Huang H-C. Hepatitis C virus NS5B polymerase exhibits distinct nucleotide requirements for initiation and elongation. Journal of Biological Chemistry 2008;283:33893-33901. 316. Estes MK, Prasad BV, Atmar RL. Noroviruses everywhere: has something changed? Curr Opin Infect Dis 2006;19:467-74. 317. Eden JS, Bull RA, Tu E, McIver CJ, Lyon MJ, Marshall JA, Smith DW, Musto J, Rawlinson WD, White PA. Norovirus GII.4 variant 2006b caused epidemics of acute gastroenteritis in Australia during 2007 and 2008. Journal of Clinical Virology 2010;49:265-71. 318. Rocha-Pereira J, Cunha R, Pinto DC, Silva AM, Nascimento MS. (E)-2- styrylchromones as potential anti-norovirus agents. Bioorganic & Medicinal Chemistry 2010;18:4195-201. 319. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 2012;9:671-5. 320. Sykes ML, Baell JB, Kaiser M, Chatelain E, Moawad SR, Ganame D, Ioset J-R, Avery VM. Identification of compounds with anti-proliferative activity against Trypanosoma brucei brucei strain 427 by a whole cell viability based HTS campaign. PLoS Neglected Tropical Diseases 2012;6:e1896. 321. Campagnola G, Gong P, Peersen OB. High-throughput screening identification of poliovirus RNA-dependent RNA polymerase inhibitors. Antiviral Research 2011;91:241-251. 192

322. Zingaretti C, Francesco R, Abrignani S. Why it is so difficult to develop an HCV preventive vaccine? Clinical Microbiology and Infection 2013. 323. Eltahla AA, Lackovic K, Marquis C, Eden JS, White PA. A fluorescence-based high- throughput screen to identify small compound inhibitors of the genotype 3a hepatitis C virus RNA polymerase. Journal of Biomolecular Screening 2013;18:1027-34. 324. Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nature Reviews Microbiology 2013;11:482-496. 325. Sarrazin C, Zeuzem S. Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology 2010;138:447-462. 326. Legrand-Abravanel F, Henquell C, Le Guillou-Guillemette H, Balan V, Mirand A, Dubois M, Lunel-Fabiani F, Payan C, Izopet J. Naturally occurring substitutions conferring resistance to hepatitis C virus polymerase inhibitors in treatment- naive patients infected with genotypes 1-5. Antiviral Therapy 2009;14:723-30. 327. Maring C, Wagner R, Hutchinson D, Flentge C, Kati W, Koev G, Liu Y, Beno D, Shen J, Lau Y. Preclinical potency, pharmacokinetic and AMDE characterization of ABT- 333, a novel non-nucleoside HCV polymerase inhibitor. Journal of Hepatology 2009;50:S347. 328. Zeuzem S, Asselah T, Angus P, Zarski JP, Larrey D, Müllhaupt B, Gane E, Schuchmann M, Lohse A, Pol S. Efficacy of the protease inhibitor BI 201335, polymerase inhibitor BI 207127, and ribavirin in patients with chronic HCV infection. Gastroenterology 2011;141:2047-2055. 329. Zeuzem S, Soriano V, Asselah T, Bronowicki J-P, Lohse AW, Müllhaupt B, Schuchmann M, Bourlière M, Buti M, Roberts SK. Faldaprevir and deleobuvir for HCV genotype 1 infection. New England Journal of Medicine 2013;369:630-639. 330. Devogelaere B, Berke JM, Vijgen L, Dehertogh P, Fransen E, Cleiren E, van der Helm L, Nyanguile O, Tahri A, Amssoms K. TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage. Antimicrobial Agents and Chemotherapy 2012;56:4676-4684. 331. Coen DM, Richman DD. Antiviral Agents. Fields Virology 2013;6:339-369. 332. Schmitt M, Scrima N, Radujkovic D, Caillet-Saguy C, Simister PC, Friebe P, Wicht O, Klein R, Bartenschlager R, Lohmann V. A comprehensive structure-function comparison of hepatitis C virus strain JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes. Journal of Virology 2011;85:2565-2581. 333. Simister P, Schmitt M, Geitmann M, Wicht O, Danielson UH, Klein R, Bressanelli S, Lohmann V. Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. Journal of Virology 2009;83:11926-11939. 334. Wagner F, Thompson R, Kantaridis C, Simpson P, Troke PJ, Jagannatha S, Neelakantan S, Purohit VS, Hammond JL. Antiviral activity of the hepatitis C virus polymerase inhibitor filibuvir in genotype 1–infected patients. Hepatology 2011;54:50-59. 335. Wyles DL, Rodriguez‐Torres M, Lawitz E, Shiffman ML, Pol S, Herring RW, Massetto B, Kanwar B, Trenkle JD, Pang PS. All‐Oral Combination of Ledipasvir, Vedroprevir, Tegobuvir, and Ribavirin in Treatment‐Naive Patients with Genotype 1 HCV Infection. Hepatology 2014. 193

336. Le Pogam S, Kang H, Harris SF, Leveque V, Giannetti AM, Ali S, Jiang WR, Rajyaguru S, Tavares G, Oshiro C, Hendricks T, Klumpp K, Symons J, Browner MF, Cammack N, Najera I. Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus. Journal of Virology 2006;80:6146-54. 337. Jones G, Willett P, Glen RC. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. Journal of Molecular Biology 1995;245:43-53. 338. Glide, version 6.2. Schrödinger, LLC, New York, NY 2014. 339. SiteMap, version 2.9. Schrödinger, LLC, New York, NY 2013. 340. Bleicher KH, Böhm H-J, Müller K, Alanine AI. Hit and lead generation: beyond high-throughput screening. Nature Reviews Drug Discovery 2003;2:369-378. 341. Macarron R, Banks MN, Bojanic D, Burns DJ, Cirovic DA, Garyantes T, Green DV, Hertzberg RP, Janzen WP, Paslay JW. Impact of high-throughput screening in biomedical research. Nature Reviews Drug Discovery 2011;10:188-195. 342. Keserű GM, Makara GM. Hit discovery and hit-to-lead approaches. Drug Discovery Today 2006;11:741-748. 343. Hughes J, Rees S, Kalindjian S, Philpott K. Principles of early drug discovery. British Journal of Pharmacology 2011;162:1239-1249. 344. Drummond A, Ashton B, Buxton S, Cheung M, Cooper A. Geneious v6.1 created by Biomatters. Available: http://www.geneious.com: Accessed, 2013.

194