Discovery of safe anti dengue virus drugs from libraries of FDA approved drugs and plants through screening against viral RNA dependent RNA polymerase activity

DISCOVERY OF SAFE ANTI-DENGUE VIRUS DRUGS FROM LIBRARIES OF FDA-APPROVED DRUGS AND PLANTS THROUGH SCREENING AGAINST VIRAL RNA-DEPENDENT RNA POLYMERASE ACTIVITY EMELYNE QUEK JIANG LI (B.SC. HONS., NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________________________________ Emelyne Quek Jiang Li 12 September 2013 ACKNOWLEDGEMENTS I would like to extend my utmost heartfelt gratitude to my supervisors, Professor Naoki Yamamoto and Dr Youichi Suzuki for their guidance, patience, support and encouragement. Their passion and drive for research have truly been inspirational. I am especially thankful to Dr Koji Ichiyama for his advice and perpetual energy that has been a constant source of motivation, as well as Ms Chikako Takahashi and Dr Hirotaka Takahashi for their invaluable suggestions and help. I would also like to show appreciation to everyone at the Translational Infectious Disease Laboratory who made it an amazingly convivial place to work in. In particular, I would like to thank Beng Hui, Qi ‘En and Wei Xin for being such strong pillars of support and a bundle of joy throughout this entire journey. Last but not least, my deepest gratitude goes to my family – my father, Quee Huat, mother, Yeow Hiang, Aunt Catherine, my sisters Angeline and Jacqueline as well as Matthew and Raymond, for their unflagging love, staying up late nights with me to make sure I was never alone and always ensuring my emergency food stash remained bountiful. I would like to dedicate this thesis to my mother, who gave me the ambition to reach for the stars and provided opportunities and unwavering support in all my endeavours. Although she is no longer with us, I am sure she shares our joy from up above. i CONTENTS SUMMARY ..…………………………………………………………….…….….. VIII LIST OF ABBREVIATIONS …………………………………...……...…………... IX LIST OF FIGURES ....………..………………………………………………..…..… X LIST OF TABLES …………..…...……………………………………………..….. XII CHAPTER 1. 1.1 INTRODUCTION .............................................................................. 1 Dengue .............................................................................................................. 1 1.1.1 Burden of disease .......................................................................................... 2 1.1.2 Dengue virus ................................................................................................. 3 1.1.3 Dengue infection pathogenesis ..................................................................... 3 1.2 1.1.3.1 Dengue fever ......................................................................................... 3 1.1.3.2 Dengue hemorrhagic fever and dengue shock syndrome ..................... 4 Characteristics of DENV and DENV genome .................................................. 5 1.2.1 Structural proteins ......................................................................................... 7 1.2.1.1 Capsid (C) protein ................................................................................. 7 1.2.1.2 Pre-membrane (prM) and membrane (M) proteins............................... 7 1.2.1.3 Envelope (E) protein ............................................................................. 8 1.2.2 Non-structural (NS) proteins ........................................................................ 9 1.2.2.1 NS1 protein ........................................................................................... 9 1.2.2.2 NS2A protein ........................................................................................ 9 ii 1.3 1.2.2.3 NS2B protein ...................................................................................... 10 1.2.2.4 NS3 protein ......................................................................................... 11 1.2.2.5 NS4A protein ...................................................................................... 12 1.2.2.6 NS4B protein ...................................................................................... 13 1.2.2.7 NS5 protein ......................................................................................... 14 DENV replication cycle .................................................................................. 16 1.3.1 Entry and uncoating .................................................................................... 17 1.3.2 Translation and further processing ............................................................. 18 1.3.3 RNA replication .......................................................................................... 18 1.3.4 Assembly and release.................................................................................. 19 1.4 Anti-dengue efforts ......................................................................................... 19 1.4.1 Ideal characteristics of dengue antiviral drug ............................................. 22 1.5 NS5: An attractive anti-dengue drug target .................................................... 23 1.6 Types of NS5 RdRp inhibitors........................................................................ 25 1.7 Conceptualization of project ........................................................................... 26 1.7.1 Current state of in vitro RdRp assays ......................................................... 26 1.7.2 Current state of in vitro DENV NS5 protein production ............................ 28 1.7.3 Current state of pharmaceutical industry .................................................... 30 1.7.4 Recent advances in anti-DENV drug discovery ......................................... 34 1.8 Specific aims of project .................................................................................. 37 CHAPTER 2. 2.1 MATERIALS AND METHODS ..................................................... 38 Wheat germ cell-free protein expression ........................................................ 38 iii 2.1.1 Construction of template plasmid DNAs .................................................... 38 2.1.2 In vitro transcription ................................................................................... 38 2.1.3 In vitro translation ....................................................................................... 38 2.1.4 Protein affinity purification ........................................................................ 39 2.1.5 Buffer exchange .......................................................................................... 39 2.1.6 Protein concentration .................................................................................. 39 2.1.7 CBB analysis............................................................................................... 40 2.1.8 Western blotting analysis ............................................................................ 40 2.2 Preparation of drugs and compounds .............................................................. 41 2.2.1 Drug/Compound libraries for primary screening assay .............................. 41 2.2.2 Drugs for validation studies ........................................................................ 41 2.3 Fluorescence-based in vitro DENV NS5 RdRp assay .................................... 42 2.4 Cell culture ...................................................................................................... 43 2.4.1 General growth and maintenance ............................................................... 43 2.4.2 Viruses preparation ..................................................................................... 44 2.5 Validation of inhibition of DENV replication by drug/compound in cell-based infection system .............................................................................................. 45 2.5.1 Cell viability assay ...................................................................................... 45 2.5.2 Infection assay: Reduction of viral titer by drug/compound ...................... 45 2.5.3 Calculation of selectivity index (SI) ........................................................... 46 2.6 Cytopathic effect (CPE)-based anti-dengue assay .......................................... 46 2.7 Plaque reduction assay .................................................................................... 47 iv 2.8 Time of addition assay .................................................................................... 48 2.9 Binding assay .................................................................................................. 48 2.10 DENV replicon luciferase assay ..................................................................... 49 CHAPTER 3. 3.1 RESULTS .......................................................................................... 50 Production of DENV-2 NS5 protein by wheat germ cell-free protein synthesis system ............................................................................................................. 50 3.2 Development of fluorescence-based DENV NS5 RdRp assay using wheat germ cell-free system-produced NS5 proteins ................................................ 54 3.3 Screening of FDA-approved drug and natural compound libraries in fluorescence-based in vitro NS5 RdRp assay ................................................. 57 3.4 Summary of primary in vitro NS5 RdRp screening study and in vitro validation of hits ............................................................................................. 61 3.5 Secondary screening of top 8 inhibitors in in vitro NS5 RdRp assay using RdRp domain mutant protein .......................................................................... 65 3.6 Validation of inhibition of DENV replication by drug/compound in cell-based system ............................................................................................................. 69 3.7 Effects of kusunoki in CPE-based anti-dengue assay ..................................... 75 3.8 Inhibitory effect of kusunoki against 4 DENV serotypes ............................... 77 3.9 Mechanistic inhibitory action of kusunoki ..................................................... 80 CHAPTER 4. 4.1 DISCUSSION .................................................................................... 84 Production of NS5 protein using wheat germ cell free system ....................... 84 v 4.2 Development of fluorescence-based NS5 RdRp assay using wheat germ cellfree system-produced NS5 proteins ................................................................ 86 4.3 Screening libraries of FDA-approved drugs and natural compounds............. 88 4.4 Primary screening of libraries of FDA-approved drugs and natural compounds in in vitro NS5 RdRp assay............................................................................. 90 4.5 Secondary screening of libraries of FDA-approved drugs and natural compounds with RdRp domain mutant .......................................................... 93 4.6 Validation of inhibition of DENV replication by drug/compound in cell-based system ............................................................................................................. 95 4.7 Effects of kusunoki in CPE-based anti-dengue assay ..................................... 99 4.8 Inhibitory effect of kusunoki against 4 DENV serotypes ............................. 101 4.9 Mechanistic inhibitory action of kusunoki ................................................... 102 CHAPTER 5. FUTURE DIRECTIONS ................................................................ 106 5.1 Extension of screening libraries .................................................................... 106 5.2 Determination of active antiviral components in kusunoki PA extract ........ 106 5.3 Verification of RdRp inhibition .................................................................... 107 5.4 Determination of antiviral effects against other flaviviruses ........................ 107 5.5 Combination treatment ................................................................................. 108 CHAPTER 6. CONCLUSION ............................................................................... 109 6.1 Summary of study findings ........................................................................... 109 6.2 Future perspectives ....................................................................................... 110 vi CHAPTER 7. REFERENCES................................................................................ 111 vii SUMMARY Dengue virus (DENV), belonging to the Flaviviridae family and Flavivirus genus, is an arthropod-borne virus with four serotypes. Causing 390 million human infections annually, DENV infection can lead to life-threatening diseases such as dengue hemorrhagic fever or dengue shock syndrome, resulting in 200,000 deaths a year. This has been further exacerbated by the lack of DENV human vaccines and antivirals. DENV NS5 RNA-dependent RNA polymerase (RdRp), a viral-specific and highly conserved protein, is a promising drug target. In this study, DENV2 NS5 protein synthesized using the eukaryotic wheat germ cell-free protein synthesis system will be presented as an alternative to other present protein synthesis methods that balances cost, efficiency and physiological relevance. The recombinant NS5 proteins were then successfully applied in the development of a fluorescence-based in vitro NS5 RdRp assay. Against a background of failed clinical trials due to safety and pharmacokinetic concerns, an emerging importance has been placed on drug repositioning to develop novel uses for existing drugs. Hence, libraries of FDA-approved drugs and natural compounds, highly regarded as safer alternatives compared to experimental synthetic compounds, were screened. Compared to other similar studies, this study achieved a significantly higher hit rate of 1.2% using a conservative cut-off criterion, suggesting that the choice to screen these safer (i.e. less cytotoxic) drugs and compounds could be a more efficient way to identify RdRp inhibitors and could expedite the clinical trial process. Eight drugs/compounds were shortlisted by the primary screen, and their anti-DENV potential was further evaluated in a cell-based DENV infection system by exploring their cytotoxicity and capacity to reduce viral titers. Of these, 62.5% demonstrated antiDENV activity in cultured cells. Of these, kusunoki, a polyphenol-enriched extract rich in oligomeric proanthocyanidins derived from the bark of the Japanese cinnamon tree, reflected the highest SI, and was chosen for further downstream validation experiments. The antiviral effect of kusunoki is demonstrated to be reproducible in a cell-type and assay-independent manner. In addition, its broad-spectrum inhibition against all four DENV serotypes is shown. Insights into the mechanistic action of kusunoki suggest that in addition to being a RdRp inhibitor, it may also further inhibit DENV by preventing viral attachment to host cells prior to entry. Kusunoki therefore holds great potential as an anti-DENV compound for further development into an antiviral drug. viii LIST OF ABBREVIATIONS DENV NTPase Dengue virus Nucleotide triphosphatase MTase Methyltransferase BSA Bovine serum albumin CBB Coomassie brilliant blue CIAP Calf intestinal alkaline phosphatase CIAP Intestinal alkaline phosphatase CMV Cytomegalovirus DHFR Dihydrofolate reductase DMSO Dimethyl sulfoxide FDA Food and Drug Administration FPLC Fast protein liquid chromatography HBV Hepatitis B virus HIV Human immunodeficiency virus HSV Herpes simplex virus MOI Multiplicity of infection NGC New Guinea C NME New molecular entities NS Non-structural PA Proanthocyanidins PBMC Peripheral blood mononucleated cell PBS Phosphate buffered saline PFU Plaque forming units R&D Research and development RdRp RNA-dependent RNA polymerase SDS Sodium dodecyl sulphate SPA Scintillation proximity assay ix x LIST OF FIGURES Figure 1.1 | Global distribution of dengue (Adapted from World Health Organization) . 1 Figure 1.2 | The DENV genome (Adapted from Yap et al., 2007) ................................... 6 Figure 1.3 | DENV replication cycle (Adapted from Stiasny and Heinz, 2006) ............ 17 Figure 1.4 | Schematic representation of DENV proteolytic processing (Adapted from Natarajan, 2010).............................................................................................................. 18 Figure 1.5 | Scintillation proximity assay for measurement of RdRp activity (Adapted from Yap et al., 2007) ..................................................................................................... 27 Figure 1.6 | BBT-ATP (Modified from Jena Bioscience)............................................... 28 Figure 1.7 | Illustration of the wheat germ cell-free protein synthesis system technology (Adapted from CellFree Sciences, Japan)....................................................................... 29 Figure 1.8 | Plot of new chemical entities against R&D spend by the pharmaceutical industry in the USA. (Adapted from Samanen, 2012) .................................................... 31 Figure 1.9 | The clinical trial cliff (Adapted from Ledford, 2011) ................................. 32 Figure 3.1 | Production of GST-NS5, GST-RdRp and GST-DHFR proteins by wheat germ cell-free protein synthesis system .......................................................................... 52 Figure 3.2 | Evaluation of fluorescence-based RdRp assay using DENV-2 NS5 and DENV-2 RdRp produced by wheat germ cell-free system............................................. 55 Figure 3.3 | Screening of FDA-approved drug and natural compound libraries in fluorescence-based DENV NS5 RdRp assay.................................................................. 60 Figure 3.4 | Summary and validation of hit compounds obtained primary screening .... 63 Figure 3.5 | Validation of top 8 inhibitors in in vitro RdRp assay using NS5 RdRp domain mutant ................................................................................................................ 67 xi Figure 3.6 | Validation of inhibition of DENV replication by drug/compound in cellbased infection system .................................................................................................... 73 Figure 3.7 | Effects of kusunoki in CPE-based anti-dengue assay ................................. 76 Figure 3.8 | Effect of kusunoki in plaque reduction assay across 4 DENV serotypes .... 78 Figure 3.9 | Mechanistic inhibitory action of kusunoki .................................................. 82 xii LIST OF TABLES Table 1.1 | A comparison of various in vitro protein synthesis systems......................... 30 Table 1.2 | Summary of recently discovered anti-DENV small molecules and drugs ... 35 xiii Chapter 1. INTRODUCTION 1.1 Dengue Dengue is a disease caused by dengue virus (DENV) infections and transmitted by mosquitoes. Tropical and sub-tropical regions around the world are among the most afflicted by the burden of this disease. The World Health Organization (WHO) ranks dengue as one of the most important infectious diseases in the world, with serious implications on international public health (Guzman and Kouri, 2002). Despite global efforts to curb dengue transmissions, both geographical disease distribution and transmission rates have been on the rise (Farrar et al., 2007) (Figure 1.1). Figure 1.1 | Global distribution of dengue (Adapted from World Health Organization) 1 1.1.1 Burden of disease Dengue incidence has brought a significant economic and disease burden. A substantial economic burden in endemic countries, the disease has cost countries US$950 million and US$2.1 billion annually in Southeast Asia and the Americas respectively. As the study in the Americas have not included components such as cost for vector control, the economic consequences of dengue remains significantly underestimated (Shepard et al., 2011; Shepard et al., 2013). The incidence of dengue has also increased globally in recent decades. Presently, estimates by the WHO places well over 2.5 billion people (approximately 40% of the world's population) at risk of dengue, with as many as 50 – 100 million dengue infections worldwide every year (Special Programme for Research and Training in Tropical Diseases. and World Health Organization., 2009). However, in a recent report by Bhatt and colleagues, the global dengue burden was demonstrated to be more than three times that of WHO’s estimates, with a staggering 390 million infection cases occurring annually (Bhatt et al., 2013). Up to the 1970s, only nine countries had experienced critical dengue epidemics. In dramatic contrast, dengue is now endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-east Asia and the Western Pacific (Chaturvedi and Shrivastava, 2004). The evident increase in DENV epidemic activity has been attributed to several factors. Firstly, the unprecedented population growth in developing areas coupled to the lack of reliable water systems have exacerbated this condition by the need to collect and store water, increasing mosquito breeding potential. The advent of modern day transportation has also increased movement of viruses in infected humans, contributing to the geographic spread of the virus. Moreover, ineffective control of its mosquito vector, Aedes aegypti, can also be ascribed for the continued viral spread and maintenance of the virus reservoir and (Mackenzie et al., 2004). Lastly, being the only known arbovirus 2 to have fully adapted to humans, DENV are no longer dependent on an enzootic cycle for maintenance (Gubler, 2002). 1.1.2 Dengue virus The Flavivirus genus, belonging to the family Flaviviridae, contains 73 viruses, and many of which are arthropod-borne, or arboviruses, a term that depicts the necessity of a blood-sucking arthropod to complete their life cycle. Of these, pathogenic flaviviruses include the DENV, West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV) that pose major public health threats worldwide. These have been known to be causative agents of emerging infectious diseases, a phenomenon epitomized by the escalating prevalence of DENV, especially in the tropical and subtropical regions of the world (Malet et al., 2008). 1.1.3 Dengue infection pathogenesis Four related but antigenically distinct DENV (DENV serotypes 1 – 4 [DENV-1 to -4]) infect approximately 390 million people annually (Bhatt et al., 2013). In most cases, after an incubation period of 4 – 7 days, clinical manifestations of DENV infection vary, and risk factors that determine severity of the disease include age, ethnicity and existing chronic diseases (Bravo et al., 1987; Guzman et al., 2002; Guzman et al., 2000). 1.1.3.1 Dengue fever Generally, DENV infections are asymptomatic or result in dengue fever (DF), a mild, undifferentiated and self-limiting disease associated with fever and malaise. Other symptoms may include a severe headache with retro-orbital pain, severe joint and muscle aches, nausea and vomiting and body rash (Simmons et al., 2012). Less than 10% of symptomatic dengue cases are reported, and a prospective cohort study of elementary school children in Thailand revealed that an average of approximately 53% of dengue cases were asymptomatic over a three-year period from 1998 to 2000 (Endy et al., 2002). The likelihood of symptomatic infections rises upon secondary infections, 3 as well as a longer time interval between primary and secondary infections (Anderson et al., 2013; Seet et al., 2005). 1.1.3.2 Dengue hemorrhagic fever and dengue shock syndrome Of the total number of DENV infections, a small but significant subset of cases totaling to about 500,000 annually develop to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Also known as severe dengue, clinical symptoms often resemble those of classical dengue fever during its early stages. However, in DHF/DSS cases, a persistent high fever is then further complicated by acute conditions characterized by plasma leakage and abnormal haemostasis. Evidence supporting the former includes a swift rise in haemotocrit, pleural effusion and ascites, hypoproteinaemia and reduced plasma volume (Bhamarapravati et al., 1967; Nimmannitya, 2009). Atypical haemostasis is associated with vascular changes such as capillary fragility changes, thrombocytopenia, coagulopathy and depression of bone marrow elements (Bokisch et al., 1973; Deen et al., 2006; Srichaikul and Nimmannitya, 2000). Severe dengue leads to 200,000 deaths annually, a condition which is exacerbated by the lack of intravenous fluid resuscitation facilities in some regions (Julander et al., 2011; Ngo et al., 2001). DSS occurs largely in childhood cases, and has been hypothesized to be due to increased microvascular permeability in children who are still developing compared to adults (Gamble et al., 2000). The pathogenesis behind the development of DHF/DSS remains elusive. A primary infection with any of the four DENV serotypes is known to result in a lifelong immunity to that particular serotype. In addition, this primary infection also provides a short-lived immunity to the other serotypes that lasts a few months (Gubler, 1998). While the primary infection is most often asymptomatic, subsequent infections by any of the other three serotypes generally result in more severe secondary infections, which may lead to DHF/DSS. 4 One of the leading hypotheses for this occurrence is the antibody-dependent enhancement (ADE) effect (Halstead, 1970). Halstead and his colleagues were one of the first proponents of this hypothesis after early studies in the 1950s that suggested that DHF/DSS occurs 15–80 times more commonly in secondary infections than in primary ones. Furthermore, a striking 99% of DHF cases reveal heterotypic antibodies to the dengue serotype causing the DHF (Halstead, 1982). In brief, a primary infection causes the development of homotypic neutralizing antibodies against the DENV serotype responsible. Concurrently, heterotypic antibodies against other serotypes are also generated. This confers the host the lifelong immunity against this serotype and transient cross-immunity to other serotypes (Sabin, 1952). This is explained by the observation that specific neutralizing IgG antibodies against the infecting DENV lasts decades, while heterotypic IgG antibodies decline rapidly over time (Halstead, 1974; Vaughn et al., 2008). This discrepancy could be due to the preferential survival of long-lived memory B cells producing homotypic antibodies (Guzman et al., 2007). Besides these two categories of antibodies, it is also possible for non-neutralizing heterotypic antibodies to be produced. This subset of antibodies enhances DENV entry into host cells upon onset of a secondary infection, leading to augmented infectivity. Interestingly, studies have revealed that a primary infection with DENV-1 or DENV-3 often resulted led to a more severe disease outcome compared to if DENV-2 or DENV4 (Vaughn et al., 1997). 1.2 Characteristics of DENV and DENV genome DENV are small enveloped viruses. Although widely accepted that two states of maturation exist (mature and immature virions), there have been increasing evidence of intermediate forms as well (Allison et al., 1999a; Rey et al., 1995). 5 A mature DENV virion is approximately 50 nm in diameter with a icosahedral capsid which contains a single-stranded, positive-sense RNA genome (Kuhn et al., 2002; Singh and Ruzek, 2013) that is organized with a type-I 5’ cap analog (m7GpppA) attached to the 5’-untranslated region (UTR), a single large open reading frame and the 3’UTR (Tomlinson et al., 2009). The DENV RNA genome (approximately 11 kb in length) encodes for 10 proteins (Figure 1.2), of which three are structural (capsid [C], premembrane [prM] and envelope [E]) and the remaining seven are non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins. In general, the structural and non-structural proteins function at distinct steps in virus replication: structural proteins for virion formation and non-structural proteins for RNA replication, respectively (Kummerer and Rice, 2002). Supporting this, the simultaneous expression of DENV C, E and prM proteins is ample for the secretion of virus-like particles that recapitulate the envelope structure and fusogenic ability of the mature virion (Tan et al., 2007). Also, DENV-derived subgenomic RNA replicons deficient in structural proteins retain replicative capabilities in cells, whereas they can be packaged into virions and released by trans expression of the structural proteins (Zou et al., 2011). While this still largely holds true, this view has been challenged with proteins that seem to have dual functions overarching both categories, effectively blurring the boundary between the two exclusive categories. Figure 1.2 | The DENV genome (Adapted from Yap et al., 2007) 6 1.2.1 Structural proteins DENV particles are made up of a host-derived lipid bilayer embedded with heterodimers of the E glycoprotein and the M protein which interact at their C-terminal ends (Allison et al., 1999b; Kuhn et al., 2002). Within the virion core, a nucleocapsid of about 40Å in diameter that is assembled of multiple C proteins encapsulates its RNA genome. 1.2.1.1 Capsid (C) protein The C protein is a relatively small protein of about 9 kDa. Multiple C proteins assemble to form the viral nucleocapsid that is within the virion core. A high proportion of amino acids found in the C protein are basic in nature. This suggests a likely function of the C protein in packaging negatively charged viral RNA, possibly through electrostatic interactions (Ma et al., 2004; Rawlinson et al., 2006). An internal signal sequence located at the C-terminal end of the protein enables the attachment of C protein to the endoplasmic reticulum (ER) membrane, initiating the site of nucleocapsid assembly (Nowak et al., 1989). The C protein has also been implicated to have a role in viral RNA replication. While some studies have concurred that the first 20 amino acids of the C protein are important for efficient viral replication, others have also demonstrated that the sequences slightly upstream of the C protein gene are involved in cyclization with the 3’ UTR region to enable full-length genome synthesis (Ditursi et al., 2006; Wu et al., 2005). 1.2.1.2 Pre-membrane (prM) and membrane (M) proteins The glycosylated prM protein is approximately 18 kDa and can be found in immature virions located intracellularly. It then undergoes changes to form the M protein of about 7 kDa and is located in mature virions. 7 The function of E protein is highly dependent on prM protein as co-expression of the two has been shown to be required for correct folding, maturation and proper assembly of E protein (Wu et al., 2005). The primary function of prM is to prevent the premature rearrangement of the E protein under mildly acidic conditions of the trans-Golgi network before virion release (Botting and Kuhn, 2012). By forming a heterodimer with E protein, the prM/E complex effectively conceals the fusion peptide situated on the E protein, thereby preventing premature fusion during the assembly process prior to release(Zhang et al., 2003). Host protease furin has been reported to cleave prM to yield fusion-competent mature virions with M proteins (Zybert et al., 2008). More recently, the prM protein has also been shown to play a role during virus entry. The interaction of prM to claudin-1, a tight junction membrane protein, has been suggested to facilitate internalization of DENV into cells (Zhou et al., 2007). 1.2.1.3 Envelope (E) protein The E protein is approximately 55 kDa and is a major glycoprotein found on the surface of the virion. It has been found to be glycosylated in most flaviviruses (Winkler et al., 1987). It is vital for cell receptor attachment and consequently, subsequent infections. Some of these receptors include GRP78 (glucose regulating protein 78), Hsp70, Hsp90 (heat shock protein 70/90), CD14, laminin receptor, mannose receptor and DC-SIGN (Chen et al., 1999; Jindadamrongwech et al., 2004; Miller et al., 2008; Reyes-Del Valle et al., 2005; Tassaneetrithep et al., 2003; Thepparit and Smith, 2004). Following which, it then facilitates fusion of the virus to host cell membrane within infected cells. As it also bears neutralization epitopes, it is often the target of antibodies (Mukhopadhyay et al., 2005). On a mature virion, E proteins are present as a homodimer with each subunit organized in a head-to-tail manner (Kuhn et al., 2002; Rey et al., 1995). These are anchored in the viral membrane by a stem anchor region that extends from the end of the dimer (Allison 8 et al., 1999a). It is estimated that about 90 E proteins can be found on the virion surface (Kuhn et al., 2002). 1.2.2 Non-structural (NS) proteins The seven NS proteins are components of the viral replication complexes (Mackenzie et al., 1998). However, in addition to their role in the replication of the RNA genome, they are also implicated in various other processes such as virion assembly and evasion of innate immune response (Elliott, 2011). 1.2.2.1 NS1 protein NS1 protein is a 50 kDa glycoprotein that contributes to different stages of the viral life cycle (Gutsche et al., 2011; Mackenzie et al., 1996). Although not present in viral particles, it has been observed to accumulate in both in the supernatant and on plasma membranes during infection (Avirutnan et al., 2007). A portion of NS1 protein is retained intracellularly, and is understood to play a crucial role in viral RNA replication (Avirutnan et al., 2006; Lindenbach and Rice, 1997). NS1 protein is also secreted by DENV infected cells into the blood stream (Flamand et al., 1999). The amount of NS1 protein found circulating in human sera has been found to be higher in patients suffering from DHF compared to those with milder dengue fever (Wu et al., 2005). Immune recognition of NS1 protein has been postulated to be a possible mechanism for vascular leakage, contributing to DHF (Avirutnan et al., 2006). Both membrane-associated and secreted forms of NS1 protein are also implicated in the immune response against DENV infection. This may be via the regulation of complement activation pathways through the creation of immune complexes or association with host proteins such as clusterin (Yap et al., 2007). 1.2.2.2 NS2A protein NS2A protein is a hydrophobic protein of approximately 22kDa known to be associated with the membrane of the endoplasmic reticulum with five membrane-spanning 9 segments (Ditursi et al., 2006; Wu et al., 2005). Apart from the full length NS2A protein, another truncated form of NS2A protein, NS2Aα protein, has also been reported in virus infected cells. NS2Aα protein results from the C-terminal cleavage of 34 amino acids by viral NS2B-3 protease after K190 at the sequence QK↓T within NS2A protein (Wu et al., 2005). A multi-faceted protein, NS2A/NS2Aα protein has been recognized to have four main roles. Firstly, it is important for viral RNA synthesis as it has been observed to be part of the replication complex, together with double-stranded (ds) form of viral RNA, NS3 and NS5 proteins (Mackenzie et al., 1998). Secondly, it is also involved in viral assembly. In particular, amino acid residue Arg84 has been found to be critical for both RNA synthesis and viral assembly (Ditursi et al., 2006). This is also supported by the study by Kummerer and Rice which found that a lysine mutation (K190S) in NS2A blocked the production of both NS2Aα protein and infectious virus particles (Kummerer and Rice, 2002). Thirdly, expression of NS2A alone is sufficient to subvert the host immune response by disruption of interferon α/β response such as through the blocking of dsRNA-activated protein kinase PKR (Munoz-Jordan et al., 2003; Niyomrattanakit et al., 2011). In addition, NS2A protein is also engaged in virus-induced membrane formation (Leung et al., 2008). 1.2.2.3 NS2B protein NS2B protein is approximately 14 kDa and primarily functions as a cofactor for NS3 protease (Falgout et al., 1991). Although DENV NS3 protein is shown to have NS2Bindependent protease activity for certain substrates (eg. N-benzoyl-L-arginine-pnitroanilide), its enzymatic activity was significantly increased with the NS2B cofactor (Zhou et al., 2007). In earlier studies, the minimal requirement of NS2B protein as a NS3 protease cofactor was found to be a 40-residue central hydrophilic region spanning from amino acid 10 residue 54 to 93, a conserved region amongst flaviviruses (Ditursi et al., 2006). Deletion analysis in later reports then contributed to existing knowledge by revealing that this conserved portion was only sufficient for basal cofactor activity, as the optimal NS2B cofactor function was highly dependent on the hydrophobic flanking regions of the protein, presumably acting as an anchor of the protease complex to the ER membrane (Fryxell, 1980; Ng et al., 2007) 1.2.2.4 NS3 protein NS3 protein, a multifunctional 618 amino acid long protein of about 70 kDa, is known to have demonstrated serine protease function, along with RNA helicase and nucleotide triphosphatase (NTPase) activities (Warrener et al., 1993; Wengler et al., 1991). Mutagenesis studies in the both the protease and helicase domains resulted in the abrogation of infectious virus particles, demonstrating the absolute requirement of these enzymes for viral replication (Lescar et al., 2008; Matusan et al., 2001). In addition, NS3 protein has also been implicated in viral assembly (Kummerer and Rice, 2002; Patkar and Kuhn, 2008). The protease domain is located in the N-terminal 186 amino acids of the NS3 protein (Lescar et al., 2008). It contains a characteristic canonical catalytic triad of His51, Asp75 and Ser135 that is well conserved both within the 4 serotypes of DENV as well as other flaviviruses (Chambers et al., 1990; Valle and Falgout, 1998). Together with its cofactor NS2B, the heterodimeric NS2B-NS3 protease complex (NS2B3) is essential for the viral replication cycle due to its ability to proteolytically process the precursor polyprotein (Chambers et al., 1990; Falgout et al., 1991). The NS2B3 serine protease is responsible for NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junction cleavages (Brinkworth et al., 1999; Chambers et al., 1990; Falgout et al., 1991; Preugschat et al., 1990) as well as cleaving within C, NS3 and NS4A proteins (Arias et al., 1993; Lin et al., 1993; Teo and Wright, 1997). 11 More recently, the NS2B3 protease complex was implicated with the inhibition of type I interferon (IFN) response by reducing the activity of IFN-β promoter. This has been shown to be achieved by the protease cleavage of human mediator of IRF3 activation (MITA), which in turn then prevents the phosphorylation needed for the activation of IRF3 (Rodriguez-Madoz et al., 2010; Yu et al., 2012). A similar phenomenon is also seen with hepatitis C virus (HCV) NS34A protease complex that proteolytically processes the IPS-1, interfering the signaling cascade that ends with the activation of IRF3 (Loo et al., 2006). The helicase/NTPase domain of NS3 lies in its C-terminal region and its enzymatic activities is ATP-driven. It is generally thought to hold apart the dsRNA intermediate that occurs during viral genome replication (Yap et al., 2007). It could also further enhance this important process by interfering with secondary structures formed by the single-stranded RNA template or oust any other factors that could potentially disrupt the replicative process (Lescar et al., 2008). Mutations in the helicase domain resulted in the abolished viral infectivity, demonstrating essentiality of this enzymatic activity in the viral replication cycle (Matusan et al., 2001). In addition, the third enzymatic domain of NS3 protein is the C-terminal RNA-5’triphosphatase (RTPase) domain. This has been proposed to be involved in RNA capping for the recognition by host translation machinery (Bartelma and Padmanabhan, 2002). More recent studies have also suggested that NS3 NTPase and RTPase activities could possibly share a common active site (Benarroch et al., 2004). 1.2.2.5 NS4A protein Comparatively less is known about the 16 kDa small hydrophobic integral membrane protein NS4A, which associates with membranes with 4 internal hydrophobic regions (Miller et al., 2007). The N-terminal region of NS4A protein is exposed in the 12 cytoplasm by cleavage from the NS2B3 protease complex. The C-terminal product of about 23 amino acid residues, on the other hand, has been suggested to act as a signal sequence that is crucial for the translocation of NS4B protein into the lumen of the ER. This signal sequence is called the 2K fragment and is then removed from the N-terminal region of NS4B protein by host signalase (Miller et al., 2007). This removal is in turn dependent on the prior cleavage at the NS4A/2K junction by NS2B3 protease (Lin et al., 1993). In studies with Kunjin virus, the pre-cleaved full-length NS4A protein has been shown to induce intracellular membrane rearrangements, a process which could be essential in the formation of a unique scaffold to support the viral replication complex (Norman et al., 1983). The removal of the C-terminal 2K fragment led to a smaller degree of membrane rearrangement (Norman et al., 1983). Indeed, this observation was supported in a recent study by Miller et al., which showed that during DENV infection, NS4A protein was primarily localized in ER-derived cytoplasmic dot-like structures that contain dsRNA and other DENV proteins, further reinforcing the notion that NS4A protein as a constituent of the membrane-bound viral replication complex (Miller et al., 2007). The expression of N-terminal NS4A lacking the 2K fragment led to the induction of membrane alterations, as observed in Norman’s study, however, the expression of full-length NS4A could not. This highlights the importance of the proteolytic processing and removal of the 2K fragment in the induction of cytoplasmic membrane alterations for the formation of virus-induced structures to support the viral replication complex (Miller et al., 2007; Norman et al., 1983). 1.2.2.6 NS4B protein NS4B protein, like NS4A protein, is a small hydrophobic protein made up of 248 amino acids. NS4B proteins of the various DENV serotypes share approximately 82% similarity, while NS4B proteins of other flaviviruses such as WNV and HCV are only 13 35% similar and completely dissimilar to DENV NS4B protein respectively (Umareddy et al., 2006). Despite the vast differences in sequences, the general topology of NS4B protein of different flaviviruses comprising of numerous endoplasmic reticular and cytoplasmic domains divided by transmembrane regions bear great resemblance to each other, proposing a conserved function of NS4B protein in the viral replication cycle (Lundin et al., 2003; Miller et al., 2006). Both deletion of NS4B and mutations in its sequence are reported to inhibit replication of bovine viral diarrhea virus (BVDV) (Balint et al., 2005). The role of NS4B protein in viral replication has also been suggested by the observation that NS4B protein interacts with NS3 to enhance helicase activity and NS5 protein (Piccininni et al., 2002; Qu et al., 2001; Umareddy et al., 2006), as well as a study reporting the NS4B-induced morphological changes in endoplasmic reticulum membrane (Egger et al., 2002). However, a conflicting account by Westaway and colleagues, who reported the inability to pull-down NS4B protein with dsRNA seemed to dispel the hypothesis of NS4B protein as a part of the viral replication complex (Westaway et al., 2003). In addition, NS4B protein has also been implicated to be engaged in the formation of a scaffold for the viral replication complex (Welsch et al., 2009), as well as its ability to subvert host immunity by the dampening of IFN-α/β response (Munoz-Jordan et al., 2005). 1.2.2.7 NS5 protein The multifunctional DENV NS5 protein is the largest DENV protein at 104 kDa. It is also the most conserved DENV protein, with NS5 proteins across the 4 DENV serotypes sharing almost 70% in amino acid sequence homology (Xu and Wang, 1991). The N-terminal region of NS5 protein has a S-adenosyl-methionine methyltransferase (SAM) domain, or simply known as a methyltransferase (MTase) domain. This domain is approximately 300 amino acids long and is known to facilitate the capping of viral 14 RNA and internal RNA methylation (Dong et al., 2012; Egloff et al., 2002). NS5 MTase has demonstrated methylase activity at both N7 position of guanine and 2’-OH position of ribose (Egloff et al., 2002). More recently, it has also been recently purported to have guanylyltransferase activity (Issur et al., 2009). These methylation events are essential for the capping of viral RNA, which in turn is vital for ribosomal recognition to facilitate host-mediated translation. Besides having MTase domain, two putative nuclear localization signals (NLSs), designated the βNLS and αβNLS, have also been uncovered in DENV NS5. The latter plays a more major role in nuclear transport facilitated by interaction with the cellular nuclear transport receptor -importin (Johansson et al., 2001). A high proportion NS5 protein accumulates in the nuclei during DENV-2 infection, similar to that of NS5 protein in YFV infection (Buckley et al., 1992). Although nuclear accumulation was thought to be directly correlated to efficient replication previously, a shift of paradigm has occurred with a study that uncoupled both processes through mutagenesis studies (Kumar et al., 2013). To date, the exact reason for NS5 protein nuclear accumulation during infection remains elusive. It is interesting to note, however, that this phenomenon is neither conserved amongst all 4 DENV serotypes nor other flaviviruses. NS5 protein of WNV, for example, does not demonstration nuclear localization during infection (Mackenzie et al., 2007). These stark differences have been hypothesized to reflect the differences in strategies used by viruses to achieve efficient replication and could even correlate with pathogenesis in vivo (Kumar et al., 2013). Another possible consequence of NS5 protein nuclear accumulation is enhanced production of the immunomodulatory cytokine IL-8, the secretion of which seems to be impacted by the former (Medin et al., 2005; Pryor et al., 2007). NS5 protein also has a RNA-dependent RNA polymerase (RdRp) domain at its Cterminus, which is characterized by the existence of the highly conserved motif C 15 (glycine, aspartate, aspartate) and similar to those found in RdRps of other RNA viruses. This domain is primarily responsible for the synthesis of viral RNA genome (Nomaguchi et al., 2003). The DENV RdRp is able to initiate RNA synthesis de novo (Ackermann and Padmanabhan, 2001). Mutagenesis studies involving the disruption of motif C have also been shown to abrogate the replication ability of flaviviruses (Khromykh et al., 1998), underscoring the significance of this enzyme to the viral life cycle. Besides these enzymatic activities, the role of NS5 protein in the evasion of innate immune response by binding to STAT2 and mediating its degradation is also well characterized (Ashour et al., 2009). More recently, UBR4, a 600 kDa member of the Nrecognin family was identified as an interacting partner of NS5 protein as data suggested that NS5 protein aided bridging between STAT2 and UBR4. The study also revealed that UBR4 promoted STAT2 degradation mediated by DENV infection, and was necessary for efficient viral replication in type-I IFN competent cells, thereby adding to existing knowledge by identifying UBR4 as a host protein exploited by DENV to inhibit IFN signaling via STAT2 degradation (Morrison et al., 2013). Another noteworthy interaction of NS5 protein is to NS3 protein. Interestingly, the region of NS5 protein binding to NS3 protein is thought to be the same region that binds to β-importin (Johansson et al., 2001). In a study by Yon and colleagues, the crosstalk between both NS3 and NS5 protein domains were demonstrated as NS5 protein stimulated the NTPase and RTPase activities of NS3 protein (Yon et al., 2005). 1.3 DENV replication cycle The DENV replication cycle can be divided into several key steps (Figure 1.3). 16 Figure 1.3 | DENV replication cycle (Adapted from Stiasny and Heinz, 2006) 1.3.1 Entry and uncoating DENV infects and replicates in a myriad of mammalian cells, including monocytes, macrophages, dendritic cells, endothelial cells, B and T leukocytes, heptocytes and kidney-derived cells (Alhoot et al., 2011). To kick-start its replication cycle, E/prM protein on the viral envelope attaches to a cell receptor such as GRP78 (glucose regulating protein 78), Hsp70, Hsp90 (heat shock protein 70/90), CD14, laminin receptor, mannose receptor and DC-SIGN (Chen et al., 1999; Jindadamrongwech et al., 2004; Miller et al., 2008; Reyes-Del Valle et al., 2005; Tassaneetrithep et al., 2003; Thepparit and Smith, 2004). The DENV particle is then internalized into the cell cytoplasm by membrane fusion and enters the host cell by endocytosis. Following the acidification of endocytic vesicles, the exposed fusion loop causes the viral and cell membrane to be in close proximity, enabling the trimerization of E protein and thereby mediating virus and cell membrane fusion (Allison et al., 1995; Modis et al., 2003). The nucleocapsid enters the cytoplasm, uncoats, and releases the viral RNA genome (Tomlinson et al., 2009). 17 1.3.2 Translation and further processing After the single-stranded, positive-sense viral genomic RNA is released into the cytoplasm, the 5’UTR directs the RNA strand to host ribosomes for translation into a single long polyprotein. This is then co- and posttranslationally processed by a combination of both viral and host proteases (Figure 1.4). The NS2B3 serine protease is responsible for NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junction cleavages (Brinkworth et al., 1999; Chambers et al., 1990; Falgout et al., 1991; Preugschat et al., 1990) as well as cleaving within C, NS3 and NS4A proteins(Arias et al., 1993; Lin et al., 1993; Teo and Wright, 1997). The cleavage of the remaining protein junctions C/prM, prM/E, E/NS1 and NS4A/NS4B are facilitated by host signal peptidases (Nowak et al., 1989; Speight et al., 1988), while prM protein is cleaved by furin to produce the mature M protein during virus maturation (Stadler et al., 1997). Figure 1.4 | Schematic representation of DENV proteolytic processing (Adapted from Natarajan, 2010) 1.3.3 RNA replication Viral RNA replication is catalyzed in a specialized structure known as the replication complex. Although the DENV replication complex is hypothesized to form when NS1, NS2A, NS3 and NS4A join NS5 in the vicinity of the 3’ stem loop after translation of viral RNA (Chambers, 2003), the main components of the replication complex are NS5 and NS3. This structure is thought to be anchored to the trans-Golgi network membrane via the viral integral membrane protein, NS4A. 18 From the positive-sense RNA genome, a negative RNA strand is synthesized. This forms a double-stranded RNA called the Replication Form (RF). Using this as a template, the replication complex produces a Replicative Intermediate (RI), which is then further processed to form single stranded positive-sense RNA. The RF is then recycled continuously as a template for asymmetric and semi-conservative RNA synthesis (Chu and Westaway, 1985). 1.3.4 Assembly and release Subsequent to RNA replication, C proteins interact with the positive-sense viral genome and are brought to the ER. The nucleocapsid is then enveloped with a lipid bilayer. Heterodimers of the E glycoprotein and the prM protein are then embedded on the nucleocapsid that form projections (Kuhn et al., 2002), facilitating the viral assembly process. The primary function of prM is to prevent the premature rearrangement of the E protein under mildly acidic conditions of the trans-Golgi network before virion release (Botting and Kuhn, 2012). Several of the DENV NS proteins like NS2A and NS3 (Liu et al., 2002) have also been reported to play a role in this process. For example, NS2A have been implicated in the biogenesis of virus-induced membranes, while NS3 has been postulated to play a role as a linker between structural proteins and RNA (Leung et al., 2008; Liu et al., 2002). Immature virions from the endoplasmic reticulum are then transported onward to the trans-Golgi network, where prM protein is cleaved by cellular furin to allow M/E rearrangement, resulting in virus maturation and release of the mature virion contained in vesicles through exocytosis (Li et al., 2008). 1.4 Anti-dengue efforts DENV infection has become a growing global health concern by the sheer number of people estimated to be at risk of this infection and startling fatality rates annually. This 19 has also placed a heavy financial burden on health care systems around the world as many infected individuals seek treatment. To keep the disease at bay, governments in endemic regions worldwide have focused on mosquito vector control. These efforts may be divided into three main categories: Biological control (e.g. the introduction of larvivorous organisms such as fish into water containers and the release of transgenic vectors with the aim of reducing wild-type vectors with those of a reduced capacity to reproduce and transmit disease), environmental management (e.g. covering and screening of water containers and reduction of human-vector contact by the use of screening doors and insecticide-treated nets), as well as chemical control (spraying of insecticides) (Erlanger et al., 2008; Simmons et al., 2012). The use of insecticides such as dichlorodiphenyltrichloroethane, malathion and pyrethroids, however, has been laden with challenges. These include environmental contamination, in particular that of aquatic ecosystems as pyrethroids are known to be toxic to aquatic life even at low levels (Friberg-Jensen et al., 2003). In addition, bioaccumulation of toxins and human toxicity, especially relating to the presence of insecticide in drinking water containers have also been of concern (Curtis and Lines, 2000). With evidence supporting the bioaccumulation of pyrethroids in mammalian tissue and possible maternal transfer of pyrethroids revealed by its presence in human breast milk (Alonso et al., 2012; Sereda et al., 2009), it is worrying that studies have also linked these same compounds to breast cancer (Go et al., 1999). Also, in a recent study by Luz et. al., it was reported that adult mosquito control was 8.2 times higher the cost of larval control and therefore the authors propagated the use of the latter. Another severe limitation of the effectiveness of chemical vector control was noted to be the short-term reduction in dengue burden of merely 2 – 4 years: Of 43 insecticide-based vector control strategies they examined, all interventions caused the emergence of insecticide resistance, which will increase the magnitude of future dengue epidemics when coupled to loss of herd immunity (Luz et al., 2011). In essence, dengue vector control is effective when interventions use a community-based and integrated 20 approach that is tailored to local conditions and combined with educational programs (Erlanger et al., 2008). In addition, anti-dengue therapeutics have also been developed in tandem with the use of vector control methods in the fight against dengue. These can broadly be divided into 2 categories: Vaccines and antiviral drugs. Dengue vaccines are an attractive approach as a prophylactic measure to prevent disease progress upon infection. In addition, the possibility of using vaccination to achieve herd immunity to reduce the risk of outbreaks is also an attractive one (Durham et al., 2013). However, among other challenges, detractors have warned about the lack of understanding of issues and risks regarding the prevalence and mechanism of ADE associated with DENV infections, as well as the possibility of a recombination between attenuated live vaccine strains and wild-type viruses leading to the former reverting to a virulent form. The search for dengue antivirals has been gaining momentum due to significant progress in the structural biology of dengue virus. The primary aim for the development of an antiviral drug against dengue is to substantially lower the viral load in patients upon administration. In light of the fact that clinical studies have shown that patients who suffer from DHF/DSS is significantly higher than that of patients suffering from mild dengue fever (Vaughn et al., 2000), it is hoped that the early administration of an antiviral that targets essential steps in virus replication can lower viremic levels and prevent disease progress and morbidity. As neither prophylactic vaccines nor antivirals are available for treatment of dengue infection, medical intervention has focused largely on the provision of haemodynamic support to compensate for plasma leakage, as well as the relief of clinical symptoms. However, considering the increasing global burden of dengue disease, there is thus an urgent need for the development of anti-dengue therapeutics. In this study, the discovery of antiviral drugs against dengue will be focused upon. 21 1.4.1 Ideal characteristics of dengue antiviral drug The development process of a good antiviral drug for dengue maximizes several factors: 1. Inhibits viral replication significantly The ultimate goal of an antiviral drug would be the complete eradication of virus from its host. However, in practice, a 50 – 90% inhibition of viral replication could be sufficient enough to bring the viral load down to a controllable level for host immune system clearance (Botting and Kuhn, 2012). 2. Specific to virus Non-specific targeting of the drug can lead to detrimental adverse effects, or even cell death. The targeting of a unique viral-specific protein that is usually not present in uninfected host cells could be a possible way to avoid such complications. 3. Targets well-conserved regions The targeting of well-conserved regions in the virus minimizes the risk of viral escape mutations arising and consequent resistance against the drug. 4. Broad-spectrum inhibition The ability of the drug to effectively inhibit numerous viruses from the same Flaviviridae family would fill the void caused by the lack of antivirals for flavivirus infections. 5. Readily enters cells An ideal drug should readily be brought into cells, either through passive diffusion or active transport. As most drugs are absorbed by passive diffusion across a biological barrier (eg. cell membranes), drugs that are small molecules with low molecular weights are preferable (Brenner and Stevens, 2010). 6. Fast-acting A major challenge facing the implementation of anti-dengue drugs is the short duration window of viremia (Low et al., 2011). 22 1.5 NS5: An attractive anti-dengue drug target With new knowledge added to our understanding of the molecular clockwork of DENV, prospective drug targets continue to emerge, especially for DENV NS proteins. This could possibly be due to a greater conservation of NS proteins compared to structural proteins as studies have revealed that 95.5% of conserved sequences in DENV are from NS proteins (Khan et al., 2008). This is further backed up by the trend that small molecules targeting NS proteins have increasingly been tested on both mouse and cell culture models (Bente and Rico-Hesse, 2006). Of all the NS proteins encoded by the DENV genome, both NS3 and NS5 proteins have been highly regarded as sound targets for drug intervention. This may be attributed to 2 reasons. Firstly, the functional domains located in these two proteins are essential for the viral life cycle. Secondly, as these two proteins are also desirable for the development of high throughput assays as they possess enzymatic activity. Antiviral efforts in counteracting human immunodeficiency virus (HIV) infections have brought to light two important lessons for the development of antivirals. With a myriad of drug classes available against HIV (reverse transcriptase inhibitors and protease inhibitors), the importance of targeting an essential viral gene critical for the viral life cycle is stressed. This criterion is met in both NS3 and NS5 as targets as both proteins exhibit multiple enzymatic activities that are indispensable for DENV replication. Furthermore, the limited success in antiretroviral therapy has been attributed to acquired drug resistance of HIV (Zdanowicz, 2006). As this occurs due to the high rate of viral replication coupled to the lack of proof-reading ability of reverse transcriptase which result in high mutation rates of the virus, attempts to take advantage of functional and structural constraints of the virus has led to the search for sequence conservation in viruses for exploitation (Snoeck et al., 2011). When brought into the context of choosing a more appropriate between NS3 and NS5 proteins as a viral target, NS5 is clearly a more preferable choice as it is the most conserved DENV protein, with NS5 23 proteins across the 4 DENV serotypes sharing almost 70% in amino acid sequence homology (Xu and Wang, 1991). A study by Khan et. al. revealed that out of 44 panDENV sequences (highly conserved sequences in all 4 serotypes of DENV) of at least 9 amino acids, approximately 40% of these were located in NS5 protein. The largest spanning pan-DENV sequence was also found in NS5, extending a total of 215 amino acids, equating to ~24% of the protein (Khan et al., 2008). As conserved sequences are likely to represent critical sites and domains, these regions are less likely to be amenable to mutations for viral escape, hence lowering the possibility of viral resistance (Valdar, 2002). A desirable property of an anti-dengue drug would be the broad-spectrum inhibition against a wide range of other flaviviruses. Out of 44 sets of highly conserved DENV sequences, 27 were found in as many as 64 other viruses in the family Flaviviridae (representatives that are human pathogens include WNV, St. Louis encephalitis virus, JEV and YFV) and 13 of these represented sequences in NS5, suggesting that an inhibitor targeting NS5 could have a higher possibility of being a broad-spectrum antiviral. The DENV NS5 protein contains two functional domains: A N-terminal MTase and Cterminal RdRp. MTases play essential roles in both normal physiology and diseases through the methylation of nucleic acids and proteins. Due to the high homology of core domains of these enzymes, designing specific inhibitors that block only viral-specific MTases have been a great challenge as problems with selectivity have arisen because of the close resemblance of viral and endogenous MTases. In this aspect, the RdRp would be a more practical selection as a drug target since no mammalian homologues of this enzyme have been found (Sugiyama et al., 2005). Being a viral-specific protein, drugs acting on this protein are less likely to interfere with the normal physiology of host cells, or affect them adversely. Moreover, of all conserved sequences found in NS5 across all 4 DENV serotypes, 60% were found in the RdRp domain, an evident increase of 30% from its MTase counterpart (Khan et al., 2008). 24 Over the years, inhibitors against HIV reverse transcriptase, as well as inhibitors against polymerases of hepatitis B virus (HBV), cytomegalovirus (CMV) and herpes simplex virus (HSV) have been approved as drugs for treatment of associated viral infections (Wu et al., 2005). More importantly, this trend is also apparent for inhibitors of the closely related HCV NS5B polymerase as Ribavirin has already been approved for use, with many others such as Sofosbuvir (completing phase III) in the pipeline currently at late phases of clinical trials (Sofia et al., 2010). The flavivirus RdRp is therefore a validated target. In conclusion, DENV NS5 RdRp presents as an attractive drug target because of its position as a viral-specific protein that is highly conserved across all 4 DENV serotypes and many flaviviruses. Additionally, this protein also has a vital role in viral RNA replication and a great potential as a target in the development of a high-throughput enzymatic assay. 1.6 Types of NS5 RdRp inhibitors NS5 RdRp inhibitors fall into two categories: a. Nucleoside analogue inhibitors These are structurally similar to nucleotides and they work as antimetabolites by being incorporated into growing RNA strands. Unlike actual nucleosides, nucleoside analogues lack a 3´-hydroxyl group on the deoxyribose moiety, which is usually hydrolyzed to form a phosphodiester bond with the next nucleic acid. These nucleoside analogues thereby act as chain terminators. However, mitochondrial toxicity and other adverse events have been reported as a common side effects of the usage of nucleotide analogues, due to structural similarities to endogenous nucleosides as well as differential tissue-specific levels of pro-drug activation (Lund et al., 2007; Noble et al., 2010). 25 b. Non-nucleoside inhibitors This class of inhibitors may not necessarily bear a structural resemblance to nucleosides. Binding to allosteric sites of the NS5 protein, these inhibitors change the conformation of the active site such that the protein is no longer competent to carry out its RdRp activities (Wang et al., 2003). 1.7 Conceptualization of project 1.7.1 Current state of in vitro RdRp assays Traditional cell-based drug screening approaches offer benefits in the pursuit of the discovery inhibitors as they are usually regarded as one of the closest conditions one can get to an in vivo environment. As general screens include numerous targets within a single screen, one may even uncover novel targets in the process. However, the key drawback associated with this methodology is the time taken for screening, as well as meticulous care and great cost into ensuring avoidance of contamination in cell cultures with the use of tissue culture facilities. The scaling up of these assays for the use in high throughput screening assays has also been a major challenge. Moreover, subsequent follow-up screens to further clarify hits are often complex and both labour and resource intensive (Westby et al., 2005). For these reasons, in vitro enzymatic biochemical assays have long been favored over traditional cell-based assays as they allow the concentration on a single target and a set of well-defined assays for follow-up purposes (Westby et al., 2005). The relative ease in the application of these in high throughput studies has also been a major advantage. Following the consensus that DENV NS5 RdRp is a promising drug target, a wide myriad of RdRp enzymatic assays in a cell-free setting have been developed. Due to the biochemical nature of RdRp in elongating RNA strands, most RdRp assays have utilized radioactive labels to track the incorporation of nucleotides. A popular radioactive assay format is the scintillation proximity assay (SPA), based on the quantification of 26 radioactivity present on newly transcribed RNA incorporated with radioactive nucleotides (Figure 1.5) (Yap et al., 2007). Figure 1.5 | Scintillation proximity assay for measurement of RdRp activity (Adapted from Yap et al., 2007) Although these radioactivity-based assays are still widely used, there has been a growing inclination to move away from the use of radioactive labels. The motivation behind this shift is that radioactive assays for use in high throughput screening studies have the aptitude to produce large amounts of waste that can cause health and safety concerns. In addition, both the acquirement and disposal of these reagents are costly (Seethala and Fernandes, 2001). A solution to this is the use of fluorophore-conjugated nucleotides as a substitute to radioactive-labeled nucleotides. An example of this is 2'-[(2-benzothiazoyl)-6'hydroxybenzothiazole]-ATP (BBT-ATP) (Figure 1.6) (Niyomrattanakit et al., 2011). Fluorescence readings can then be taken after nucleotide incorporation which in turn 27 results in the release of fluorophore tags. These measurements would therefore be proportional to NS5 RdRp activity. Figure 1.6 | BBT-ATP (Modified from Jena Bioscience) 1.7.2 Current state of in vitro DENV NS5 protein production DENV NS5 proteins used in in vitro have traditionally been produced by Escherichia coli (E. coli) or baculovirus-based systems (Tan et al., 1996). Although NS5 proteins yielded from both systems have been shown to have RdRp activity (Niyomrattanakit et al., 2010), both protein synthesis systems have many shortcomings. For instance, although the E. coli system produces a high yield of proteins rapidly at a low cost, a major inherent weakness of it is the lack of post-translational modifications present due to it being a prokaryotic-based system. Conversely, although a euykaryotic baculovirus system allows for some degree of post-translational modifications, it is both an expensive and time-consuming process of up to 10 days. Furthermore, being a cellbased technique, it is highly susceptible to contamination. In recent years, an increasing number of post-translational modifications have been discovered for DENV NS5. These include phosphorylation, myristoylation, glycosylation and amidation (Khan et al., 2008). With some of these ascertained to be important for its function, such as in the study by Bhattacharya et. al. that uncovered that the phosphorylation of NS5 at amino acid residue 449 modulates DENV replication, there is a pressing need to seek out an alternative NS5 protein synthesis system (Bhattacharya et al., 2009). 28 In this study, eukaryotic wheat germ cell-free protein synthesis system is employed to produce DENV NS5 protein. Despite being a fairly new system, this protein production system is well-established, having generated 13,364 human proteins (encompassing 70% of the 22,000 human genes). Additionally, 77% of the phosphatases synthesized by this system have also showed biological activity (Goshima et al., 2008). The wheat germ cell-free protein synthesis system is a rapid method to produce proteins, taking one day to produce a large and consistent yield of proteins in a costefficient manner. The technology of this system is outlined in Figure 1.7. Figure 1.7 | Illustration of the wheat germ cell-free protein synthesis system technology (Adapted from CellFree Sciences, Japan) Being a eukaryotic system, the wheat germ system has also been shown to allow post translational modifications such as glycosylation, acetylation, phosphorylation, isoprenylation, myristoylation, complex protein folding and proteolytic processing (Martin et al., 1997; Nakamura, 1993; Rubenstein and Chappell, 1983; Zagorski et al., 1983). Table 1.1 shows a comparison of the various in vitro protein synthesis methods. 29 Table 1.1 | A comparison of various in vitro protein synthesis systems In this study, DENV-2 NS5 protein synthesized using the wheat germ cell-free protein synthesis system will be presented as an alternative to other present protein synthesis methods that balances cost, efficiency and physiological relevance. 1.7.3 Current state of pharmaceutical industry In the pharmaceutical industry, it is well-known that productivity has not kept pace with increasing research and development (R&D) investments. The industry has seen an exponential increase in R&D costs over the decades. However, this has not equated to a proportional increase in approved new molecular entities (NMEs) (Figure 1.8). This increasing gap between the number of approved NMEs and R&D expenditure is termed as the “innovation gap” (Samanen, 2012). 30 Figure 1.8 | Plot of new chemical entities against R&D spend by the pharmaceutical industry in the USA. (Adapted from Samanen, 2012) In an increasingly risk-averse world, growing R&D expenses are largely spent in the drug discovery and development process which ensues in three overarching stages: Discovery phase (new compounds are screened and identified), the preclinical phase (compounds are tested in vitro and in animal models), and lastly, the clinical phase (drug candidates are tested in humans in trials) (Padhy and Gupta, 2011). This increase in R&D costs has been further exacerbated by the high attrition rates of drugs from clinical trials, dubbed “the clinical trial cliff”, with the number of drugs terminated in Phase III of clinical development doubling in 5 years, reaching 55 compounds in a 2 year period of 2008 - 2010 (CMR, 2012). The primary reason for drop-outs in Phase I and II is safety-related issues, while withdrawals in Phase III are ascribed to a lack of efficacy (Figure 1.9) (Novac, 2013). 31 Figure 1.9 | The clinical trial cliff (Adapted from Ledford, 2011) To combat these problems, the idea of recycling late-phase-failed compounds or already marketed drugs to a new indication was sprung. The process of identifying or developing new uses for existing or abandoned drugs or compounds is called “drug repositioning” (Ashburn and Thor, 2004). This is an appealing prospect from various standpoints. Firstly, from the industrial standpoint, this course of action can potentially bring in more revenue to maximize R&D expenditure. Also, proven formulation and manufacturing routes can further reduce additional related investment expenses. From an ethical stance, the information generated by existing patient data in clinical trials for future indications should be capitalized on to its full potential. 32 Lastly, from the scientific perspective, compounds with established safety and bioavailability profiles would undoubtedly get a good head-start in the R&D process. These can probably enter clinical trials quicker than NMEs and would less likely fail due to human safety and pharmacokinetic issues (Stuart-Kregor, 2007). In addition, a well-characterized pharmacology could aid studies into the mechanistic analysis of new indications. The success stories of several repositioned drugs such as sildenafil (angina to erectile dysfunction) and thalidomide (morning sickness to multiple myeloma) have been well chronicled (Novac, 2013). More recently, mifepristone, initially approved as an abortifacient, has also been repurposed as treatment for Cushing’s syndrome in early 2012 (Castinetti et al., 2012). These case studies are indicative of the recognition of the increasing importance of drug repositioning today. Brought into the context of this study, these principles were a guide into the selection of screening library to utilize. For the various reasons aforementioned, a library of the US Food and Drug Administration (FDA)-approved drugs was opted for. In addition, to further expand this screening library, natural compounds, some of which are used as health supplements, were also appended. Natural compounds, often used in traditional medicine, were chosen for their better safety and tolerance profiles compared to synthetic compounds or general NMEs (Patwardhan and Vaidya, 2010). This is probably the reason why over 60% of anticancer and 75% of anti-infective drugs approved from 1981 - 2002 can be traced back to natural origins (Gupta et al., 2005). The screening of FDA-approved drugs and natural compounds in an in vitro assay presents as a safer starting point as FDA-approved drugs, for example, would have passed all in vivo toxicity testing successfully. The choice of using safe drugs and compounds for screening would ideally accelerate the follow-up cell-based evaluation and drug repositioning process significantly. The importance of this sentiment has been 33 echoed in recent efforts as several promising anti-DENV compounds exhibited unfavourable high toxicity in vivo (Chen et al., 2010; Yin et al., 2009) (Section 1.7.4 ). 1.7.4 Recent advances in anti-DENV drug discovery Much of anti-dengue drug discovery still remains in its infant stages of identification potential antiviral candidates. Small molecules or repositioned drugs that have been recently uncovered in anti-DENV drug discovery efforts are as summarized in Table 1.2. 34 Name Entry inhibitors ST-148 LCTA-949 1662G07 SA-17 NITD-448 NS3 inhibitors ST-610 Ivermectin Compound 23i Aminobenzamide Compound 32 BP2109 NS5 inhibitors Compound 10 Balapiravir NITD-107 NITD-2 NITD-449 NITD-203 NITD-008 Target Cellular activity CC50 (µM) EC50 (µM) Reference C protein Unspecified >100 >25 0.016 6.9 (Byrd et al., 2013a) (De Burghgraeve et al., 2012) (Schmidt et al., 2012) (Kaptein et al., 2010) (Poh et al., 2009) E protein E protein E protein >100 43 48.7 16.9 0.52 9.8 Helicase Helicase >100 3.8 0.272 0.7 Protease Protease >100 No data 24.7 No data Protease Protease >100 29.28 50 2.61 (Lim et al., 2011) (Nguyen et al., 2013) (Noble et al., 2013) (Niyomrattanakit et al., 2010) (Chen et al., 2010) RdRp >100 0.64 (Yin et al., 2009) (Byrd et al., 2013b) (Mastrangelo et al., 2012) (Deng et al., 2012) (Aravapalli et al., 2012) (Steuer et al., 2011) (Yang et al., 2011) Table 1.2 | Summary of recently discovered anti-DENV small molecules and drugs In particular, RdRp inhibitors are of significant interest in this study and will be further discussed. Most recently, balapiravir, originally developed for HCV treatment and later terminated due to adverse hematological effects in patients (Nelson et al., 2012), was repositioned as an anti-DENV drug for a Phase II trial after its anti-DENV efficacy in both DENV replicon and human peripheral blood mononucleated cells (PBMCs) were 35 established in vitro (Nguyen et al., 2013). Unfortunately, balapiravir was not shown to improve the clinical and virological parameters of patients, and a possible reason could be that higher plasma levels of the drug are required for the drug to exert antiviral effects. A large proportion of anti-dengue drug discovery has been led by Novartis Institute for Tropical Diseases (NITD), an effort that has led to the development of many high throughput assays to screen their library of 1.8 million compounds. Not surprisingly, a significant number of RdRp inhibitors have been discovered. The most promising DENV NS5 RdRp inhibitors (NITD-008, NITD-449 and NITD-203) were found to have high inhibitory effects and low cytotoxicity in vitro. However, all exhibited high toxicity in in vivo studies and advancement to drug development were halted. For instance, although the treatment of DENV-infected mice with NITD-008 suppressed peak viremia significantly, toxicological testing through extended oral dosing for 2 weeks in rats and dogs resulted in severe side effects (Chen et al., 2010; Yin et al., 2009). As high toxicity of antiviral molecules can form significant stumbling blocks to the development of anti-DENV RdRp inhibitors, this further lends weight to the approach of this study to screen libraries of FDA-approved drugs and natural compounds as a safer alternative. 36 1.8 Specific aims of project i. Synthesis of high quality DENV-2 NS5 full-length and RdRp domain mutant proteins by utilizing the wheat germ cell-free protein synthesis system ii. Application of a fluorescence-based in vitro NS5 RdRp screening assay to wheat germ cell-free system-derived NS5 protein iii. Identification of inhibitors against DENV NS5 by screening libraries of FDA-approved drugs and natural compounds using primary in vitro NS5 RdRp assay iv. Further validation of candidate drugs/compounds from primary screening assay in secondary in vitro assay v. Evaluation of inhibitory effect of candidate drugs/compounds in cell-based DENV infection system by exploring their cytotoxicity and capacity to reduce viral titers vi. Demonstration that anti-DENV effects of best candidate drug/compound are reproducible in a cell-type and assay-independent manner vii. Investigation into the mechanistic details of inhibition of best candidate drug/compound 37 Chapter 2. Materials and Methods 2.1 Wheat germ cell-free protein expression 2.1.1 Construction of template plasmid DNAs The cDNA encoding the full-length DENV-2 NS5 protein (900 amino acids) or Cterminal RdRp domain mutant (amino acid 405 - 900 of DENV-2 NS5) were obtained by amplification from a plasmid with a pDVWS601 backbone containing the replicon cDNA of DENV subtype 2, New Guinea C (NGC) strain via PCR (Ng et al., 2007). The resulting cDNA were then inserted into the pEU-E01-GST-TEV-MCS vector (CellFree Sciences, Japan), generating the expression plasmids for N-terminal GST-fused NS5 (pEU-GST-NS5) and GST-fused RdRp (pEU-GST-RdRp) respectively. As a negative protein control, a bacterial protein, the dihydrofolate reductase (DHFR) (GST-DHFR) was used. 2.1.2 In vitro transcription Cell-free protein expression was executed using the wheat germ cell-free protein expression system (CellFree Sciences). Using the template plasmids obtained (pEUGST-NS5, pEU-GST-RdRp, pEU-GST-DHFR), in vitro transcription (IVT) to obtain mRNA was carried out according to the manufacturer’s protocols. In brief, IVT was carried out in a 250 µl reaction mixture (1X Transcription buffer, 2.5 mM NTP mix, 1U/µl RNase inhibitor, 1U/µl SP6 RNA polymerase, 100 ng/µl respective cell-free expression plasmid) and incubated for 6 h. Following which, the mRNA quality was checked by method of gel electrophoresis by resolving samples with a 1% agarose gel. 2.1.3 In vitro translation In vitro translation via a bilayer method was then performed with the mRNA obtained. The bottom layer reaction mixture (500 µl) including in vitro transcribed mRNA, 120 OD/ml WEPRO®1240G, and 40 ng/µl creatine kinase was first made and later added to 38 a well of a 6-well plate containing 5.5 ml 1X SUB-AMIX®. The bilayer reaction was incubated for 16 h at 16°C (CellFree Sciences). 2.1.4 Protein affinity purification Affinity purification of cell-free recombinant proteins was performed using Protemist DTII, an automated robotic protein synthesizer (CellFree Sciences). Firstly, the crude protein (cell-free translation mixture after 16 h incubation at 16°C) was mixed with 200 mM NaCl and 10 mM DTT and incubated with Glutathione Sepharose Fast Flow beads (GE Healthcare) for 2 h with mild rocking. After 3 rounds of washing by PBS, the GSTtagged recombinant proteins were then eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM reduced glutathione, 10% glycerol) yielding the purified protein. 2.1.5 Buffer exchange As the presence of reduced glutathione in the purified protein could affect downstream assays, purified proteins by Protemist DTII were further subjected to buffer exchange by fast protein liquid chromatography (FPLC) using a HiTrap Desalting column (GE Healthcare). These were eluted with a protein buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol). 2.1.6 Protein concentration Proteins were then concentrated approximately 20-fold with Amicon Ultracentrifugal filters (Millipore). Next, protein concentration was determined using QuickStart Bradford Protein Assay (Bio-Rad) using bovine serum albumin (BSA) as a protein standard. Proteins were then aliquoted and stored at -80°C until use. 39 2.1.7 CBB analysis To determine the purification efficiency of the recombinant proteins, samples from various purification steps were subjected to Coomassie brilliant blue (CBB) staining. The samples were added to an equal volume of sample of 2X sodium dodecyl sulfate (SDS) buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol) and boiled at 98°C for 10 min. The samples were then resolved by SDS-polyacrylamide gel electrophoresis (SDSPAGE) with a 10% acrylamide gel. Electrophoresis was performed using running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 25 mA for 1 h. After SDS-PAGE concluded, the gel was washed briefly with water and fixed by incubation with CBB fixing solution (50% methanol, 10% glacial acetic acid) for 10 min with gentle shaking. This was repeated three times, followed by staining with Quick CBB Plus (Wako Pure Chemical Industries, Japan) for 30 min. The gel was then destained in water with gentle shaking and brief microwaving every 30 min to facilitate the process. Pre-stained and unstained molecular weight markers (Thermo Fisher Scientific) were also loaded to track SDS-PAGE protein migration and CBB staining efficiency respectively. 2.1.8 Western blotting analysis The samples were added to an equal volume of sample of 2X SDS buffer (125 mM TrisHCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM βmercaptoethanol) and boiled at 98°C for 10 min. The samples were then resolved by SDS-PAGE with a 10% acrylamide gel. Electrophoresis was performed using running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 25 mA for 1 h. Resolved proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore) at 120mA/membrane for 70min. After blocking with 5% 40 BSA dissolved in 0.05% phosphate-buffered saline-Tween 20 solution (PBS-T, 0.05% Tween-20 in PBS), immunoblots were performed using monoclonal rabbit anti-NS5 antibody (1:2000, GeneTex) dissolved in 3% BSA/PBS-T. Secondary horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibodies (1:2000, Cell Signaling Technology) dissolved in PBS-T were then used to probe primary antibody binding, followed by detection with Western Lightning® Pro enhanced chemiluminescence (ECL) reagent (Perkin-Elmer). Protein band imaging was performed by ImageQuant LAS4000 mini (GE Healthcare) and analysis was conducted using ImageQuant™ TL software (GE Healthcare). 2.2 Preparation of drugs and compounds 2.2.1 Drug/Compound libraries for primary screening assay The FDA-approved drug library comprised of 640 drugs (ENZO Life Sciences) and was kindly provided from Professor Chang Young-Tae (Department of Chemistry, NUS). The natural compound library was a combination compounds extracted by Dr. Yoshiyuki Yoshinaka (Tokyo Medical and Dental University, Japan) and health supplements from Goodcare Pharma. All drugs and compounds in the libraries were kept at a stock concentration of 400 µg/ml in 100% dimethyl sulfoxide (DMSO, HybriMax, Sigma-Aldrich) at -20°C for storage. All drugs/compounds were used at a final concentration of 20 µg/ml with 5% DMSO in the primary screen using in vitro DENV NS5 RdRp assay. 2.2.2 Drugs for validation studies As drugs in the FDA-approved drug library was limited in quantity and only available in low concentrations, hits derived from the primary screening assay were commercially obtained and used for further validation studies. In validation studies, several compounds were commercially obtained, including oxiconazole nitrate (AK Scientific), itraconazole (AK Scientific), tolcapone (AK Scientific), butoconazole nitrate (AK 41 Scientific), lomofungin (ENZO Life Sciences), pinacidil (ENZO Life Sciences) and entacapone (Tocris Bioscience). All drugs were diluted to a stock concentration of 50 mM with 100% DMSO, aliquoted and stored at -20°C. All drugs were freshly diluted for each use. 2.3 Fluorescence-based in vitro DENV NS5 RdRp assay A linear RNA template, 3’UTR-U30 (5’-U30-ACC AGG UUC UAG AAC CUG UU-3’ [(Niyomrattanakit et al., 2011), was diluted in TE buffer and incubated at 60°C for 5 min. Next, it was left at room temperature for a further 60 min to induce the formation of an intramolecular short hairpin structure. This hairpin RNA template mimics the 3’ untranslated (UTR) region of the DENV genome and is a substrate of DENV NS5 protein. To kick-start the in vitro assay, 100 nM hairpin RNA template and 25 nM GST-NS5, GST-RdRp or negative control protein (GST-DHFR) were pre-incubated in RdRp assay buffer (50 mM Tris-HCl, pH 7.0, 2 mM DTT, 10 mM KCl, 1 mM MnCl2, 0.01% Triton-X-100) in a total volume of 10 µl for 30 min in individual wells of a black 384well plate (Corning) to allow binding of protein to RNA template. After the pre-incubation, drugs/compounds from the screening libraries were added. This was followed by the further addition of 2 µM BBT-ATP (Jena Bioscience, Germany) to initiate hairpin RNA template-dependent RNA polymerization. After addition of all reagents, the plate was shaken at high speed for 20 s using iMark Microplate Reader (Bio-Rad). This template-dependent RNA polymerization was carried out in a total volume of 20 µl for 2 h at room temperature. Next, 10 µl of 1 ng/µl calf intestinal alkaline phosphatase (CIAP, Promega) buffered in 150 mM Tris-HCl pH 9.3, 300 µM MgCl2 was added to the reaction mixture. The plate was shaken for 20 s at high speed using iMark Microplate Reader and incubated in the 42 dark for 1 h for the liberation of BBT from BBT-PPi, a by-product of the incorporation of the nucleotide analogue into the growing RNA strand. Liberated BBT was measured by Synergy H1 hybrid microplate reader (Biotek) at an excitation wavelength of 422 nm and an emission wavelength of 566 nm (Niyomrattanakit et al., 2011; Takahashi et al., 2012). 2.4 Cell culture 2.4.1 General growth and maintenance Human hepatocellular carcinoma cells, Huh-7.5 (Nakabayashi et al., 1982), was maintained in Dulbecco's modified Eagle medium (DMEM) (Biowest) supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen) and antibiotics (100 U/ml penicillin and streptomycin, Invitrogen). Baby hamster kidney cell, BHK-21, was maintained in RPMI 1640 (Biowest) supplemented with 10% FBS and antibiotics. Rhesus monkey kidney cells, LLC-MK2, was maintained in Eagle's minimal essential medium (EMEM) (Sigma-Aldrich) supplemented with 10% FBS, antibiotics and 2 mM L-glutamine (Sigma). BHK-21 cells harbouring a DENV subgenomic replicon with a luciferase reporter (Ng et al., 2007) were maintained in DMEM supplemented with 10% FBS, antibiotics, and 3 µg/ml puromycin (Invitrogen). Aedes albopictus mosquito cells, C6/36 cells, were maintained in RPMI 1640 supplemented with 8% FBS and antibiotics. The cells were cultured in a 37°C incubator with a 5% CO2 atmosphere, with the exception of C6/36 cells in a 28°C incubator. The culture medium was refreshed every three days. When confluence was reached, cells were washed once with phosphate buffered saline (PBS) before trypsinization. One milliliter of 0.25% trypsin/1 mM EDTA solution (Invitrogen) was added for trypsinization, before 9 ml of complete medium was combined to inactivate trypsin. Cells were spun down at 1,500 rpm for 5 min at room temperature, resuspended in complete medium, and passaged at a 1:10 ratio. 43 2.4.2 Viruses preparation A DENV-2 strain (Singapore isolate EDEN2) isolated in Singapore(Low et al., 2006), which was kindly provided by Professor Subhash Vasudevan from Duke-NUS, was inoculated into C6/36 cells at multiplicity of infection (MOI) 0.1 for amplification and the cells were maintained in RPMI 1640 containing 8% FBS and antibiotics. At 5 days post-inoculation, the supernatant was filtered to remove cell debris, aliquoted and stored at -80°C. For the titration of virus stock, plaque assay was performed. BHK-21 cells were seeded at a density of 8 x 105 cells in each well of a 6-well plate. The next day, serial dilutions of 10 µl of viral supernatant were used to infect the BHK cells in 1 ml serum-free RMPI media. Infection was carried out for 1 h at 37°C with gentle rocking every 15 min. After which, the infection media was replaced by plaque assay overlay media (0.8% methylcellulose [Sigma], 2% FBS, 100 U/ml penicillin and streptomycin in RPMI 1640). After 5 days, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% paraformaldehyde (PFA, Wako Pure Chemical Industries, Japan) for 20 min at room temperature, washing in running water for 5 min and staining with 1% crystal violet (Sigma) for another 20 min at room temperature. After a further washing step and drying of plates, plaques were enumerated and viral titers were determined. This virus stock was used for the infection of Huh-7.5 cells. For the infection of LLC-MK2 cells, all four serotypes of DENV including DENV-1 (Singapore isolate S144), DENV-2 (NGC strain), DENV-3 (Singapore isolate EDEN 130/05) and DENV-4 (Singapore isolate S8976) were kindly provided by Dr. Justin Chu (Department of Microbiology, NUS). Likewise, these viruses were propagated in C6/36 cells and the cells were maintained in RMPI 1640 (8% FBS, 100 U/ml penicillin and streptomycin). At 5 days post-inoculation, the supernatant was centrifuged to remove cell debris. If concentration of virus was necessary, the viral supernatants were loaded onto a 30% sucrose cushion and centrifuged for 16 h at 100, 000 xg at 4°C. The virus 44 fraction obtained was then re-suspended in PBS, aliquoted and stored at -80°C. The viral titers were determined by plaque assay and used for the infection of LLC-MK2 cells. 2.5 Validation of inhibition of DENV replication by drug/compound in cellbased infection system 2.5.1 Cell viability assay Huh-7.5 cells were seeded at a density of 2 x 104 cells in each well of a white, clear bottom 96-well plate (Corning). After 24 h of incubation, the culture media was removed and replaced with a wide concentration range of drugs/compounds diluted in DMEM to a final DMSO concentration of 0.5%. Forty-eight hours later, 100 µl of supernatant was removed. 100 µl of CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega) was then added and the plate was shaken at 600 rpm for 3 min. After further 10 min incubation in the dark, luminescence readings directly proportional to the amount of ATP released were taken with Synergy H1 hybrid microplate reader (Biotek). 2.5.2 Infection assay: Reduction of viral titer by drug/compound Huh-7.5 cells were seeded at a density of 2 x 104 cells in each well of a white, clear bottom 96-well plate. After 24 h of incubation, the culture media was removed and replaced with a different concentration of drugs/compounds to a final DMSO concentration of 0.5%. Simultaneously, the cells were also infected with DENV-2 at a MOI of 0.1. After an incubation of 48 h, viral supernatants were collected and stored at 80°C till use. To determine differences in viral titer in supernatants, plaque assay was performed in BHK cells. 45 BHK-21 cells were seeded at a density of 8 x 105 cells in each well of a 6-well plate. The next day, 10 µl of viral supernatant was used to infect the BHK-21 cells in 1 ml serum-free RPMI 1640 media. Infection was carried out for 1 h at 37°C with gentle rocking every 15 min. After which, the infection media was replaced by plaque assay overlay media (0.8% methylcellulose, 2% FBS, 100 U/ml penicillin and streptomycin in RPMI 1640). After 5 days, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% PFA for 20 min at room temperature, washing in running water for 5 min and staining with 1% crystal violet for another 20 min at room temperature. After a further washing step and drying of plates, plaques were enumerated and viral titers were determined. 2.5.3 Calculation of selectivity index (SI) The values obtained for both cell viability assays and viral titer determination via plaque assays were processed and expressed as a relative percentage against the readings for 0.5% DMSO control in each assay. CC50 (concentration of a drug that causes 50% cell death) and EC50 (effective concentration of a drug that inhibits 50% of viral replication) were then calculated from cell viability assays and plaque assays respectively. From these values, the selectivity index (SI = CC50/ EC50) of the various drugs were derived. Selectivity index of an effective drug/compound should be greater than 1. The higher a drug’s SI is, the wider the window is between an effective dose from a cytotoxic dose, and hence, the greater its potential as a drug. 2.6 Cytopathic effect (CPE)-based anti-dengue assay This assay was developed based on the cytopathic effect (CPE) of DENV-2 infection seen in LLC-MK2 cells and anti-dengue activity of a drug/compound is characterized by the rescue of cells from infection-induced cell death (Ichiyama et al., 2013). 46 LLC-MK2 cells were seeded in a 96-well plate at a density of 2 x 103 cells per well one day prior to the start of the assay. After 24 h, cell culture media was replaced with a range of concentrations of the test drug/compound diluted in EMEM. Following which, half the set of cells were inoculated with DENV-2 at MOI 0.5, while the other half remained as a mock infected control. The cells were then allowed to proliferate. After 5 days of infection, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the number of viable cells present (Mosmann, 1983). In brief, 20 µl of 5 mg/ml stock MTT solution (Sigma) was added into each well and incubated for 1 h at 37°C. Next, acid-isopropanol (0.04 N HCl in isopropanol) was added, and further incubated at room temperature for 1 h with shaking. Fluorescence readings were then taken using Infinite 200 Microplate Reader (Tecan). 2.7 Plaque reduction assay The plaque reduction assay tests the ability of a drug/compound to reduce viral titer at non-cytotoxic doses in LLC-MK2 cells. LLC-MK2 cells were seeded in a 6-well plate at a density of 3 x 105 cells per well one day prior to the start of the assay. After 24 h, cell culture media was replaced with an inoculation of 100 plaque forming units (PFU) of DENV, followed by a range of concentrations of drugs/compounds (final DMSO concentration of 0.5%) diluted in plaque assay overlay media (0.8% methylcellulose, 10% FBS and antibiotics in EMEM). At 5 days post-inoculation, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% PFA for 20 min at room temperature, washing in running water for 5 min and staining with 1% crystal violet for another 20 min at room temperature. After a further washing step and drying of plates, plaques were enumerated and viral titers were determined. 47 2.8 Time of addition assay LLC-MK2 cells were seeded in a 6-well plate at a density of 3 x 105 cells per well one day prior to the start of the assay. Twenty-four hours later, the cells were inoculated with 100 PFU of DENV-2 at 4°C for 1.5 h. After the cells were washed with ice cold PBS thrice, the plates were shifted for culturing in a 37°C incubator. Kusunoki (50 µg/mg) diluted in plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the cells at different time points pre- and postinfection (-1.5, 0, 1, 2, 3, 4 and 5 h). Heparin (100 µg/mg), a known entry inhibitor of DENV, was used as a positive control (Lin et al., 2002), while DMSO was used as a negative control. At 5 days post-inoculation, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% PFA for 20 min at room temperature, washing in running water for 5 min and staining with 1% crystal violet for another 20 min at room temperature. After a further washing step and drying of plates, plaques were enumerated. 2.9 Binding assay LLC-MK2 cells were seeded in a 6-well plate at a density of 3 x 105 cells per well one day prior to the start of the assay. Twenty-four hours later, the cells were inoculated with 200 PFU of DENV-2 at 4°C or 37°C for 1.5 h with gentle rocking every 5 min in the presence of kusunoki (50 µg/mg). DMSO was used as a negative control. After the cells were washed with ice cold PBS thrice, plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the wells. At 5 days post-inoculation, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% PFA for 20 min at room temperature, washing in running water for 5 min and staining with 1% crystal violet for another 20 min at room temperature. After a further washing step and drying of plates, plaques were enumerated. 48 2.10 DENV replicon luciferase assay BHK-21 cells containing a DENV subgenomic replicon with a luciferase reporter gene (Ng et al., 2007) were seeded in a 6-well plate at a density of 2.5 x 105 cells per well one day prior to the start of the assay and weaned off the puromycin usually included in culturing media to maintain the DENV replicon. Twenty-four hours later, the cells were treated with kusunoki (40µg/ml) diluted in DMEM. NITD-008 (10, 20, 50 µg/ml), a known DENV RdRp inhibitor, was used as a positive control (Yin et al., 2009). After an incubation of 48 h, the cells were washed briefly with PBS and harvested. After a lysis step using M-PER (mammalian protein extraction reagent, Thermo Fisher Scientific) with shaking for 5 min, the cells were spun down at 14,000 xg for 15 min. The supernatant was then transferred to a new tube and used for analysis. For luciferase assay, the renilla luciferase assay system (Promega) was utilized. In brief, 100 µl of reagent was added to 20 µl of cell lysate. Luciferase activity was measured using Infinite 200 Microplate Reader. 49 Chapter 3. Results 3.1 Production of DENV-2 NS5 protein by wheat germ cell-free protein synthesis system The first aim of the project is to present DENV-2 NS5 protein made using the wheat germ cell-free protein synthesis system as a useful alternative to conventional protein production methods (eg. E. coli, baculovirus systems) that balances cost, efficiency and physiological relevance. Full-length NS5 (GST-NS5), its C-terminal RdRp domain mutant (GST-RdRp) and a negative control protein (GST-DHFR) were synthesized using the wheat germ cell-free system. A schematic illustration (Figure 3.1a) outlines the protein production method and the downstream purification steps taken. To determine the purification efficiency of the recombinant proteins, GST-NS5 protein samples from various purification steps (crude, purified, purification flow-through and desalted) were subjected to CBB staining. A representative CBB staining (Figure 3.1b) is shown. In the crude sample, a protein band of the correct size (133 kDa) was seen (lane 2). However, in addition to the protein of interest, many unspecific protein bands were seen, most possibly due to the abundance of endogenous proteins present in the wheat germ endosperm. After affinity purification using glutathione sepharose beads, the purified sample showed a single band of approximately 133 kDa (lane 3), representing high purification efficiency, although some protein of interest could possibly have been lost, as detected by a band of the same size in the purification flowthrough (lane 4). As the presence of reduced glutathione in the purified protein could affect downstream assays, purified GST-NS5 proteins were further purified using a desalting column. This process did not affect the purity of the sample, as seen in the single band of the purified sample (lane 5). However, as the purified sample was diluted in the purification buffer (faint band), concentration of the protein was performed. In 50 addition, western blotting analysis also showed specificity of the NS5 protein produced (Figure 3.1c). 51 Figure 3.1 | Production of GST-NS5, GST-RdRp and GST-DHFR proteins by wheat germ cell-free protein synthesis system a) Schematic illustration of the protein production procedure. pEU-based plasmid DNAs (pEU-GST-NS5, pEU-GST-RdRp and pEU-GST-DHFR) were used as templates for in vitro transcription step of wheat germ cell-free protein synthesis system. mRNA transcripts obtained were then used for in vitro translation via a bilayer method, yielding crude protein mixtures. The crude proteins were then subject to affinity purification first by glutathione sepharose beads (GE Healthcare), subsequently eluted with reduced glutathione in an automated robotic system, Protemist DTII (CellFree Sciences). As the presence 52 of reduced glutathione in the protein sample could affect downstream assays, purified proteins were further subjected to buffer exchange using a desalting column. Proteins were then concentrated. b) CBB staining analysis of GST-NS5 from various purification steps. Protein samples including GST-NS5 from various purification steps (crude [lane 2], purified [lane 3], purification flow-through [lane 4] and buffer-exchanged [lane 5]) were subjected to SDS-PAGE analysis and stained with CBB. c) Western blotting analysis of GST-NS5. Purified GST-NS5 was subjected to SDS-PAGE and blotted with anti-NS5 antibody (lane 1). d) CBB staining analysis of GST-DHFR. Purified GST-DHFR (negative control protein) was subjected to SDS-PAGE analysis and stained with CBB (lane 2). 53 3.2 Development of fluorescence-based DENV NS5 RdRp assay using wheat germ cell-free system-produced NS5 proteins The fluorescence-based in vitro assay measures the RdRp activity of DENV NS5 protein (Niyomrattanakit et al., 2011; Takahashi et al., 2012). A hairpin RNA mimicking the 3’ UTR region of DENV was first prepared. This hairpin RNA served as a template for NS5 to bind to, and elongate the RNA strand with an available nucleotide pool containing a nucleotide analogue, BBT-ATP. After an incubation period to allow for RNA synthesis, CIAP was added to liberate of BBT from BBT-PPi, a by-product of the incorporation of the nucleotide analogue into the synthesizing RNA strand. The liberated BBT was measured by Synergy H1 hybrid microplate reader. To clarify the specificity of the NS5 RdRp assay, core assay constituents were removed separately. As shown in Figure 3.2a, this included reactions with no proteins (i.e. protein buffer alone was added in place of GST-NS5), no MnCl2 (RdRp cofactor) and no nucleotide analogue BBT-ATP respectively. These were then compared to the reaction with the full reagent mix (GST-NS5). Both the no protein and no BBT-ATP reactions display minimal background RdRp activity at approximately 10% and 1% respectively. Without the cofactor of RdRp, MnCl2, NS5 RdRp activity was decreased to about 40% (Figure 3.2a). Fifty micromolar of NITD-008, a reported DENV RdRp inhibitor (Yin et al., 2009), was also added to the full NS5 RdRp reaction mix as a control and inhibited approximately 40% of NS5 RdRp activity. To further ensure that the measured fluorescence levels in the in vitro RdRp assay correlated to enzymatic activity specific to NS5 protein, the assay was carried out using increasing concentration of GST-NS5 (1.56 - 25 nM). A dose-dependent increase in fluorescence intensity was seen as the concentrations of GST-NS5 were increased (Figure 3.2b). The relative RdRp activities of DENV-2 full-length NS5 (GST-NS5) were explored using RdRp domain mutant of NS5 (GST-RdRp) and a negative protein control (GSTDHFR). As expected, DHFR only produced background fluorescence levels of about 54 10%, comparable to the no protein control (Figure 3.2c). Although equal concentrations of wild-type (i.e. full-length) and RdRp domain mutant of NS5 proteins were used, the RdRp domain mutant protein only achieved approximately 80% of RdRp activity compared to full-length NS5 (Figure 3.2c). Figure 3.2 | Evaluation of fluorescence-based RdRp assay using DENV-2 NS5 and DENV-2 RdRp produced by wheat germ cell-free system a) Specificity of NS5 RdRp assay. In addition to the reaction with the full reagent mix (GST-NS5), core assay constituents were removed. This included reactions with no proteins (desalting buffer was added in place of GST-NS5), no MnCl2 and no nucleotide analogue BBT-ATP, respectively. A reported DENV RdRp inhibitor, NITD-008, was also added (50 µM) to the full NS5 RdRp reaction mix 55 as a control. All readings were expressed relative to that of full NS5 RdRp reaction mix. Results are the mean of 3 independent experiments with error bars ± sd. b) Dose-dependency of NS5 RdRp assay. To ensure that the measured fluorescence levels in the in vitro NS5 RdRp assay correlated to RdRp activity specific to NS5 protein, increasing GST-NS5 concentrations (1.56 - 25 nM) were used in separate reactions. Results are the mean of 3 independent experiments with error bars ± sd. c) RdRp activities of full-length and RdRp domain mutant of NS5. Twenty five nanomolar of GST-NS5 (full-length), GST-RdRp (domain mutant) and GSTDHFR (negative control), was used in the in vitro RdRp assay. Results are the mean of 3 independent experiments with error bars ± sd. 56 3.3 Screening of FDA-approved drug and natural compound libraries in fluorescence-based in vitro NS5 RdRp assay After the fluorescence-based RdRp assay using DENV-2 NS5 synthesized by wheat germ cell-free system was established, the primary screening of a total of 648 drugs, which were composed of 640 FDA-approved drugs (Figure 3.3a) and 8 natural compounds (Figure 3.3b), was conducted. These are regarded as safer alternatives to experimental synthetic compounds. Inhibitors against RdRp activity of RNA viruses developed so far can fall into two categories: nucleoside inhibitors and non-nucleoside inhibitors. Nucleoside inhibitors are administered as pro-drugs for increased safety, bioavailability and efficacy (Peterson and McKenna, 2009). Since they are nucleoside analogues, these have to be successively phosphorylated by cellular enzymes such as phosphoglycerate kinase and creatine kinase (Cihlar and Ray, 2010) . These are structurally similar to nucleosides and they work as antimetabolites by being incorporated into growing RNA strands. Unlike actual nucleotides, nucleotide analogues lack a 3´-hydroxyl group on the deoxyribose moiety, which is usually hydrolyzed to form a phosphodiester bond with the next nucleic acid. These nucleoside analogues thereby act as chain terminators. However, mitochondrial toxicity and other adverse events have been reported as a common side effects of the usage of nucleoside analogues, due to structural similarities to endogenous nucleotides as well as differential tissue-specific levels of pro-drug activation (Lund et al., 2007; Noble et al., 2010). Non-nucleoside inhibitors, on the other hand, may not necessarily bear a structural resemblance to nucleotides. Binding to allosteric sites of the NS5 protein, these inhibitors change the conformation of the active site such that the protein is no longer competent to carry out its RdRp activities (Wang et al., 2003). As majority of the drugs and compounds screened in this study belonged to the FDAapproved drug library, any inhibitors picked up by the RdRp assay are more likely to be non-nucleoside inhibitors. This is because nucleotide analogues in the FDA-approved 57 library would be pro-drugs, and would not have been able to be converted to an active triphosphate nucleotide form in an in vitro setting. A limitation in using a FDA-approved library of drugs in an in vitro assay would therefore be the inevitable inability to pick up drugs which require processing in an in vivo setting, such as nucleotide analogues. All drugs and compounds were screened at a final concentration of 20 µg/ml in 5% DMSO and measurements were expressed relative to 5% DMSO diluent control without drugs or compounds. Any drug or compound which inhibited NS5 activity would show a lower fluorescence intensity compared to the control reaction containing 5% DMSO without drugs/compounds. 58 59 Figure 3.3 | Screening of FDA-approved drug and natural compound libraries in fluorescence-based DENV NS5 RdRp assay Screening results of 640 FDA-approved drugs (a) and 8 natural compounds (b) in in vitro NS5 RdRp assay. After pre-incubation of RNA template and GST-NS5 protein, drugs or compounds were added to the reaction mixture at a final screening concentration of 20 µg/ml in 5% DMSO. BBT-ATP was then added to initiate template RNA-dependent de novo RNA polymerization. After an incubation of 2 h at room temperature, CIAP was added and further incubated in the dark for 1 h for the liberation of BBT from BBT-PPi, a by-product of the incorporation of the nucleotide analogue into the growing RNA strand. Liberated BBT was measured by Synergy H1 hybrid microplate reader. All readings are expressed relative to 5% DMSO diluent control without drugs or compounds. Measurements were taken over 2 independent experiments, denoted by red and blue circles. 60 3.4 Summary of primary in vitro NS5 RdRp screening study and in vitro validation of hits After the completion of the primary screening study, summary statistics were consolidated (Figure 3.4a). Out of 648 FDA-approved drugs and natural compounds screened against DENV-2 NS5 RdRp activity, a total of 25 drugs and compounds showed at least a 30% inhibition of RdRp activity over two independent experiments. These compounds were grouped into 6 groups in terms of relative percentage inhibition of RdRp activity compared to the 5% DMSO diluent control without drugs or compounds (Figure 3.4a). An inclusion criteria set for downstream validation studies was set at a minimal inhibition of 50% RdRp activity over two independent experiments in the primary screening assay (Figure 3.4a). Using this criterion, the overall hit rate was 1.2%. A total of seven FDA-approved drugs and one natural compound proceeded to the next validation step. In descending order of percentage inhibition of RdRp activity, these are: Oxiconazole nitrate (compound #507), itraconazole (#469), tolcapone (#136), entacapone (#157), butoconazole nitrate (#619), lomofungin (#7), pinacidil (#19) and the proanthocyanidin-rich oligomeric polyphenol fraction of the extract from the Japanese cinnamon tree (Cinnamomum camphora), kusunoki (#648). As the drugs in the FDA-approved drug library were available in limited quantities and low concentrations, commercial drugs were purchased for further validation studies. Natural compounds were not commercially obtainable. These eight drugs and compounds were subjected to the same in vitro NS5 RdRp assay conditions to verify their inhibitory activity in varying concentrations (2.5 µM, 25 µM and 250 µM for FDA-approved drugs; 0.025 mg/ml, 0.25 mg/ml and 2.5 mg/ml for natural compound kusunoki). All drugs and compounds showed a dose-dependent 61 inhibition of in vitro RdRp activity to varying degrees. The least inhibition was detected for oxiconazole nitrate and pinacidil (Figure. 3.4b). 62 Figure 3.4 | Summary and validation of hit compounds obtained primary screening a) Summary statistics of the primary in vitro NS5 RdRp screening study with 640 FDA-approved drugs and 8 natural compounds. All readings were expressed relative to 5% DMSO diluent control without drugs or compounds. A total of 25 drugs and compounds showed at least a 30% inhibition of RdRp activity over two independent experiments. b) Validation experiment of top 8 inhibitors from the primary screening assay. The top 8 inhibitors (7 FDA-approved drugs and 1 natural compound) from the 63 primary screening assay showed at least a 50% inhibition of RdRp activity relative to 5% DMSO diluent control without drugs or compounds. The 7 drugs were obtained commercially, and together with the natural compound, were put through the same in vitro NS5 RdRp assay in varying doses (2.5 µM, 25 µM and 250 µM for drugs obtained commercially; 0.025 mg/ml, 0.25 mg/ml and 2.5 mg/ml for natural compound kusunoki). All readings were expressed relative to 5% DMSO diluent control without drugs or compounds. Results are the mean of 3 independent experiments with error bars ± sd. 64 3.5 Secondary screening of top 8 inhibitors in in vitro NS5 RdRp assay using RdRp domain mutant protein DENV-2 NS5 is a bifunctional protein made up of 2 functional domains: N-terminal MTase domain and C-terminal RdRp domain (Ackermann and Padmanabhan, 2001; Egloff et al., 2002). The additional validation of the top eight inhibitors in in vitro RdRp assay using NS5 RdRp domain mutant (Figure 3.5a) was conducted with two aims: 1. The RdRp domain mutant protein exhibited in vitro RdRp activity (Figure 3.2c). To ensure that the inhibition by drugs seen in the primary screening assay was indeed due the action of drugs on RdRp activity of NS5, a RdRp domain mutant protein (i.e. lacking MTase domain) was used in the same RdRp assay in an attempt to attribute the dose-dependent inhibition of RdRp activity by the drug or compound to the RdRp domain specifically. 2. This experiment could give insights into the binding region of the drug to NS5. As possible non-nucleoside inhibitors, drugs or compounds could bind in an allosteric site either within the RdRp domain, or in a region elsewhere on the NS5 protein (eg. MTase domain). Binding to an allosteric site in or outside the RdRp could cause a conformational change in the NS5 protein specficially within the RdRp active site, affecting RdRp activity (Wang et al., 2003). The top eight drugs and compounds were subjected to an in vitro RdRp assay using a NS5 RdRp domain mutant (GST-RdRp, Figure 3.5a). Varying concentrations of drugs and compounds were applied (2.5 µM, 25 µM and 250 µM for FDA-approved drugs; 0.025 mg/ml, 0.25 mg/ml and 2.5 mg/ml for natural compound kusunoki). All drugs and compounds again showed a dose-dependent inhibition of in vitro RdRp activity exhibited by the domain mutant. While most drugs and compounds showed similar extents inhibition compared to when full-length NS5 protein was used (Figure 3.5b), itraconazole exhibited a greater degree of inhibition of RdRp activity when RdRp mutant protein was used. This was in contrast to pinacidil and kusunoki, which showed 65 evidence of smaller magnitude of inhibition in the validations assay when RdRp domain mutant was used compared to full-length NS5 protein. 66 Figure 3.5 | Validation of top 8 inhibitors in in vitro RdRp assay using NS5 RdRp domain mutant a) A schematic representation of full-length (GST-NS5) and RdRp domain mutant (GST-RdRp) of NS5 67 b) Activity of top 8 inhibitors in in vitro NS5 RdRp assay using RdRp domain mutant. The top 8 inhibitors from the primary screening assay showed at least a 50% inhibition of RdRp activity relative to 5% DMSO diluent control without drugs or compounds. These 8 drugs and compounds were subjected to a secondary in vitro RdRp assay using GST-RdRp. Varying concentrations of drugs and compounds were applied (2.5 µM, 25 µM and 250 µM for FDAapproved drugs; 0.025 mg/ml, 0.25 mg/ml and 2.5 mg/ml for natural compound kusunoki). All readings were expressed relative to 5% DMSO diluent control without drugs or compounds. Results are the mean of 3 independent experiments with error bars ± sd. 68 3.6 Validation of inhibition of DENV replication by drug/compound in cellbased system The top 8 inhibitors from the in vitro NS5 RdRp assay were next tested in a cell-based assay to verify if they had the ability to inhibit DENV replication. This validation step comprised of 2 experiments: A cell viability assay to measure the cytotoxicity of a drug/compound and a plaque assay to monitor the reduction of viral titer by the drug/compound. Huh-7.5 cells were seeded in a 96-well plate and infected with DENV-2 at MOI 0.1 while simultaneously treated with a wide concentration range of drugs/compounds diluted in DMEM to a final DMSO concentration of 0.5%. After an incubation of 48 h, viral supernatants were collected and the viral titer of each was determined in a plaque assay using BHK-21 cells. In parallel experiments, cell viability was analyzed using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega) to measure the cytotoxicity of a drug/compound, which measures metabolically active cells through quantification of ATP via luciferase activity. The values obtained for both cell viability assays (blue) and viral titer (red) determination via plaque assays were processed and expressed relative to 0.5% DMSO diluent control without drugs or compounds. Results are the mean of 3 independent experiments with error bars ± sd. CC50 (concentration of a drug that causes 50% cell death) and EC50 (concentration of a drug that inhibits 50% of viral replication) were then calculated from cell viability assay and plaque assay data respectively. In addition to these values, the selectivity index (SI = CC50/ EC50) of the various drugs were derived. As viral titer (and therefore EC50) is affected by both drug/compound cytotoxicity and actual inhibition of drug/compound, the selectivity index of an effective drug/compound should be greater than 1 to demonstrate that the reduction of viral titer is due to the action of a drug/compound inhibiting viral replication. The higher 69 a drug’s SI is, the wider the window is between an effective dose from a cytotoxic dose, and hence, the greater its potential as a drug. With the exception of oxiconazole nitrate (data not shown), treatment of Huh-7.5 cells with the rest of the shortlisted drugs and compounds generally showed a dose-dependent decrease in both cell viability and viral titer ( Figure a - g). In contrast, oxiconazole nitrate showed significant decrease in neither cell viability nor viral titer up to 250 µM. Figure h shows a summary of the cell-based validation study, together with brief additional information known about the drugs. 5 out of 8 drugs (itraconazole, entacapone, butoconazole nitrate, pinacidil and kusunoki) and compounds showed an SI of higher than 1, showing that 62.5% of the shortlisted drugs and compounds demonstrated anti-DENV activity in cultured cells ( Figure h). A possible inhibitor candidate that may be suitable for future anti-viral use is pinacidil ( Figure f). After an initial decrease in cell viability at low concentrations, cell viability was still maintained at approximately 80% despite increasing pinacidil concentrations. Even though CC50 was not reached, viral titer was almost entirely abolished at 300 µM with respect to 0.5% DMSO control. Interestingly, of the 5 drugs and compounds that showed anti-DENV activity, the proanthocyanidin extract from the Japanese cinnamon tree (Cinnamomum camphora), kusunoki ( Figure g) reflected the highest SI value of 1387.3 with a non-cytotoxic dose of 145.9 ng/ml needed to reduce viral titer by 50%, suggesting a wide window for therapeutic anti-DENV use. 70 Due to the high SI recorded, kusunoki was therefore subjected to further experiments to validate its potential as an anti-viral therapeutic. 71 72 73 Figure 3.6 | Validation of inhibition of DENV replication by drug/compound in cell-based infection system To measure cytotoxicity of drugs and compounds, Huh-7.5 cells were treated with a wide concentration range (indicated in each graph) of drugs/compounds diluted in DMEM to a final concentration of 0.5%. A cell viability assay was performed using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega) after 48 h. To measure the ability of the drugs/compounds to reduce viral titer, Huh-7.5 cells were infected with DENV-2 at MOI 0.1 while simultaneously treated with drugs/compounds. Forty-eight hours after infection, viral supernatants were collected and the viral titer of each was determined in a plaque assay using BHK-21 cells. a – g) Cell viability and viral titer measurements after treatment of Huh-7.5 cells with itraconazole (a), tolcapone (b), entcapone (c), butoconazole nitrate (d), lomofungin (e), pinacidil (f) and kusunoki (g), respectively. Cell viability readings expressed relative to 0.5% DMSO diluent control without drugs or compounds. Viral titer measurements expressed as PFU/ml. Results are the mean of 3 independent experiments with error bars ± sd. 74 h) Figure 3.6 | Validation of inhibition of DENV replication by drug/compound in cell-based infection system h) Summary of anti-DENV validation in cell-based infection system with additional information on drugs/compounds 75 3.7 Effects of kusunoki in CPE-based anti-dengue assay To ensure that kusunoki inhibition of DENV was not a cell-line or assay-specific phenomenon, the effects of kusunoki in a CPE-based anti-dengue assay in LLC-MK2 (monkey kidney) cells were explored. This assay was developed based on the CPE of DENV-2 infection seen in LLC-MK2 cells and anti-dengue activity of a drug/compound is characterized by the rescue of cells from infection-induced cell death (Ichiyama et al., 2013). In a 96-well plate format, LLC-MK2 cells were treated with range of concentrations of kusunoki diluted in EMEM. Following which, half the set of cells were inoculated with DENV-2 at MOI 0.5, while the other half remained as a mock infected control. The cells were then allowed to proliferate for 5 days. After 5 days of incubation, a MTT assay was performed to determine the number of viable cells present (Mosmann, 1983). Fluorescence readings were then taken using Infinite® 200 Microplate Reader (Tecan). Readings were expressed as a relative percentage of controls, where 100% cell viability represented maximum cell growth without infection and kusunoki treatment and 0% cell viability represented cell death caused by DENV-2 infection. Results are the mean of 3 independent experiments with error bars ± sd. Kusunoki was found to be similarly non-cytoxtoxic in both LLC-MK2 cells and Huh7.5 cells (CC50 in LLC-MK2 cells: 0.183 mg/ml; CC50 in Huh-7.5 cells: 0.202 mg/ml) (Figure 3.7, Figure g). However, its anti-DENV properties were less apparent in LLC-MK2 cells compared to Huh-7.5 cells (EC50 in LLC-MK2 cells: 0.0108 mg/ml; EC50 in Huh-7.5 cells: 0.000146 mg/ml). With a SI of 16.8 in LLC-MK2 cells, the ability of kusunoki to inhibit DENV replication was established to be independent of the choice of cell lines or anti-DENV assay used. 76 Figure 3.7 | Effects of kusunoki in CPE-based anti-dengue assay LLC-MK2 cells treated with range of concentrations of kusunoki diluted in EMEM. Following which, half the set of cells were inoculated with DENV-2 at MOI 0.5, while the other half remained as a mock infected control. The cells were then allowed to proliferate. After 5 days of incubation, a MTT assay was performed to determine the number of viable cells present. Fluorescence readings were then taken and expressed as a relative percentage of controls, where 100% cell viability represented maximum cell growth without infection and kusunoki treatment and 0% cell viability represented cell death caused by DENV-2 infection. Results are the mean of 3 independent experiments with error bars ± sd. [CC50: 0.183 mg/ml; EC50: 0.0108 mg/ml; SI: 16.8] 77 3.8 Inhibitory effect of kusunoki against 4 DENV serotypes There are four serologic types of dengue virus, DENV-1 - 4. An ideal property of an anti-viral compound for dengue would be the ability to protect the host against all the 4 serotypes. Thus, the capacity of kusunoki to inhibit the 4 serotypes of DENV was examined by plaque reduction assay. A suitable non-cytotoxic concentration range of kusunoki in LLC-MK2 cells was first determined using results gathered from CPE-based anti-dengue assay (Figure 3.7). LLC-MK2 cells were inoculated with 100 PFU of each serotype of DENV, followed by 0 - 100 µg/ml of kusunoki (final DMSO concentration of 0.5%) diluted in plaque assay overlay media. At 5 days post-inoculation, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using 4% paraformaldehyde and staining with 1% crystal violet. No significant cytotoxic effects were observed with 0 - 100 µg/ml of kusunoki in the CPE-based anti-dengue assay (Figure 3.7), indicating that a reduction of plaques was solely attributed to the direct anti-DENV action of kusunoki, and not cytotoxicity of the compound. Plaques were enumerated (Figure 3.8a) and expressed as a relative percentage to the number of plaques produced for incubation with 0.5% DMSO without kusunoki (Figure 3.8b). The concentration needed to reduce the number of plaques by 50%, EC50, was then calculated (Figure 3.8c). Kusunoki inhibited DENV in the plaque reduction assays in a dose-dependent manner (Figure 3.8a - b). This trend was apparent across all 4 DENV serotypes with comparable EC50 values (DENV-1: 32.8 µg/ml; DENV-2: 39.0 µg/ml; DENV-3: 16.5 µg/ml; DENV-4: 30.2 µg/ml, Figure 3.8b – c). 78 Figure 3.8 | Effect of kusunoki in plaque reduction assay across 4 DENV serotypes LLC-MK2 cells were inoculated with 100 PFU of each serotype of DENV, followed by 0 – 100 µg/ml of kusunoki (final DMSO concentration of 0.5%) diluted in plaque assay 79 overlay media. At 5 days post-inoculation, the overlay media was removed and cells were washed twice with PBS. This was followed by fixation using paraformaldehyde and staining with crystal violet. After a further washing step and drying of plates, number of plaques were enumerated. These were expressed as a relative percentage to the number of plaques produced for incubation with 0.5% DMSO without kusunoki. a) Representative plaque reduction assay for enumeration. Plaque reduction assay plate image from infection with DENV-2 and simultaneous treatment with 0 - 100 µg/ml kusunoki. b) Plaque reduction assay results. Plaque reduction assay results from infection with 4 DENV serotypes respectively, with simultaneous treatment with 0 – 100 µg/ml kusunoki. (DENV-1: blue, DENV-2: red, DENV-3: green, DENV-4: purple). Plaques enumerated expressed as a relative percentage to the number of plaques produced for incubation with 0.5% DMSO without kusunoki. c) Summary of plaque reduction assay results. 80 3.9 Mechanistic inhibitory action of kusunoki To give mechanistic insights into the anti-DENV action of kusunoki, a time of addition assay was first performed. This assay broadly dichotomizes inhibitors by distinguishing their mode of action, differentiating attachment/entry inhibitors from intracellular inhibitors that work against any other step in the rest of the viral replication cycle, by adding the inhibitor in question at different time points before and after viral inoculation and examining the extent of viral inhibition. Twenty-four hours after seeding of LLC-MK2 cells, 100 PFU of DENV-2 was inoculated at 4°C for 1.5 h. After washing with PBS, the plates were shifted for culturing in a 37°C incubator. Kusunoki (50 µg/mg) diluted in plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the cells at different time points pre- and post-infection (-1.5, 0, 1, 2, 3, 4 and 5 h). Heparin (100 µg/mg), a known DENV entry inhibitor, was used as a positive control, while DMSO was used as a negative control. After 5 days, cells were fixed and stained. Plaques were then enumerated. Supporting its role as a RdRp inhibitor discovered by the in vitro RdRp assay (Figure 3.4b, Figure 3.5b), viral inhibition was apparent in early stages of the infection (Figure 3.9a, >1 h post infection), at time points consistent with steps in the viral replication cycle which occur intracellularly after entry and internalization of the viral particle (Wang et al., 2011b). Next, to further support the hypothesis that kusunoki is a possible RdRp inhibitor, a DENV subgenomic RNA replicon system containing the NS genes was used. Twentyfour hours after BHK-21 cells containing a DENV replicon with a luciferase reporter gene were seeded, the cells were treated with kusunoki (40 µg/ml) diluted in DMEM. NITD-008 (10, 20, 50 µg/ml), a known DENV RdRp inhibitor, was used as a positive control. After an incubation of 48 h, the cells were harvested and lysed. The cell lysate was then subjected to luciferase assay. 81 At a low concentration of 40 µg/ml, kusunoki was sufficient to reduce luciferase activity by 50% (Figure 3.9b), consistent with its ability to inhibit RdRp activity (Figure 3.4b). Surprisingly, a higher degree of viral inhibition was evident when kusunoki was added at even earlier time points (-1.5 to 1 h) post infection in the time of addition assay (Figure 3.9a). The inhibitory profile of kusunoki at these time points was found to be similar to that of heparin, a known DENV entry inhibitor (Figure 3.9a). This suggests that although kusunoki can inhibit intracellular viral RNA replication, it also inhibits attachment/entry of DENV. In an attempt to clarify if kusunoki inhibits DENV at either the viral attachment or internalization step, a binding assay was performed. This assay is based on early findings that distinguished both processes. While viral attachment is a temperatureindependent process which occurs at both 4 and 37°C, viral internalization can only proceed at 37°C (Salas-Benito and del Angel, 1997). LLC-MK2 cells were inoculated with 200 PFU of DENV-2 at 4°C or 37°C for 1.5 h in the presence of kusunoki (50 µg/mg) or DMSO (negative control). After the cells were washed with PBS, plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the wells. After 5 days, cells were fixed and stained. Plaques were then enumerated. Ninety-eight percent viral inhibition was attained at 4°C, while a comparable 100% inhibition was achieved at 37°C (Figure 3.9c). This suggests that kusunoki possibly inhibits DENV attachment to cells. In conclusion, kusunoki seems to inhibit DENV at multiple stages of its replication cycle: viral attachment step as well as RNA replication process that is facilitated by NS5 RdRp. 82 Figure 3.9 | Mechanistic inhibitory action of kusunoki a) Time of addition assay Twenty-four hours after LLC-MK2 cells were seeded, 100 PFU of DENV-2 was 83 inoculated at 4°C for 1.5 h. After the cells were washed with PBS, the plates were shifted for culturing in a 37°C incubator. Kusunoki (50 µg/mg) diluted in plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the cells at different time points pre- and post-infection (-1.5, 0, 1, 2, 3, 4 and 5 h). Heparin (100 µg/mg), a known entry inhibitor of DENV, was used as a positive control, while DMSO was used as a negative control. After 5 days, cells were fixed and stained. Plaques were then enumerated. Number of plaques attained with respective compound treatments was expressed relative to the number of plaques for DMSO, and finally expressed as percentage inhibition. b) DENV replicon luciferase assay Twenty-four hours after BHK-21 cells containing a DENV subgenomic replicon with a luciferase reporter were seeded, the cells were treated with kusunoki (40 µg/ml) diluted in DMEM. NITD-008 (10, 20, 50 µg/ml), a known DENV RdRp inhibitor, was used as a positive control. After an incubation of 48 h, the cells were harvested and lysed. The cell lysate was then subjected to luciferase assay. Luminescence intensity is expressed relative to 0.5% DMSO control without kusunoki. c) Binding assay Twenty-four hours after LLC-MK2 cells were seeded, 200 PFU of DENV-2 was inoculated at 4°C or 37°C for 1.5 h in the presence of kusunoki (50 µg/mg) or DMSO (negative control). After the cells were washed with PBS, plaque assay overlay medium (0.8% methylcellulose, 10% FBS and antibiotics in EMEM) was added to the wells. After 5 days, cells were fixed and stained. Plaques were then enumerated. Plaques enumerated are expressed as a relative percentage to the number of plaques produced for incubation with 0.5% DMSO without kusunoki. Results are the mean of 3 independent experiments with error bars ± sd. 84 Chapter 4. Discussion The aims of the present project may be divided into three broad categories: 1. Synthesis of high quality DENV-2 NS5 full-length and RdRp domain mutant proteins by utilizing the wheat germ cell-free protein synthesis system 2. Application of these proteins in a fluorescence-based in vitro NS5 RdRp screening assay 3. Identification of inhibitors against DENV NS5 by screening libraries of FDAapproved drugs and natural compounds using the in vitro NS5 RdRp assay Conceptually, this is the first report to exploit the wheat germ cell-free protein synthesis system for an in vitro NS5 RdRp assay to our knowledge. Through conducting these studies, some candidate drugs/compounds were obtained and additionally validated in the primary and secondary in vitro screening assays respectively. Their inhibitory effect was further evaluated in a cell-based DENV infection system by exploring their cytotoxicity and capacity to reduce viral titers. Investigations to reveal the extent and mechanistic details of inhibition of the best candidate were then carried out. 4.1 Production of NS5 protein using wheat germ cell free system DENV NS5 proteins used in in vitro assays have traditionally been produced by E. coli and baculovirus-infected insect cell-based systems (Niyomrattanakit et al., 2010; Tan et al., 1996). Although NS5 proteins yielded from the both system have been shown to have RdRp activity, they have many shortcomings. For instance, although the E. coli system produces a high yield of proteins rapidly at a low cost, a major inherent weakness of it is the lack of post translational modifications present due its nature of being a prokaryotic-based system. Conversely, although a euykaryotic baculovirus system allows for some degree of post-translational modifications, it is both an expensive and time-consuming protein synthesis process of 85 up to 10 days. Furthermore, being a cell-based technique, it may be highly susceptible to contamination. In recent years, an increasing number of post translational modifications have been discovered for DENV NS5. These include phosphorylation, myristoylation, glycosylation and amidation (Khan et al., 2008). With some of these ascertained to be important for its function, such as in the study by Bhattacharya et. al. that uncovered that the phosphorylation of NS5 at amino acid residue 449 modulates DENV replication, there is a pressing need to seek out an alternative NS5 protein synthesis system (Bhattacharya et al., 2009). In this study, eukaryotic wheat germ cell-free protein synthesis system was employed to produce DENV NS5 protein. Being a eukaryotic system, the wheat germ system has also been shown to allow post-translational modifications such as glycosylation, acetylation, phosphorylation, iso-prenylation, myristoylation, complex protein folding and proteolytic processing (Martin et al., 1997; Nakamura, 1993; Rubenstein and Chappell, 1983; Zagorski et al., 1983). This ability would enable recombinant NS5 synthesized to be a more physiologically relevant protein. Despite being a fairly new system, this protein production system is well-established, having generated 13,364 human proteins (encompassing 70% of the 22,000 human genes). Additionally, 77% of the phosphatases synthesized by this system have also showed biological activity (Goshima et al., 2008). In addition to high quality functional proteins, the wheat germ cell-free protein synthesis system is also rapid, taking only one day to produce a large and consistent yield of proteins in a cost-efficient manner. The wheat germ cell free system was successfully utilized to synthesis full-length NS5, RdRp domain mutant and DHFR proteins, all which were fused with N-terminal GSTtag for affinity purification. A representative CBB staining of GST-NS5 samples from 86 the various purification steps is shown (Figure 3.1b). In the crude protein sample after in vitro translation (Lane 2, Figure 3.1b), a protein band of the correct size (133 kDa) was seen, showing that protein production was a success. Importantly, proteins produced were also soluble, an advantage over the use of the E. coli system as it is known to result in the accumulation of misfolded heterogenous proteins into insoluble aggregates which are biologically non-functional (Ventura, 2005). After successive purification steps of high efficiency, GST-NS5 was further subjected to buffer exchange chromatography as the presence of reduced glutathione could potentially affect downstream assays. This process did not affect the purity of the sample, as seen in the single band in the final preparation (Figure 3.1b, lane 5). In addition, western blotting analysis also showed specificity of the NS5 protein produced (Figure 3.1c, lane 1). In addition, the full-length and RdRp domain mutant proteins of NS5 also demonstrated RNA polymerization activity in vitro (Figure 3.2c), lending credibility to the study’s proposition of NS5 synthesized using the eukaryotic wheat germ cell-free protein synthesis system as a viable alternative to other present protein synthesis methods that balances cost, efficiency and physiological relevance. 4.2 Development of fluorescence-based NS5 RdRp assay using wheat germ cell-free system-produced NS5 proteins Following the increasing recognition of DENV NS5 RdRp as a promising drug target, a wide myriad of RdRp enzymatic assays in a cell-free setting have been developed, most of which utilize radioactive labels to track the incorporation of nucleotides. A popular radioactive assay format is SPA, which is based on the quantification of radioactivity present on newly transcribed RNA incorporated with radioactive nucleotides (Yap et al., 2007). Although these radioactivity-based assays are still widely used, there has been a growing inclination to move away from the use of radioactive labels. The motivation 87 behind this shift is that radioactive assays for use in high-throughput screening studies have the aptitude to produce large amounts of waste that can cause health and safety concerns. In addition, both the acquirement and disposal of these reagents are costly (Seethala and Fernandes, 2001). A solution to this is the use of fluorophore-conjugated nucleotides as a substitute to radioactive-labeled nucleotides. An example of this is BBT-ATP (Niyomrattanakit et al., 2011). Fluorescence readings can then be taken after nucleotide incorporation which in turn results in the release of fluorophore tags. These measurements would therefore be proportional to NS5 RdRp activity. To clarify the specificity of the fluorescence-based NS5 RdRp assay constituents, core assay constituents were removed separately. This included reactions with no proteins (protein buffer was added in place of GST-NS5), no RdRp cofactor MnCl2 and no nucleotide analogue BBT-ATP respectively (Figure 3.2a). These were then compared to the reaction with the full reagent mix. Both the no protein and no BBT-ATP reactions display an acceptable minimal background RdRp activity at approximately 10% and 1% respectively. Without the cofactor of RdRp, MnCl2, NS5 RdRp activity was decreased to about 40%. While the divalent metal ion greatly enhances RdRp activity as a cofactor, it does not seem to be obligatory for the reaction to occur. This observation has also been reported with the HCV RdRp, NS5B (Ranjith-Kumar et al., 2004). In addition, a recently reported DENV RdRp inhibitor, NITD-008 (Yin et al., 2009), also inhibited approximately 40% of NS5 RdRp activity at 50 µM, lending weight to the functionality of the enzyme assay. The measured fluorescence levels in the in vitro RNA polymerization assay correlated to RdRp activity specific to NS5 protein, as a dose-dependent increase in fluorescence intensity was also observed with the increase of NS5 (Figure 3.2b). The relative RdRp activities of DENV-2 full-length NS5, its RdRp mutant protein and an unrelated protein, DHFR, which was used as a negative protein control, were 88 explored (Figure 3.2c). As expected, DHFR only produced background fluorescence levels of about 10%, which was comparable to the no protein control. Although equal concentrations of full-length NS5 and RdRp domain mutant proteins were used, the RdRp domain mutant protein only achieved approximately 80% of RNA polymerization activity compared to full-length NS5 (Figure 3.2c). The results attained in this study raise the possibility of the MTase domain contributing to RdRp activity of NS5. Interaction between the two functional domains MTase and RdRp is currently unknown as the DENV NS5 protein crystal structure has not been solved in its entirety. While some reports suggest that these domains are independent and functionally irrelevant to each other (Bussetta and Choi, 2012), the results in this study seem to suggest otherwise. This notion is echoed in other reports which show genetic evidence of crosstalk between both domains (Malet et al., 2007). More recently, interaction between both domains (amino acid residues 46-49 in the MTase αA3-motif and residue 512 in RdRp) were confirmed using pull-down assays and mutagenesis studies of these interacting regions have been found to completely abolish DENV viability (Tan et al., 2013). 4.3 Screening libraries of FDA-approved drugs and natural compounds Productivity has not kept pace with increasing R&D investments in the pharmaceutical industry. Despite exponential increase in R&D costs over the decades, a proportional increase in approved drugs has not been met. This increase in R&D costs has been further exacerbated by the high attrition rates of drugs from clinical trials, dubbed “the clinical trial cliff”, with the number of drugs terminated in Phase III of clinical development doubling in 5 years, reaching 55 compounds in a 2 year period of 2008 2010 (CMR, 2012). The primary reason for drop-outs in Phase I and II is safety-related issues, while withdrawals in Phase III are ascribed to a lack of efficacy (Novac, 2013). To combat these problems, the idea of recycling late-phase-failed compounds or already marketed drugs to a new indication was sprung. The process of identifying or 89 developing new uses for existing or abandoned drugs or compounds is called “drug repositioning” (Ashburn and Thor, 2004). This is an appealing prospect from various standpoints. Firstly, from the industrial standpoint, this course of action can potentially bring in more revenue to maximize R&D expenditure. Also, proven formulation and manufacturing routes can further reduce additional related investment expenses. From an ethical stance, the information generated by existing patient data in clinical trials for future indications should be capitalized on to its full potential. Lastly, from the scientific perspective, compounds with established safety and bioavailability profiles would undoubtedly get a good head-start in the R&D process. These can probably enter clinical trials quicker than NMEs and would less likely fail due to human safety and pharmacokinetic issues (Stuart-Kregor, 2007). In addition, a well-characterized pharmacology could aid studies into the mechanistic analysis of new indications. The success stories of several repositioned drugs such as sildenafil (angina to erectile dysfunction) and thalidomide (morning sickness to multiple myeloma) have been well chronicled (Novac, 2013). More recently, mifepristone, initially approved as an abortifacient, has also been repurposed as treatment for Cushing’s syndrome in early 2012 (Castinetti et al., 2012). These case studies are indicative of the recognition of the increasing importance of drug repositioning today. Brought into the context of this study, these principles were a guide into the selection of screening library to utilize. For the various reasons aforementioned, a library of FDAapproved drugs was opted for. In addition, to further expand this screening library, natural compounds, some of which are used as health supplements, were also appended. Natural compounds, often used in traditional medicine, were chosen for their better safety and tolerance profiles compared to synthetic compounds or general NMEs 90 (Patwardhan and Vaidya, 2010). This is probably the reason why over 60% of anticancer and 75% of anti-infective drugs approved from 1981 - 2002 can be traced back to natural origins (Gupta et al., 2005). The screening of FDA-approved drugs and natural compounds in an in vitro assay presents as a safer starting point. FDA-approved drugs, for example, would have passed all in vivo toxicity testing successfully. The choice of using safe drugs and compounds for screening would ideally accelerate the follow-up cell-based evaluation and drug repositioning process significantly, and is thus the approach taken in this study. The importance of this sentiment has been echoed in recent efforts. Much of antidengue drug discovery still remains in its infant stages of identification potential antiviral candidates. A large proportion of anti-dengue drug discovery has been led by Novartis Institute for Tropical Diseases and that has led to the development of many high throughput assays to screen their library of 1.8 million compounds. The most promising NS5 RdRp inhibitors (NITD-008, NITD-449 and NITD-203) were found to have high inhibitory effects and low cytoxicity in vitro. However, all exhibited high toxicity in in vivo studies and advancement to drug development were halted (Chen et al., 2010; Yin et al., 2009), further lending weight to the approach of this study to screen libraries of FDA-approved drugs and natural compounds as a safer alternative. 4.4 Primary screening of libraries of FDA-approved drugs and natural compounds in in vitro NS5 RdRp assay After the fluorescence-based NS5 RdRp assay using DENV-2 NS5 synthesized by wheat germ cell free system was established, the primary screening of a total of 648 drugs and compounds from libraries of FDA-approved drugs (Figure 3.3a) and natural compounds (Figure 3.3b) was conducted. Polymerase inhibitors against RNA viruses developed to date fall into two categories: nucleoside inhibitors and non-nucleoside inhibitors. Nucleotide analogues are administered as pro-drugs for increased safety, bioavailability and efficacy (Peterson 91 and McKenna, 2009). Since they are nucleotide analogues, these have to be successively phosphorylated by cellular enzymes such as phosphoglycerate kinase and creatine kinase (Cihlar and Ray, 2010). These are structurally similar to nucleosides and they work as antimetabolites by being incorporated into growing RNA strands. Unlike actual nucleotides, nucleotide analogues lack a 3´-hydroxyl group on the deoxyribose moiety, which is usually hydrolyzed to form a phosphodiester bond with the next nucleic acid. These nucleoside analogues thereby act as chain terminators. Non-nucleoside inhibitors, on the other hand, may not necessarily bear a structural resemblance to nucleotides. An HCV study has revealed that by binding to allosteric sites of the NS5B protein, these inhibitors change the conformation of the active site such that the NS5B is no longer competent to carry out its RdRp activities (Wang et al., 2003). As majority of the drugs and compounds screened belonged to the FDA-approved library, any inhibitors picked up by the assay were more likely to be non-nucleoside inhibitors. This is because any FDA-approved nucleoside inhibitors would be pro-drugs, and would not have been able to be converted to an active triphosphate nucleotide form in an in vitro setting. A limitation in using a FDA-approved library of drugs in an in vitro assay would therefore be the inevitable inability to identify hits that are administered as pro-drugs (e.g. nucleoside analogues) as they require processing in vivo. This shortcoming, however, may be seen as a better alignment towards the search for safe anti-dengue drugs as mitochondrial toxicity and other adverse events have been reported as a common side effects of the usage of nucleotide analogues, due to structural similarities to endogenous nucleotides as well as differential tissue-specific levels of pro-drug activation (Lund et al., 2007; Noble et al., 2010). Out of 648 FDA-approved drugs and natural compounds screened against DENV-2 NS5 RdRp activity, a total of 20 drugs and five natural compounds showed at least a 30% inhibition of RdRp activity over two independent experiments (Figure 3.4a). 92 Two interesting trends were noted. Of the 20 top drug inhibitors, 6 were azole antifungal drugs (compounds #507, #469, #619, #490, #376 and #565). This class of antifungal drugs inhibits the enzyme lanosterol 14-α-demethylase which is the enzyme necessary to convert lanosterol to ergosterol. This is turn results in the depletion of ergosterol in fungal membranes, disrupting its structure and function that leads to the eventual inhibition of fungal growth. This could imply a possible structural similarity between DENV RdRp and the region where these drugs bind to lanosterol 14-αdemethylase. If proven to be effective RdRp inhibitors, this can potentially impact endeavours in rational drug design as attempts so far have been hampered due to the lack of extensive knowledge of the entire NS5 protein structure (Ditursi et al., 2006). Another fascinating observation was that five (#136, #157, #473, #485, #94) out of the top 20 drug inhibitors were used for the treatment of Parkinson’s disease. This degenerative disorder of the central nervous system is caused by the death of dopaminegenerating cells in the substantia nigra of the brain. Levodopa, a precursor of dopamine, is usually administered to relief disease symptoms. In addition, this is usually coupled to a dopa decarboxylase inhibitor and catechol-O-methyl transferase (COMT) inhibitor to decrease side effects and increase bioavailability of levadopa (Kaakkola, 2000). This combination of three classes of drugs is commercially available as a “triple therapy in a pill” such as Stalevo (levodopa/entacapone/carbidopa). As levodopa (#473) and entacapone (#157, COMT inhibitor), together with another dopa decarxylase inhibitor, benserazide (#233), were all found to independently inhibit RdRp activity in the primary screen, the potential of using anti-parkinsonian dopaminergic combination medication such as Stalevo as an antiviral drug against DENV could bring about synergistic effects and seems promising and worthy of further exploration. An inclusion criteria set for downstream validation studies was set at a minimal inhibition of 50% RdRp activity over two independent experiments in the primary screening assay (Figure 3.4a). Using this criterion, the overall hit rate was 1.2%. In comparison to other high-throughput screening assays to identify RdRp inhibitors that 93 typically use a 25% - 50% RdRp activity inhibition cut-off to yield hit rates ranging from 0.001% to 0.7%, the conservative criterion of 50% used in this study has a significantly higher hit rate, suggesting that the approach taken by the study in terms of choice of screening libraries could be a more efficient way to identify DENV NS5 RdRp inhibitors (Niyomrattanakit et al., 2011; Niyomrattanakit et al., 2010; Wang et al., 2011a). A total of 7 FDA-approved drugs and 1 natural compound proceeded to the next validation step. In descending order of percentage inhibition of RdRp activity, these are: oxiconazole nitrate (compound #507), itraconazole (#469), tolcapone (#136), entacapone (#157), butoconazole nitrate (#619), lomofungin (#7), pinacidil (#19) and the proanthocyanidin-rich oligomeric polyphenol fraction of the extract from the Japanese cinnamon tree (Cinnamomum camphora), kusunoki (#648). As the drugs in the FDA-approved drug library were available in limited quantities and low concentrations, commercial drugs were purchased for further validation studies. Natural compounds were not commercially obtainable. These eight drugs and compounds were subjected in varying concentrations to the same primary in vitro NS5 RdRp assay conditions once again for verification. All drugs and compounds showed a dose-dependent inhibition of in vitro RdRp activity to varying degrees (Figure 3.4b), corroborating the data from the primary screen. 4.5 Secondary screening of libraries of FDA-approved drugs and natural compounds with RdRp domain mutant DENV-2 NS5 is a bifunctional protein made up of N-terminal MTase domain and Cterminal RdRp domain (Ackermann and Padmanabhan, 2001; Egloff et al., 2002). The secondary screening of the top eight inhibitors from the in vitro NS5 RdRp assay using RdRp domain mutant (Figure 3.5b) was conducted with following two aims. 94 Firstly, the RdRp domain mutant was shown to demonstrate RNA polymerization activity (Figure 3.2c). To ensure that the inhibition by compounds seen in the primary screening assay was indeed due the action of the compounds on RdRp region of NS5, the RdRp domain mutant was used in the same assay in place of full-length NS5 protein in an attempt to attribute the dose-dependent inhibition of RNA polymerization activity by the drug or compound to the RdRp domain specifically. Secondly, this experiment could also give insights into the binding region of the drug to NS5. As possible non-nucleotide analogue inhibitors, drugs or compounds could bind in an allosteric site either within the RdRp domain, or in a region elsewhere on the NS5 protein (eg. MTase domain). Binding to an allosteric site in or outside the RdRp could cause a conformational change in the NS5 protein specficially within the RdRp active site, affecting RdRp activity (Wang et al., 2003). All drugs and compounds showed a dose-dependent inhibition of in vitro activity of the RdRp domain mutant. Most drugs and compounds showed similar extents inhibition compared to when full-length NS5 protein was used (Figure 3.5b), suggesting that they bind to a region within the RdRp domain, directly interfering with enzymatic activity of NS5. Conversely, itraconazole exhibited a greater degree of inhibition of RdRp activity when the RdRp mutant was used instead of wild type NS5, while in contrast, pinacidil and kusunoki showed evidence of smaller magnitude of inhibition in the secondary screening assay (Figure 3.4b and 3.5b). These may be indicative that itraconazole, pinacidil and kusunoki bind to multiple sites within NS5, with the major inhibitory site of itraconazole being within the RdRp domain and those of pinacidil and kusunoki being outside this domain. In addition, differences in inhibitory effects of compounds when wild type NS5 and domain mutant are used are also consistent with data shown earlier (Figure 3.2c) that suggests interaction between the MTase (or elsewhere on NS5) and RdRp domains, a phenomenon supported by other recent studies (Malet et al., 2007; Tan et al., 2013). This possible interaction seems to affect RdRp activity. 95 4.6 Validation of inhibition of DENV replication by drug/compound in cellbased system The top eight inhibitors from the in vitro RdRp assay of NS5 were next tested in a cellbased assay to verify if they had the ability to inhibit DENV replication. This validation step comprised of 2 experiments: A cell viability assay to measure the cytotoxicity of a drug/compound and a plaque assay to monitor the reduction of viral titer by the drug/compound. CC50 and EC50 were then calculated from cell viability assay and plaque assay data respectively. In addition to these values, the selectivity index (SI = CC50/ EC50) of the various drugs were derived. As viral titer (i.e. EC50) is a combined consequence by both drug/compound cytotoxicity and actual inhibition of drug/compound, the selectivity index of an effective drug/compound should be greater than 1 to demonstrate that the action of a drug/compound inhibiting viral replication plays a more major role in the contribution to viral titer reduction. The higher a drug’s SI is, the wider the window is between an effective dose from a cytotoxic dose, and hence, the greater its potential as a drug. With the exception of oxiconazole nitrate (data not shown), treatment of Huh-7.5 cells with the rest of the shortlisted drugs and compounds generally showed a dose-dependent decrease in both cell viability and viral titer ( Figure a - g). In contrast, oxiconazole nitrate showed significant decrease in neither cell viability nor viral titer up to 250 µM. Figure h shows a summary of the cell-based validation study, together with brief additional information known about the drugs. Five out of 8 shortlisted drugs/compounds (itraconazole, entacapone, butoconazole nitrate, pinacidil and kusunoki) showed an SI of higher than 1, showing that 62.5% of the shortlisted drugs and compounds demonstrated anti-DENV activity in cultured cells ( Figure h). 96 97 The discrepancy between the effectiveness of the compounds inhibiting RdRp activity in the RdRp enzymatic assay and the low potency of DENV inhibition in cell-based assays is apparent through the higher doses of drugs needed in cell-based assays to exert an antiviral effect. This may be attributed to two main reasons. 1. As metabolism of majority of the shortlisted drugs occur primarily in the liver, the use of human liver cells (i.e. Huh-7.5) for cell-based testing could have resulted in the rapid breakdown of the active drug. This would have lowered the effective drug concentration during cell-based antiviral screening, and consequently, concentrations of drug to achieve an antiviral effect could not be reached. This is held in contrast to enzymatic assays where the drugs would not be broken down quickly. To combat this, the use of a non-liver DENV-relevant cell line could have been used for testing to lengthen the half-life of the drugs. Additionally, additional dosing to maintain the effective drug concentration in the culture media could also be considered. 2. The indications these drugs were approved for by the FDA were not for the treatment of DENV infection. As such, doses that achieve antiviral effects in cell-based assays may be much higher than that of approved doses for their original indications. This could translate to toxicity of the drugs when used at high concentrations in cell-based assays, lowering drug potency significantly. A possible strategy to counteract this is the chemical modification of the shortlisted drugs to mediate better interaction with binding sites on DENV NS5 RdRp to disrupt its activity. Itraconazole and butoconazole nitrate, two of the five validated hits, are both azole antifungal drugs, as previously mentioned in Section 4.4 The inhibitory action of azoles against DENV in Vero cells has previously been reported (Barradas et al., 2008; McDowell et al., 2010), and may be regarded as an independent validation of this study’s results. In addition, one of these azole compounds, ETAR, has also been shown to exhibit antiviral efficacy against a range of flaviviruses in vitro (McDowell et al., 98 2010), raising the possibility that these azole drugs could perform well as broadspectrum inhibitors. This could also suggest that the binding site of these azole antifungal drugs to NS5 lie in one or more of the 13 highly conserved sequences of DENV NS5 which is also found in 64 other flaviviruses (Khan et al., 2008). A possible inhibitor candidate that may be suitable for future antiviral use is pinacidil ( Figure f). Pinacidil is an anti-hypertensive drug that was approved for clinical use by FDA in 1989. A cyanoguanidine drug that opens ATP-sensitive potassium channels, the administration of this drug results in the relaxation of vascular smooth muscle to produce peripheral vasodilatation and hence, a consequent reduction in blood pressure (Friedel and Brogden, 1990; Gollasch et al., 1995). After an initial decrease in cell viability at low concentrations, cell viability was maintained at approximately 80% despite increasing pinacidil concentrations. Even though CC50 was not reached, viral titer was almost entirely abolished at 300 µM with respect to 0.5% DMSO diluent control. This could possibly pose as a challenge as the concentration of 300 µM of pinacidil would be unattainable in vivo by the current standard clinical dose of the drug used for anti-hypertensive purposes. Illustrated in the study by Thuillex et al. which administered a standard dose of 25 mg of pinacidil to healthy volunteers, the peak plasma concentration of pinacidil was approximately 60 ng/ml (0.25 µM) 2 hours after oral administration (Thuillez et al., 1991). Although this big difference in drug concentration might seem daunting, a 2-pronged approach may be taken to overcome this. Firstly, it is important to realize that it is not necessary for a drug to fully eradicate viremia. For example, it has been suggested that a mere 50% inhibition of viral replication could be sufficient enough to bring the viral load down to a controllable level for subsequent host immune system clearance (Botting and Kuhn, 2012). Hence, a much lower concentration of pinacidil could be sufficient for an antiviral effect in an immune competent in vivo system compared to an in vitro cell culture system as employed in this study. Secondly, plasma concentration of pinacidil may also be 99 increased as no adverse effects have been observed for up to 300 ng/ml in healthy volunteers (Ward et al., 1984). The plasma concentration may be further elevated by (i) Increasing the standard dose of 25 mg: this may be adjusted upwards to 0.535 mg/kg (DIS, 2012); (ii) Utilizing intravenous delivery of drug instead of oral administration as this has been known to increase the plasma concentration of pinacidil by at least 5 times (McBurney et al., 1985). With these strategies, pinacidil would make a feasible inhibitor against DENV for further in vivo testing. Interestingly, of the five drugs and compounds that showed anti-DENV activity, the proanthocyanidin-rich oligomeric polyphenol extract from the Japanese cinnamon tree (Cinnamomum camphora), kusunoki ( Figure g), reflected the highest SI value of 1387.3 with a non-cytotoxic dose of 145.9 ng/ml needed to reduce viral titer by 50%, suggesting a wide window for therapeutic anti-DENV use. The antiviral property of cinnamon is not surprising. Cinnamon, a spice obtained from the inner bark of several trees from the genus Cinnamomum, has traditionally been used in traditional medicine for a myriad of aliments. The most commonly studied cinnamon plant species include C. verum, C. zeylanicum and C. cassia. Antiviral effects of many cinnamon-derived compounds have already been discovered. In an in vitro screening study using an assortment of plants utilized in traditional Indian medicine, a C. cassia extract was shown to have an inhibitory effect on HIV (Premanathan et al., 2000). In the same year, yet another group reported the in vitro and in vivo anti-herpes ability of eugenol, a compound commonly found in essential oils distilled from cinnamon (Benencia and Courreges, 2000). More recently, the compound cinnzeyanine attained from C. zeylanicum, was revealed to have antiviral properties in a silkworm baculovirus model. Cinnezeylanine was then later confirmed to have inhibitory effects on the proliferation of HSV in cultured Vero cells (Orihara et al., 2008). The kusunoki extract used in the study was a polyphenol-enriched extract rich in oligomeric proanthocyanidins (PAs). The positive health effects associated with the 100 intake of cinnamon have been partially attributed to its phenolic composition of PAs. PAs are a class of polyphenolic compounds often found in various species of plants. These are oligomers of flavan-3-ol monomer units. The most common monomeric units consist of the classes procyanidins (chains of catechin, epicatechin, as well as their gallic acid esters) and prodelphinidins (gallocatechin, epigallocatechin, and their galloylated derivatives) (Lazarus et al., 1999). Indeed, mass spectroscopy techniques in recent studies have revealed that PAs derived from cinnamon bark include combinations of (epi)catechin, (epi)catechingallate, (epi)gallocatechin, and (epi)afzelechin (MateosMartin et al., 2012). In addition to their cardioprotective, anti-tumor, anti-inflammatory, anti-carcinogenic and anti-allergic activities, PAs have also been known to possess antiviral activity (Frankel, 1995; Gescher et al., 2011a; Leung et al., 2001). These include viral inactivation against feline calicivirus, which are experimental surrogates for noroviruses, and coxsackievirus, which was used as a representative enteric virus (Iwasawa et al., 2009). In addition, in vitro antiviral activity against HSV has also been uncovered (Xu et al., 2010). More recently, the anti-HSV activity by PAs has been elucidated to be by the inhibition of viral adsorption and penetration (Gescher et al., 2011b). Due to the high SI recorded, kusunoki was therefore subjected to further experiments to validate its potential as an anti-viral therapeutic. 4.7 Effects of kusunoki in CPE-based anti-dengue assay Antiviral activities of compounds have a possibility to occur in a cell-line or assayspecific dependent manner. As sound and reproducible in vitro studies form the premise and foundation of further studies, this has dire implications on the ultimate goal of translational studies to bring the compound into both in vivo efficacy studies and finally, a clinical setting. To ensure that kusunoki inhibition of DENV was not a cell-line or assay-specific phenomenon, the effects of kusunoki in a CPE-based anti-dengue assay in monkey 101 kidney (LLC-MK2) cells were explored. This assay was developed based on the CPE of DENV-2 infection seen in LLC-MK2 cells and anti-dengue activity of a drug/compound is characterized by the rescue of cells from infection-induced cell death (Ichiyama et al., 2013). The choice of this assay was based on the interest to assess the extent to which kusunoki could exert cytoprotective effects, a desirable trait of an anti-DENV drug as virus-induced cellular apoptosis is hypothesized to play a role in the pathogenesis of DENV infections since local tissue damage or transient homeostatic imbalances could trigger additional deleterious events (Marianneau et al., 1998). LLC-MK2 cells, a cell line originating from monkeys, were employed with two broad aims. Firstly, the antiDENV effects of kusunoki had to be verified to be reproducible in as many DENVrelevant cell types as possible. With the initial verification in Huh-7.5 cells (human liver cells), the study was extended to kidney cells as DENV infection has been known to be permissible to a wide multitude of cell types, including liver cells, kidney cells, spleen cells, macrophages, monocytes and lymphocytes (Jessie et al., 2004). Moreover, it was interesting to be able to reveal if the inhibitory activities of kusunoki was speciesspecific, the impetus behind the use of monkey cells in this experiment. Kusunoki was found to be similarly non-cytoxtoxic in both LLC-MK2 cells and Huh7.5 cells (CC50 in LLC-MK2 cells: 0.183 mg/ml; CC50 in Huh-7.5 cells: 0.202 mg/ml) (Figure 3.7 and Figure g). This is in line with the knowledge of different kinds of cinnamon being safely consumed throughout various cultures for a large gamut of reasons ranging from culinary flavouring and as traditional medical remedies. Its unique position as being widely perceived to be safe for consumption would undoubtedly facilitate the clinical trial process of any drug or compound derived from the common spice, cinnamon, as it is unlikely to fail in toxicity studies. Kusunoki’s anti-DENV properties, however, were less apparent in LLC-MK2 cells compared to Huh-7.5 cells (EC50 in LLC-MK2 cells: 0.0108 mg/ml and EC50 in Huh-7.5 cells: 0.000146 mg/ml) (Figure 3.7). This result is acceptable, considering different 102 assays of varying sensitivities have been used. Therefore, a general consistent antiviral trend (both SIs sigificantly higher than 1) is sufficient for comparison. Furthermore, the inhibitory effecrts of kusunoki is much more potent in the human cell line, consistent with its future intended use. With a SI of 16.8 in LLC-MK2 cells, the ability of kusunoki to inhibit DENV replication was established to be independent of the choice of DENV-relevant cell lines (liver or kidney cells), species (human or monkey) and assay system used. 4.8 Inhibitory effect of kusunoki against 4 DENV serotypes There are four serologic types of dengue virus, DENV-1 – 4. An ideal property of an antiviral inhibitor for dengue would be the ability to protect the host against all the 4 serotypes. Thus, the capacity of kusunoki to inhibit the 4 serotypes of DENV was examined by plaque reduction assay. No significant cytotoxic effects were observed with 0 - 100 µg/ml of kusunoki in the CPE-based anti-dengue assay (Figure 3.7), indicating that a reduction of plaques was solely attributed to the direct anti-DENV action of kusunoki, and not cytotoxicity of the compound. Plaques were enumerated after the plaque reduction assay was performed (Figure 3.8a) and expressed as a relative percentage to the number of plaques produced for incubation with 0.5% DMSO without kusunoki (Figure 3.8b). The concentration needed to reduce the number of plaques by 50%, EC50, was then calculated for comparison (Figure 3.8c). Kusunoki inhibited DENV in the plaque reduction assays in a dose-dependent manner (Figure 3.8a – b). This trend was apparent across all 4 DENV serotypes with comparable EC50 values (DENV-1: 32.8 µg/ml, DENV-2: 39.0 µg/ml, DENV-3, 16.5 µg/ml, DENV-4: 30.2 µg/ml, Figure 3.8b - c). From this result, DENV-3 seems to display a higher susceptibility to kusunoki with 2fold lower EC50 than the other serotypes. If the trend apparent in this set of data is 103 reproducible in vivo, it would be prudent to examine the genetic differences that distinguish DENV-3 from the others as this may give an insight into the mechanistic action of kusunoki for further understanding and exploitation for the drug discovery process against DENV. The broad-spectrum inhibition of kusunoki against all four DENV serotypes is an extremely important trait of an anti-DENV drug to be used in a global setting. Although the dominant DENV serotype in Singapore has been serotype 2 since 2007, it is important to comprehend that the dynamics of the disease remains in constant flux. For example, within a small local community like Singapore, the proportions of DENV-1 and DENV-3 infections have increased from 5% and 12% in September 2012 to 25% and 20% in February 2013 respectively (NEA, 2013). In a global context, although DENV serotypes used to be limited by geographical boundaries 30 years ago, disease transmission of all four serotypes have since been found circulating in tropical and subtropical regions of the world, including Asia, Africa and the Americas, a significant portion of the present globalized world (Guzman et al., 2010). Coupled to incidences of heterologous DENV infections, it is imperative for the development of a drug which can be used in the universal treatment of DENV regardless of its serotype in a rapid manner, without having to additionally consider the DENV serological differences in infected patients. 4.9 Mechanistic inhibitory action of kusunoki To give mechanistic insights into the anti-DENV action of kusunoki, a time of addition assay was first performed. This assay broadly dichotomizes inhibitors by distinguishing their mode of action, differentiating attachment/entry inhibitors from intracellular inhibitors that work against any other step in the rest of the viral replication cycle, by adding the inhibitor in question at different time points before and after viral inoculation and examining the extent of viral inhibition. 104 Supporting its role as a RdRp inhibitor discovered by the in vitro RNA polymerization assay (Figure 3.4b and Figure 3.5b), viral inhibition was apparent in early stages of the infection (Figure 3.9a, >1 h post infection), at time points consistent with steps in the viral replication cycle which occur intracellularly after entry and internalization of the viral particle (Wang et al., 2011b). Next, to further support the hypothesis that kusunoki is a possible RdRp inhibitor, a DENV subgenomic replicon encoding the NS proteins was used. At a low concentration of 40 µg/ml, kusunoki was sufficient to reduce replicon activity by 50% (Figure 3.9b), consistent with its ability to inhibit in vitro RdRp activity (Figure 3.4b). This possibility is reminiscent of previous reports that describe the effective inhibition of PAs isolated from blueberry leaves on HCV subgenomic replicon expression, which is a flavivirus closely related to DENV (Takeshita et al., 2009). Surprisingly, a higher degree of viral inhibition was evident when kusunoki was added at even earlier time points (-1.5 to 1 h) post infection in the time of addition assay (Figure 3.9a). The inhibitory profile of kusunoki at these time points was found to be similar to that of heparin, a known DENV entry inhibitor (Figure 3.9a). This suggests that kusunoki can inhibit attachment/entry of DENV, in addition to inhibiting intracellular viral RNA replication. In an attempt to clarify if kusunoki inhibits DENV at either the viral attachment or internalization step, a binding assay was performed. This assay is based on early findings that distinguished both processes. While viral attachment is a temperatureindependent process which occurs at both 4 and 37°C, viral internalization can only proceed at 37°C (Salas-Benito and del Angel, 1997). High levels of viral inhibition were observed at 4°C, while a comparable 100% inhibition was achieved at 37°C (Figure 3.9c). This suggests that kusunoki possibly inhibits DENV attachment to host cells. The ability of PAs to prevent viral attachment to host cells has been exemplified in previous studies and may occur through two broad 105 levels. In the case of HIV-1, PAs from grape seeds have been shown to be able to directly downregulate the expression of the viral entry co-receptors, CCR2b, CCR3 and CCR5, although the mechanism is currently unknown (Nair et al., 2002). On the other hand, PAs have been observed to interact with the viral envelope proteins extracellularly, causing their oligodimerization and thereby blocking their attachment to receptors to host cells (Gescher et al., 2011a). As kusunoki is able to exert inhibition of RNA replication at an intracellular level, it may be postulated that the transport of kusunoki into the cell occurs adequately. The significant blocking of viral attachment could be possibly due to a combination of multiple modes of action such as those aforementioned at both intracellular and extracellular levels. Two forms of evidences may add to the credibility of the proposed multifaceted inhibitory roles of kusunoki in this study. Firstly, the phenomenon of the multiple roles of PAs has been extensively reported. For example, the PA epigallocatechin gallate (EGCG), most commonly found in green tea, has been found to inhibit multiple viral enzymes involved in stages of the HIV-1 replication cycle including reverse transcriptase, integrase and protease (Jiang et al., 2010; Yamaguchi et al., 2002). In addition, EGCG has also been implicated in the inhibition of host factors such as nitric oxide synthase, NF- B and casein kinase 2 to suppress HIV-1 infection (Haneda et al., 2000; Melgarejo et al., 2010; Yamaguchi et al., 2002). As DENV NS5 have also been shown to possess a casein kinase 2 site that has been implicated in the nuclear targeting of NS5, probably by means of a cytoplasmic retention mechanism, EGCG could also be involved in host-mediated suppression of the virus. It is interesting to note that epigallocatechin has been reported to be found in extracts isolated from cinnamon tree bark, bringing about the possibility of its esterified counterpart being present as well (Mateos-Martin et al., 2012). Secondly, kusunoki, the PA oligomer fraction of the extract contains a myriad of PAs. If a single PA such as EGCG has the ability to have multiple modes of inhibitory action on 106 HIV-1, it would be plausible for either a single or multiple PAs in this extract to exhibit the numerous inhibitory effects on DENV shown in this study. Thus, kusunoki is able to inhibit DENV replication at both the viral attachment, as well as the NS5-mediated RNA replication steps. In this study, hits were obtained through the screening of libraries of FDA-approved drugs and natural compounds in an in vitro NS5 RdRp assay. These compounds may be a distance away from ideal drugs for administration under clinical settings given their selectivity indices. Nevertheless, this may be attributed to the screening of a mere 648 drugs/compounds. Presently, the in vitro NS5 RdRp assay resulted in an overall hit rate of 1.2% using a conservative criterion of 50%. This is in comparison to other primary HTS assays utilizing random compound libraries to identify RdRp inhibitors that typically use a more liberal range of 25 - 50% RdRp activity inhibition cut-off to yield lower hit rates ranging from 0.001% to 0.7% (Niyomrattanakit et al., 2011; Niyomrattanakit et al., 2010; Wang et al., 2011a). Subsequently, 0.77% of the overall drugs/compounds were validated to have an anti-DENV effect. Thus, more ideal antiviral drugs may be uncovered if the present screening system is used with larger drug/compound libraries. This attempt can also be strengthened and made more efficient with the combination of in silico prescreening. 107 Chapter 5. Future Directions 5.1 Extension of screening libraries The overall significantly higher hit rate in this study suggests that the approach taken by the study in terms of choice of screening libraries comprising of safer alternatives in comparison to other random compound libraries could be a more efficient way to identify RdRp inhibitors. With this result, the study should be extended to a bigger library of FDA-approved drugs, health supplements that have been approved by relevant authorities and traditional medications that mostly comprise of natural compounds. The choice of these as screening material would mostly translate to non-cytotoxic hits, and would greatly expedite the clinical trial process. 5.2 Determination of active antiviral components in kusunoki PA extract The kusunoki extract used in the study was a polyphenol-enriched extract rich in oligomeric (high molecular weight) PAs. It would be interesting to compare the different molecular weight fractions of kusunoki (low, medium, high) to see differences in their DENV inhibitory profile to explore if the extent of oligomerization of the PAs affects their antiviral effect. In particular, it would be essential to further resolve and identify all the oligomeric PAs present in the extract isolated from kusunoki that was used in this study. These should then be subjected to the same tests to elucidate their potential inhibitory mechanisms in order to find out how each PA contributes to the anti-DENV effect shown by kusunoki. In addition, as the isolation process of the various PAs may not be fully efficient, there is a possibility of contaminants being present in trace amounts. These include other compounds that may be found in cinnamon bark such as camphor, safrole, dipentenes, cineole, borneole, camphene, terpeneol and linalool (Daniel, 2005). As compounds such as camphor have also been shown to have antiviral activity (Chen et al., 2013), the verified PAs should then be ideally obtained from a different source (commercially or synthesized) to decrease chances of contaminants and ensure reproducibility in results. 108 5.3 Verification of RdRp inhibition To verify if the drugs or compounds indeed inhibit RdRp, a multidimensional approach should be taken. Firstly, in silico screening and docking experiments can be utilized to reveal possible binding sites of the PA on NS5. Simultaneously, the generation of viral strains resistant to the drug or compounds should be attempted in cell culture experiments. Any escape mutations that is traced back to the RdRp domain could suggest that the drug or compound in question is an inhibitor of viral RNA polymerase. In addition, any additional mutations in other regions could imply interactions with RdRp domain of NS5. Results from both in silico and resistant virus experiments should ideally corroborate with each other and reinforce our understanding of the drug or compound. A final step would then be to generate a drug- or compound-resistant protein mutant by site-directed mutagenesis and perform both the in vitro NS5 RdRp assay and cell culture experiments to validate these results. To further substantiate these sets of data, the binding affinity of DENV NS5 protein and the drug or compound may be measured by surface plasmon resonance technology. 5.4 Determination of antiviral effects against other flaviviruses A desirable property of an anti-DENV drug would be the broad-spectrum inhibition against a wide range of other flaviviruses. Out of 44 sets of highly conserved DENV sequences across all four serotypes, 27 were found in as many as 64 other viruses in the family Flaviviridae (representatives that are human pathogens include WNV, St. Louis encephalitis virus, JEV and YFV) and 13 of these represented sequences in NS5, the viral target of this study. This suggests that an inhibitor targeting NS5 could have a higher possibility of being a broad-spectrum antiviral. 109 Drugs and compounds that are found to be inhibitors of NS5 RdRp activity should therefore be screened against other flaviviruses as they would also aid in filling the void caused by the lack of antivirals for flavivirus infections. 5.5 Combination treatment DENV NS5 RdRp presents as an attractive drug target because of its position as a viralspecific protein which plays a vital role in viral RNA replication. In addition, it is also highly conserved across all four DENV serotypes and many flaviviruses. Additionally, this protein also has a vital role in viral RNA replication. Even so, the emergence of drug resistance remains a constant problem, as seen in anti-HIV and HCV therapy (Xie et al., 2011). A strategy to minimize drug resistant viral strains has been successfully employed in antiretroviral therapy for patients afflicted with HIV. This method utilizes a combination of two to three antiviral drugs that have different inhibitory mechanisms such as two nucleoside-analogue reverse transcriptase and a protease inhibitor (Imamichi, 2004). For example, in the case of DENV, protease or entry inhibitors may be used in combination with RdRp inhibitors. Alternatively, inhibitors with novel or unconventional mechanisms may be targeted. This includes viral proteins without enzymatic activity, and has been shown for NS5A in HCV (Gao et al., 2010). Apart from preventing viral resistance, the use of combination treatment could also bring about synergistic antiviral effects and should therefore be explored. 110 Chapter 6. Conclusion 6.1 Summary of study findings With the increasing recognition of RdRp activity of NS5 as an attractive drug target, this protein was chosen as a target for anti-DENV drug screening. The synthesis of high quality DENV-2 NS5 as well as its RdRp domain mutant by utilizing the wheat germ cell-free protein synthesis system was accomplished in this study. This was an attempt to present these proteins as an alternative to other present protein synthesis methods that balances cost, efficiency and physiological relevance. These recombinant NS5 proteins were then successfully applied in the development of a fluorescence-based in vitro RdRp assay which was then used in the identification of inhibitors against DENV NS5 by screening libraries of FDA-approved drugs and natural compounds. Compared to other similar studies, this study achieved a significantly higher hit rate of 1.2% using a conservative cut off criterion, suggesting that the choice to screen drugs and compounds, a safer (i.e. less cytotoxic) alternative to newly synthesized compounds could be a more efficient way to identify RdRp inhibitors. Eight drugs/compounds were shortlisted by the in vitro screening, and their anti-DENV potential was further evaluated in a cell-based DENV infection system by exploring their cytotoxicity and capacity to reduce viral titers. Of these, five (itraconazole, entacapone, butoconazole nitrate, pinacidil and kusunoki) showed an SI of higher than 1, showing that 62.5% of the shortlisted drugs and compounds demonstrated antiDENV activity in cultured cells. As kusunoki (a polyphenol-enriched extract rich in oligomeric PAs derived from the bark of the Japanese cinnamon tree) had reflected the highest SI, this was chosen for further downstream validation experiments. The antiviral effect of kusunoki was demonstrated to be reproducible in a cell-type and assay-independent manner. In addition, its broad-spectrum inhibition against all four DENV serotypes was shown. Insights into the mechanistic action of kusunoki suggests that it not only serves as a RdRp inhibitor, but could also further inhibits DENV by 111 preventing viral attachment to host cells prior to entry. Kusunoki therefore holds great potential as an anti-DENV compound for further development into an antiviral drug. The hits from this study may be a distance away from ideal drugs for administration under clinical settings given their selectivity indices. Nevertheless, this may be attributed to the screening of a mere 648 drugs/compounds. With larger drug/compound libraries used in the present screening system, more ideal antiviral drugs may be uncovered. This attempt can also be strengthened and made more efficient with the combination of in silico prescreening. 6.2 Future perspectives A further understanding of the pathogenesis of DENV infections could possibly lead to the identification of novel drug targets. In parallel, it is presently also vital to build on existing knowledge to fully exploit known viral targets. This may be in the form of uncovering the detailed mechanisms and molecular clockwork of these viral proteins, as well as solving their crystal structure to aid rational drug design. It is also essential to systemically ensure that pharmaceutical R&D efforts to date are maximized by repositioning drugs to various other indications whenever possible. 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J Gen Virol 89, 3047-3051. 133 [...]... successfully applied in the development of a fluorescence-based in vitro NS5 RdRp assay Against a background of failed clinical trials due to safety and pharmacokinetic concerns, an emerging importance has been placed on drug repositioning to develop novel uses for existing drugs Hence, libraries of FDA- approved drugs and natural compounds, highly regarded as safer alternatives compared to experimental synthetic... 2002) 1.1.2 Dengue virus The Flavivirus genus, belonging to the family Flaviviridae, contains 73 viruses, and many of which are arthropod-borne, or arboviruses, a term that depicts the necessity of a blood-sucking arthropod to complete their life cycle Of these, pathogenic flaviviruses include the DENV, West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and tick-borne... approximately 300 amino acids long and is known to facilitate the capping of viral 14 RNA and internal RNA methylation (Dong et al., 2012; Egloff et al., 2002) NS5 MTase has demonstrated methylase activity at both N7 position of guanine and 2’-OH position of ribose (Egloff et al., 2002) More recently, it has also been recently purported to have guanylyltransferase activity (Issur et al., 2009) These... NS3 and NS4A join NS5 in the vicinity of the 3’ stem loop after translation of viral RNA (Chambers, 2003), the main components of the replication complex are NS5 and NS3 This structure is thought to be anchored to the trans-Golgi network membrane via the viral integral membrane protein, NS4A 18 From the positive-sense RNA genome, a negative RNA strand is synthesized This forms a double-stranded RNA. .. trimerization of E protein and thereby mediating virus and cell membrane fusion (Allison et al., 1995; Modis et al., 2003) The nucleocapsid enters the cytoplasm, uncoats, and releases the viral RNA genome (Tomlinson et al., 2009) 17 1.3.2 Translation and further processing After the single-stranded, positive-sense viral genomic RNA is released into the cytoplasm, the 5’UTR directs the RNA strand to host... | Mechanistic inhibitory action of kusunoki 82 xii LIST OF TABLES Table 1.1 | A comparison of various in vitro protein synthesis systems 30 Table 1.2 | Summary of recently discovered anti- DENV small molecules and drugs 35 xiii Chapter 1 INTRODUCTION 1.1 Dengue Dengue is a disease caused by dengue virus (DENV) infections and transmitted by mosquitoes Tropical and sub-tropical regions around... main roles Firstly, it is important for viral RNA synthesis as it has been observed to be part of the replication complex, together with double-stranded (ds) form of viral RNA, NS3 and NS5 proteins (Mackenzie et al., 1998) Secondly, it is also involved in viral assembly In particular, amino acid residue Arg84 has been found to be critical for both RNA synthesis and viral assembly (Ditursi et al., 2006)... increased movement of viruses in infected humans, contributing to the geographic spread of the virus Moreover, ineffective control of its mosquito vector, Aedes aegypti, can also be ascribed for the continued viral spread and maintenance of the virus reservoir and (Mackenzie et al., 2004) Lastly, being the only known arbovirus 2 to have fully adapted to humans, DENV are no longer dependent on an enzootic... exploring their cytotoxicity and capacity to reduce viral titers Of these, 62.5% demonstrated antiDENV activity in cultured cells Of these, kusunoki, a polyphenol-enriched extract rich in oligomeric proanthocyanidins derived from the bark of the Japanese cinnamon tree, reflected the highest SI, and was chosen for further downstream validation experiments The antiviral effect of kusunoki is demonstrated... disease associated with fever and malaise Other symptoms may include a severe headache with retro-orbital pain, severe joint and muscle aches, nausea and vomiting and body rash (Simmons et al., 2012) Less than 10% of symptomatic dengue cases are reported, and a prospective cohort study of elementary school children in Thailand revealed that an average of approximately 53% of dengue cases were asymptomatic ... system-produced NS5 proteins 86 4.3 Screening libraries of FDA-approved drugs and natural compounds 88 4.4 Primary screening of libraries of FDA-approved drugs and natural compounds in in vitro... why over 60% of anticancer and 75% of anti-infective drugs approved from 1981 - 2002 can be traced back to natural origins (Gupta et al., 2005) The screening of FDA-approved drugs and natural... This has been further exacerbated by the lack of DENV human vaccines and antivirals DENV NS5 RNA- dependent RNA polymerase (RdRp), a viral- specific and highly conserved protein, is a promising