Identification and characterisation of small molecule inhibitors targeted to the hepatitis C virus NS2 autoprotease. Joseph Charles Shaw (BSc (Hons)) Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Astbury Centre for Structural Molecular Biology September 2014 The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2014 The University of Leeds Joseph Charles Shaw The right of Joseph Charles Shaw to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. i Acknowledgments The completion of this thesis would not have been possible without a number of people. Firstly, I would like to thank my supervisors Prof. Mark Harris and Prof. Colin Fishwick for their continued advice, support and enthusiasm with this project. I would also like to thank past and present members of Garstang 8.61. Thanks in particular to Doug for helpful discussion and to Barney and Bruno for the same as well as occasionally taking my mind off science. Special thanks to Hazel for advice and for her relentless optimism and ability to cheer me up during the more frustrating times. I am also grateful to Katie, Martin, Sarah and Ricky for providing a great deal of help to a biologist in a chemistry lab. Thank you to my family for their support throughout the highs and lows of the last four years. Finally I would like to acknowledge the Wellcome Trust for the funding which made this project possible. ii Abstract Hepatitis C virus (HCV) is a positive-strand RNA virus present in 2-3% of the global population and commonly establishing a chronic infection, leading to long term diseases such as liver cirrhosis and hepatocellular carcinoma. Recent advances have led to the development of a range of direct-acting anti-viral drugs (DAAs), some of which are already improving outcomes in the clinic. It is clear however, that effective therapy for the treatment of HCV will most likely require a combination of DAAs to overcome the rapid onset of viral resistance. In this regard additional inhibitors of the virus lifecycle, which act through a novel molecular target, are required. The autoprotease activity encoded within the C-terminus of the non-structural 2 (NS2) protein is essential for processing of a precursor to the mature viral proteins, and as a consequence is also required for the onset of viral genome replication and the establishment of HCV infection. Despite representing an attractive target for anti-virals, no inhibitors of the NS2 autoprotease have been reported. In order to identify small molecule inhibitors of the NS2 autoprotease, two independent assays were optimised as a measure of NS2-mediated proteolysis. These assays were employed to demonstrate that inhibitors of the NS2 autoprotease were able to block HCV genome replication. The assays were subsequently used to identify a lead-like small molecule inhibitor by screening an in silico enriched library. This compound was further characterised in the context of NS2 activity in vitro and cell culture models of the virus lifecycle. The resultant series represent the first documented inhibitors capable of exerting an anti-viral effect by targeting the NS2 autoprotease. iii Table of Contents Acknowledgments ...................................................................................................................... ii Abstract ..................................................................................................................................... iii Table of Contents ...................................................................................................................... iv Table of Figures ....................................................................................................................... viii Table of Tables ........................................................................................................................... x Abbreviations ............................................................................................................................ xi Chapter 1 - Introduction ............................................................................................................... 1 1.1 Viral hepatitis ...................................................................................................................... 2 1.2 Hepatitis C virus .................................................................................................................. 2 1.2.1 HCV classification, epidemiology and genetic heterogeneity ...................................... 2 1.2.3 HCV transmission ......................................................................................................... 5 1.2.4 Disease progression ..................................................................................................... 5 1.3 Treatment of HCV ............................................................................................................... 8 1.4 Molecular virology and lifecycle of HCV ........................................................................... 13 1.4.1 Genome organisation................................................................................................. 13 1.4.2 Virus particle composition ......................................................................................... 14 1.4.3 Viral entry ................................................................................................................... 14 1.4.4 Polyprotein processing ............................................................................................... 17 1.4.5 Genome replication ................................................................................................... 18 1.4.6 Virus assembly and release ........................................................................................ 21 1.5 HCV encoded proteins ...................................................................................................... 22 1.5.1 Core ............................................................................................................................ 22 1.5.2 E1 and E2 .................................................................................................................... 22 1.5.3 p7 ............................................................................................................................... 23 1.5.4 Non-structural 2 ......................................................................................................... 24 1.5.5 Non-structural 3 ......................................................................................................... 27 1.5.6 Non-structural 4A ....................................................................................................... 30 1.5.7 Non-structural 4B ....................................................................................................... 30 1.5.8 Non-structural 5A ....................................................................................................... 30 1.5.9 Non-structural 5B ....................................................................................................... 32 1.6 Current anti-viral strategies for HCV ................................................................................. 34 1.7 HCV model systems ........................................................................................................... 35 iv 1.8 The NS2-3 autoprotease ................................................................................................... 39 1.8.1 The NS2 cysteine protease ......................................................................................... 39 1.8.2 The role of NS3 and zinc on the NS2 autoprotease ................................................... 42 1.8.3 Relevance of the post-cleaved NS2 crystal structure ................................................ 43 1.8.4 Evidence of NS2 homo-dimerisation ......................................................................... 49 1.8.5 NS2 as a target for anti-virals ..................................................................................... 50 1.9 Rational drug design and virtual high throughput screening ........................................... 53 1.9.1 De novo design ........................................................................................................... 54 1.9.2 Virtual high-throughput screening ............................................................................. 55 1.10 Project Aims .................................................................................................................... 57 Chapter 2 - Materials and Methods ........................................................................................... 59 2.1 General Materials ............................................................................................................. 60 2.1.1 Bacterial strains .......................................................................................................... 60 2.1.2 Mammalian cell lines ................................................................................................. 60 2.1.3 Virus sequences ........................................................................................................