Design, Synthesis and Analysis of FMDV 3C-Protease Inhibitors

Design, Synthesis and Analysis of FMDV 3C-Protease Inhibitors by Núria Roqué-Rosell A thesis submitted to Imperial College London in candidature for the Diploma of Imperial College and for the degree of Doctor of Philosophy. Department of Chemistry Imperial College London Exhibition Road London, SW7 2AZ June 2008 Abstract Foot-and-mouth disease virus (FMDV) causes a highly infectious disease of cloven-hoofed livestock with economically devastating consequences. The political and technical problems associated with the use of FMDV vaccines make drug- based disease control an attractive alternative. The FMDV genome is translated as a single polypeptide precursor that is cleaved into functional proteins by virally- encoded proteases. Ten of the thirteen cleavages are performed by the highly conserved 3C protease (3Cpro), making this enzyme an attractive target for anti-viral drugs (Curry et al., 2007a). pro This thesis describes the design, synthesis and evaluation of FMDV 3C inhibitors. A study of the activity of different FMDV 3Cpro mutants and a recently determined enzyme–substrate crystal structure provide an insight to the structure– activity relationships of the target and provides a basis for the inhibitor design (Curry et al., 2007b; Sweeney et al., 2007). Taking the enzyme‘s specificity as a starting point (Birtley et al., 2005), a small peptide sequence corresponding to an optimal substrate has been modified at the C-terminus, by the addition of a warhead, making it able to irreversibly react with the active site of this cysteine protease. Several synthetic routes have been developed, one allowed easy modification of the peptidic component of the inhibitor, whereas a second was designed to enable the introduction of variation on the warhead moiety. After the expression and purification of the FMDV 3Cpro, a FRET assay, developed to monitor changes on 3Cpro activity (Jaulent et al., 2007), was used to evaluate the inhibition of the enzyme by a range of potential synthetic inhibitors. The success of the inhibitor design has been demonstrated by the development of compounds that react as Michael acceptors with the enzyme active site. The investigation of the peptidic part of the inhibitor has highlighted key elements for inhibition which are supported by structural data. Finally, consideration of the bioavailability of these inhibitors, particularly in regard to membrane penetration, has led to use pro-drug approach in the further development of the inhibitors. 3 Acknowledgements I would like to thank all the people that have made this thesis possible and have contributed to make my time at Imperial College and in London the most enriching and enjoyable years (weather permitting). First and foremost, my supervisors. Robin Leatherbarrow, his valuable advice and experience have always been much appreciated, I am endebited to his patience and confidence for renewing my energies with every conversation and every silent nod; Stephen Curry, for his sheer enthusiasm, the time spent sharing all the developments at both sides of campus and for giving me the opportunity to work in his lab. Through him I would also like to thank those responsible for the antiviral assays, Graham Belsham and Thierry Jackson in particular, for the visit to the Institute for Animal Health. For an excellent working environment and the sweetest group meetings I am grateful to Robin together with the past and present members of the group. Ed Tate, who has been there all along, for the insightful discussions and warm support. Agnès Jaulent and Stephen Knox, for the time spent in and outside the lab during a fantastic first year. William Heal, for carefully reading this thesis. Rachael, Sasala, Gillian, Fran, Took and, from next door, Sarra and Steve, for all their contributions but mostly for making this journey so pleasant. For kindly hosting me and putting up with all my questions I am obliged to all the members of the biophysics group. I would like to mention James Birtley and especially Trevor Sweeney and Patricia Zunszain, working with them has not only been enlighting but also great fun. The European Union, for the financial support. Those who I left behind, for their visits and warm welcomes, and those who I met throughout my PhD, for all the moments shared. Severin, for more than what I can possibly describe. And finally, for their encouragement and unconditional love, I am most indebted to my parents and my brother. To all of you, thank you. 5 Contents Abstract .............................................................................................................. 3 Acknowledgements ........................................................................................... 5 Contents ............................................................................................................. 6 List of Figures .................................................................................................... 9 List of Tables ................................................................................................... 13 List of Abbreviations ...................................................................................... 15 1 Introduction ......................................................................................... 19 1.1 Foot-and-Mouth Disease .................................................................... 21 1.1.1 Distribution of FMD ................................................................... 21 1.1.2 Control of FMD .......................................................................... 23 1.1.3 FMD in the United Kingdom...................................................... 24 1.2 Foot-and-Mouth Disease Virus .......................................................... 27 1.2.1 FMDV serotypes......................................................................... 27 1.2.2 The FMDV genome .................................................................... 29 1.2.3 The FMDV cycle ........................................................................ 31 1.2.4 FMDV proteins ........................................................................... 34 1.3 Antiviral Agents ................................................................................. 37 1.3.1 Efforts towards FMDV inhibition .............................................. 38 pro 1.3.2 FMDV 3C as a viable drug target ........................................... 40 1.4 FMDV 3C Protease ............................................................................ 42 1.4.1 Proteases ..................................................................................... 42 1.4.2 Structure...................................................................................... 47 1.4.3 Catalytic mechanism ................................................................... 51 1.4.4 Substrate specificity .................................................................... 53 1.4.5 Protease inhibitors ...................................................................... 55 1.5 Project Outline ................................................................................... 59 1.5.1 Structure of this thesis ................................................................ 59 pro 2 FMDV 3C Structure Activity Relationships ........................ 61 2.1 Assay to Monitor FMDV 3Cpro Activity ............................................ 63 6 Contents 2.1.1 Michaelis-Menten kinetics .......................................................... 63 2.1.2 Design of fluorogenic substrates ................................................. 66 2.1.3 Synthesis of fluorogenic substrates ............................................. 70 2.1.4 Comparison of AMC and FRET substrates ................................. 73 2.1.5 The FRET assay .......................................................................... 75 pro 2.2 Recombinant FMDV 3C Mutants ................................................... 78 pro 2.2.1 Over-expression and purification of FMDV 3C ...................... 80 2.3 Structure Activity Relationships ........................................................ 87 2.3.1 Activity of 142 mutants .............................................................. 87 pro 2.3.2 Structure of the FMDV 3C –substrate complex ....................... 89 2.3.3 Discussion ................................................................................... 93 2.4 Summary ............................................................................................ 97 pro 3 Strategy toward FMDV 3C Inhibitors ................................... 99 3.1 Inhibitor Design................................................................................ 101 3.2 Synthesis of FMDV 3Cpro Inhibitors ................................................ 108 3.2.1 Synthetic routes ......................................................................... 108 3.2.2 Characterisation ........................................................................ 114 3.3 Inhibition Assays .............................................................................. 117 3.3.1 Active site modification ............................................................ 117 3.3.2 Evaluation of FMDV 3Cpro inhibition ....................................... 119 3.4 Summary .......................................................................................... 124 4 Peptide Chain Structure Activity Relationships ................... 125 4.1 The Peptide Size ..............................................................................

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