The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008
Appendix 69
ENHANCED PROSPECTS FOR FMDV ANTI-VIRALS TARGETED TO THE 3C PROTEASE S. Curry1*, P. Zunszain1, T. Sweeney1, N. Roqué-Rosell2, S. Knox2, A. Jaulent2 and R. Leatherbarrow2 1
Biophysics Section, Blackett Laboratory, Imperial College, London SW7 2AZ 2 Department of Chemistry, Imperial College, London SW7 2AZ
ABSTRACT Introduction In infected cells the RNA genome of FMDV is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally-encoded proteases. Most of these cleavages are performed by the highly-conserved 3C protease (3Cpro), making the enzyme an attractive target for antiviral drugs. Such drugs could provide a valuable prophylactic weapon during FMD epidemics in previously disease-free regions by reducing transmission in the period before vaccination takes effect. Materials and methods We have developed a soluble, active, recombinant form of FMDV 3Cpro that may be expressed at high levels in E. coli and used this material to investigate the structure and specificity of the protease with X-ray crystallography and peptide cleavage assays. Results The crystallographic analysis of FMDV 3Cpro (including the first determination of the structure of the complex of 3C with a peptide substrate) provides us with a detailed understanding of the protease structure that will be invaluable for inhibitor design. Peptide cleavage assays showed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4´), a result that is consistent with our structural analyses. We have also developed a fluorescent peptide substrate that can be used in high-throughput cleavage assays. Collectively these results establish a valuable framework for the development of FMDV 3Cpro inhibitors. I will briefly review our recent results and discuss the challenges ahead for the development of FMDV antivirals. 1. INTRODUCTION The picornavirus foot-and-mouth disease virus (FMDV) causes a serious vesicular disease of a wide range of mammalian hosts, including domesticated livestock such as cattle, pigs, sheep and goats. The disease is rarely fatal but it is extremely contagious: infected animals rapidly produce high viral loads that may be excreted or exhaled and are easily transmitted to uninfected hosts in aerosols. As a result of this pathogenic profile, FMD is endemic in many parts of the world. Although many regions, such as the EU and the USA, are officially disease-free and strive to remain so via strict import controls, the risk of sporadic epidemics is real and ongoing. The EU faces particular difficulties due to the proximity of countries suffering frequent outbreaks; for example Turkey, Israel and Egypt all reported outbreaks in early 2007. Control of FMDV outbreaks places a severe economic burden on the affected country: the last major epidemic in the EU, which occurred in the UK in 2001, inflicted total costs of around £6 billion. Although vaccination remains a powerful weapon in the struggle to control FMD, current vaccines have problems that inhibit their practical benefit in endemic and epidemic situations (see Discussion). It is therefore worthwhile to consider alternative or supplementary control measures (Grubman & Baxt, 2004; Kitching et al, 2006; Sutmoller et al, 2003). The development of such measures is clearly dependent on a fuller understanding of the molecular basis of viral pathogenesis. A potentially powerful approach, as has been adopted for HIV, is to target essential viral enzymes (e.g. proteases, polymerases) with inhibitory drugs. We propose to investigate the potential of FMDV 3C protease (3Cpro) as a viable target for drug design.
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