S. CURRY, P. ZUNSZAIN, T. SWEENEY, N. ROQUÉ-ROSELL, S. KNOX, A. JAULENT AND R. LEATHERBARROW

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-Rosell

2, S. Knox2, A. Jaulent

2 and R.

Leatherbarrow2

1Biophysics Section, Blackett Laboratory, Imperial College, London SW7 2AZ

2Department 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.

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008

2. MATERIALS AND METHODS

The 3C protease from type A1061 was mutated to enhance solubility, expressed in E. coli, purified and crystallised as described previously (Birtley & Curry, 2005; Birtley et al, 2005; Sweeney et al, 2007). A customised fluorescent peptide substrate [4-(4-dimethylaminophenylazo) benzoic acid- APAKQLLD [5-(2-amino-ethyl) amino-1-naphthalenesulfonic acid (EDANS)] FDLLK (3C-FRET4)] was synthesised and used in cleavage assays as reported (Jaulent et al, 2007b).

3. RESULTS

We have engineered a soluble, active recombinant form of the enzyme that can be expressed at high levels in E. coli (Birtley & Curry, 2005; Birtley et al, 2005), yielding around 25 mg per litre of culture. This has allowed us to determine crystal structures of 3Cpro, revealing different conformational states and providing important new insights into the catalytic mechanism and substrate recognition (Birtley et al, 2005; Curry et al, 2007; Sweeney et al, 2007). More recently we have determined the co-crystal structure of a 3Cpro-peptide complex, a first for any picornaviral protease (Fig. 1; unpublished data). The active site of the enzyme involves the participation of a flexible loop (known as the -ribbon) that folds in contact with the bound substrate; the importance of this interaction has only recently been revealed by crystallography and emphasises the need for structural information in the search for new inhibitors (Sweeney et al, 2007). We have also developed a continuous assay of 3Cpro cleavage activity—based on a quenched fluorescent peptide—with which we have begun to examine determinants of cleavage specificity, both on the enzyme and within the peptide substrate, and to probe inhibitor activity (Jaulent et al, 2007a; Sweeney et al, 2007). More recent work, performed in collaboration with Prof. Julie Frearson, (Scottish Hit Discovery Facility, Dundee University), has established that our fluorescent peptide cleavage assay can be adapted to the 384-well format, greatly reducing the enzyme load (Fig. 2; unpublished data). Under these assay conditions the %CV is 5.4 and the Z-factor is 0.83: the assay therefore comfortably meets the criteria for use in HT screening. Using synthetic peptides in our cleavage assays we have identified the peptide corresponding to the VP1-2A junction as the best substrate sequence within the viral polyprotein (Birtley & Curry, 2005). Using the results generated from these experiments we have synthesised peptide-based inhibitors that specifically block FMDV 3Cpro in in vitro assays (Fig. 3; unpublished data). Although such peptide based reagents are unlikely to be feasible drug candidates, they are valuable research tools since they permit exploration of the structural determinants of high affinity binding. With this exciting series of breakthroughs we now have the reagents and methodologies in place to forge ahead with our investigations. In particular, we can now make a systematic search for small molecule inhibitors of FMDV 3Cpro and follow up potential hits with further activity and structural analyses. But the development of effective drugs from initial hits is a long and complex process and a key question for discussion is whether there is a viable market for such drugs in today’s world.

4. DISCUSSION

As with other picornaviruses such as poliovirus (PV), human rhinovirus (HRV) and hepatitis A virus (HAV), FMDV comprises a protein capsid containing a single-strand, positive-sense RNA genome that is translated as a long polyprotein precursor shortly after cell entry. The FMDV polyprotein must be cleaved at 13 distinct locations by virally encoded proteases in order to release the proteins needed for capsid assembly and RNA replication. The proteolytic activity of FMDV 3Cpro is absolutely crucial to virus replication because it performs 10 of the 13 cleavages and is therefore an attractive target for therapeutic intervention. Despite the potential benefits of anti-viral therapy for FMD, there has so far been relatively little effort to develop drugs targeted to key viral enzymes. In part this is due to the lack of structural information and the appropriate tools to investigate enzyme inhibition. Recent crystallographic work has uncovered the structures of the viral polymerase (Ferrer-Orta et al, 2006) and the L (Guarne et al, 1998) and 3C (Birtley et al, 2005; Sweeney et al, 2007) proteases. These advances are likely to stimulate the quest for effective antiviral compounds. In our lab we have made substantial progress in investigating the structure and activity of FMDV 3Cpro. We are now in a strong position to advance these investigations and to be able to initiate the search for effective inhibitors.

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008 Current FMDV vaccines, consisting of inactivated virus particles, have significantly reduced the incidence of disease worldwide, but their use as a control measure in the event of new outbreaks, especially in previously disease-free countries, is beset by a number of problems. Initial diagnosis is complicated by the fact that FMDV is symptomatically similar to other diseases (e.g. vesicular stomatitis). Once an outbreak has been confirmed, vaccine doses must be formulated from frozen stocks, a process that takes several days; since FMDV exists in seven distinct serotypes and multiple subtypes, diverse vaccine stocks must be maintained so that doses can be formulated to protect against the outbreak strain. There is a further delay of up to a week post-injection before vaccinated animals become fully protected. Even then, vaccination does not provide sterile immunity, i.e. does not prevent infection and has no impact on the development of the carrier state, the persistence of infection in animals that have apparently recovered from the disease. In countries that strive to remain diseasefree, control by slaughter is sometimes seen as a more effective way to terminate outbreaks and has the attraction of incurring a shorter delay before the resumption of livestock trading with the rest of the world. However, pre-emptive slaughter (before diagnosis) is a difficult control strategy to implement because of farmers’ resistance; moreover, a slaughter policy can threaten the existence or valuable rare breeds. Although it is certainly vital that vaccine development work should continue, it is also important to explore alternative control measures (Follet, 2002). Until very recently the knowledge and tools did not exist to permit such exploration but structural work on key FMDV enzymes, including our studies on FMDV 3Cpro, creates an opportunity to investigate the role that antivirals could play in effective disease control. The major attraction of such compounds is that they could overcome many of the short-comings of FMDV vaccines. They could act rapidly, especially if orally bioavailable. Moreover, since the viral enzymes such as 3Cpro and 3Dpol are highly conserved across all FMDV serotypes (Carrillo et al, 2005), it is likely that inhibitors will have broad spectrum (cross-serotype) activity, a major advantage over current vaccines. In addition, a drug-treated animal that was not infected would be easy to distinguish (using existing tests) from an infected (or vaccinated animal). The prophylactic use of antivirals could provide a powerful weapon for disease control; conceivably such drugs could be used for short periods only in conjunction with vaccines to close the window of opportunity that the slow onset of the immune response offers to the virus. Moreover, it is conceivable that an antiviral might also be able to cure carrier state animals. Previous research on the 3C proteases of HAV, HRV and PV revealed that they belong to a unique class of enzymes that combines a chymotrypsin-like fold with a cysteine protease mechanism; since there are no known cellular homologues, these proteases are attractive targets for drug design. Determination of the crystal structures greatly stimulated the search for inhibitors as potential therapeutics compounds; much of the research has focused on HRV 3Cpro—since effective vaccines exist for HAV and PV infections—and has led to the development of compounds such as rupintrivir (Patick, 2006; Patick et al, 2005), which acts as a potent irreversible inhibitor of HRV 3Cpro. This inhibitor was demonstrated to protect against viral challenge, thus establishing the principle that picornaviral 3C inhibitors may provide an effective prophylactic defence against infection (Hayden et al, 2003). The progress made with HRV 3Cpro has recently led to proposals that anti-HRV 3Cpro compounds may be used to initiate development of antivirals to prevent PV infection (Katz et al, 2006) (to overcome the risks of production and use of the live poliovirus vaccine in a post-eradication world) and to combat potentially fatal SARS CoV infections in humans (Anand et al, 2003). Proof of principle of antivirals targeted to proteases has already been demonstrated for HRV 3Cpro and HIV protease. More recently antivirals to pestiviruses (designed to target the viral polymerase) have exhibited potential as a disease control measure (Paeshuyse et al, 2006; Vrancken et al, 2008). In fact it has been shown that a compounds developed against one pestivirus, bovine viral diarrhea virus (BVDV), can confer protection in pigs from challenge by a different pestivirus, classical swine fever virus (CSFV) - a disease with symptomatic similarities to FMDV. With regard to FMDV, recent reports indicate that the 3D polymerase may be a valuable drug target (Goris et al, 2006; Sakamoto et al, 2006). These data underscore the very real potential of antiviral drugs to help control infectious diseases of livestock. In a very similar vein, it is conceivable that inhibitors of FMDV 3Cpro could be developed as effective antivirals. The key question remaining is whether the international community is willing to provide the resources needed to take these efforts forward.

5. ACKNOWLEDGEMENTS

SC and RJL are grateful for grant support from the BBSRC and access to synchrotron facilities at Daresbury SRS (UK), EMBL-DESY (Germany) and ESRF (France). NRR was funded by a Marie Curie Host Fellowship for Early Stage Research Training. We thank Stuart McElroy and Prof Julie Frearson (Dundee University) for optimising HT assays and providing the data for Fig. 2.

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The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008