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

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). This thesis describes the design, synthesis and evaluation of FMDV 3Cpro 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

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate.

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Structural and Mutagenic Analysis of Foot-and-Mouth Disease Virus 3C Protease Reveals the Role of the β-Ribbon in Proteolysis

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a β-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This β-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the β-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the β-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors