UK scientists have engineered a molecular knot that inhibits an enzyme crucial to foot-and-mouth disease, an infectious disease amongst cloven-hoofed animals. 

Edward Tate, Robin Leatherbarrow and colleagues from Imperial College London have looked to natural product cyclotides to create analogues that inhibit a range of protein-digesting enzymes. Among these was the first reported peptidic inhibitor of the enzyme FMDV 3C^pro - essential for the foot-and-mouth disease virus (FMDV) to replicate.

'Cyclotides are small proteins of around 30 amino acids that contain both a head-to-tail macrolactam backbone and a cystine knot, an arrangement of three disulfide bonds between pairs of cysteine residues, whereby two bonds form a loop through which the third is threaded,' said Tate. After carrying out the total synthesis of cyclotides from the MCoTI family, the UK team re-engineered the rigid and well-defined structures to create analogues with different biological activities.

MCoTI cyclotides are potent inhibitors of trypsin, a protease enzyme that breaks down proteins in the digestive system. By re-engineering the proteins, the cyclotides' inhibitory activity can be redirected towards an alternative protease target, Tate explained. This included a protease from a completely different mechanistic class: a cysteine protease from foot and mouth disease virus.

Cyclotides contain three disulfide bonds (yellow) and form a cystine knot

Whilst it is not the first FMDV protease inhibitor reported, Tate explained, 'to my knowledge, it is the most effective reported inhibitor in a controlled assay against the enzyme in isolation.' He added that, although the cyclotides were too complex for use as an animal drug, his results could help guide future non-cyclotide based inhibitor design for the FMDV enzyme. 

'The more interesting thing is that the analogues can inhibit a cysteine protein at all, since this is a very different class of protease from that typically targeted by MCoTI cylotides,' said Tate. 'The ease with which these structurally complex cyclotides can now be synthesised and re-engineered will enable new studies into their biological activity, for example, as potential peptide-based drugs.'

David Craik, from the University of Queensland in Brisbane, Australia, who studies protein structures relevant to drug design, agreed: 'The work represents an exciting development in the field of cystine knot proteins. By demonstrating a high yielding chemo-enzymatic approach to their synthesis as well as the ability to engineer tailored enzyme inhibitory activity into them, it is likely to greatly enhance applications of this ultra-stable class of proteins as drug development scaffolds.'

Elinor Richards