Matriptase is a type-II transmembrane serine protease that is key to several important physiological functions including angiogenesis and cell growth. Impaired activity of matriptase is relevant to several diseases. One of these is cancer where deregulated matriptase activity enables angiogenesis, metastasis and tumour growth. These effects are produced through proteolytic activation of signalling pathways, such as c-Met, and also through disruption of the extracellular matrix.
We have generated variants of the naturally occurring cyclic peptides sunflower trypsin inhibitor-1 and Momordica cochinchinensis trypsin inhibitor-II to increase affinity for matriptase and to enhance specificity over trypsin, a closely related protease. Naturally occurring cyclic peptides offer great stability and scaffolds amenable to changes which are features suitable for drug design. We investigated specific amino acid interactions at the interface between the enzyme and inhibitor through an alanine scan of inhibitor variants. This revealed key residues that were not amenable to changes but also revealed residues that reduced affinity for trypsin and enhanced specificity for matriptase when modified.
The optimisation of recombinant expression of matriptase allowed generation of mutant enzymes where residues were substituted to assess their role in the putative interactions with the inhibitor. The nature and amino acid residue partners involved in these interactions were also evaluated through molecular dynamics simulations. This in silico approach also aided in evaluation of inhibitors prior to synthesis.
The specificity of matriptase for cleavage was assessed with the intention to adapt the reactive loops of the inhibitors for a better fit at the active site. This was done using a phage display library. This revealed an important feature about specificity which was implemented into the inhibitor scaffolds.
This work succeeded in generating variants with improved affinity and specificity for matriptase over trypsin.