Canning, Peter, 1984- (2009) The prolyl oligopeptidase family enzymes: structural basis of inhibition. PhD thesis, University of Warwick.

Abstract

The prolyl oligopeptidase family enzymes are a group of medically significant proteins distributed in all kingdoms of life that have several unifying structural and functional features. Members of the family hydrolyse short peptides, normally no more than 30 amino acids long. This strict substrate specificity is regulated by β-propeller domains that restrict access to the active site. Conformational changes allow access to the active site by small peptides but block access to larger, structured peptides. Mechanistic details such as these were revealed when the structure of prolyl oligopeptidase was solved by X-ray crystallography in 1998. Since then, numerous crystal structures of family members have been solved, increasing understanding of the structure-function relationships of these important enzymes. Many members of the family are either proven or potential drug targets. As such, structural characterization of these enzymes and their interactions facilitates the design of pharmaceutical compounds. With this in mind, this research was undertaken, using X-ray crystallography to further understand the interactions between particular members of the prolyl oligopeptidase family and inhibitor compounds. Four specific, potent inhibitors were visualized in complex with the active site of prolyl oligopeptidase. All four inhibitors follow a similar template which was shown to be successful when one of them advanced to phase 3 clinical trials as a treatment for Alzheimer‟s disease. The structure of oligopeptidase B from Trypanosoma brucei, which is involved in cell invasion leading to Trypanosomiasis, was solved to 2.5Å, revealing a number of interesting features and enabling various avenues of study. Finally, a plant derived glutamyl endopeptidase known to be regulated by endogenous inhibitors was overexpressed, purified to a high degree and shown to be correctly folded and active. Establishing this procedure allows for the commencement of crystal trials, as well as study by other biophysical methods.

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