Abstract:
This thesis investigates the binding affinity and selectivity of novel macrocyclic peptide inhibitors isolated against three therapeutically relevant matrix metalloproteinases (MMPs). The apparent equilibrium dissociation constant (KDapp) of individual selected macrocyclic peptide ligand towards each single MMP was determined using yeast surface display technology. This methodology combined with flow cytometry allows quantitative and high-throughput characterisation of single macrocyclic peptide variants directly as cell surface fusions, eliminating the need for additional chemical synthesis and purification steps. Thirteen yeast-encoded macrocyclic peptides ligands were analysed. The determined KDapp values are in the nanomolar range. Particularly, for all three MMPs tested, at least one macrocyclic peptide ligand that showed KDapp values below 20 nM was identified. To assess the extent of specificity of the selected macrocyclic peptides, we measured the binding of each yeast-displayed macrocyclic peptide ligand toward homologue proteins that share at least 50-60% sequence identity in their catalytic domains. No or very weak binding signals were detected for eleven out of thirteen (~85%) macrocyclic peptides analysed. Further kinetic competition studies using a molar excess of a potent broad spetrum inhibitor of MMPs revealed that most of the macrocyclic peptides isolated bind to the catalytic pocket.
These results offer valuable insights into the potential therapeutic application of these macrocyclic peptides as selective MMP inhibitors, contributing to the development of targeted treatments for MMP-related diseases.
