Development of Molecular Probes Responsive to Matrix Metalloproteinases

Abstract

Matrix metalloproteinases (MMPs) have been identified as biomarkers for cancer, offering prognostic potential. However, they lack non-invasive detection. This thesis highlights the work carried out on the design, synthesis and development of 19F MRI probes for the molecular imaging of MMP activity. The design of these MMP-activated imaging agents was based on the paramagnetic effects of gadolinium(III) on the NMR/MRI signal of a CF3 group in close proximity to the metal cation. 1st generation probes were prepared, incorporating MMP-2 selective peptide substrates SPAYYTA or SLAYYTA as a linker between gadolinium(III)-DOTA and Asp[(trifluoromethoxy)benzylamide]-OH. Attempts to monitor MMP-2 mediated peptide hydrolysis via HPLC and fluorescent assays proved problematic. The use of 68Ga and its complexation to DOTA provided a highly sensitive assay for the assessment of the peptide compounds. Using this method, assay conditions were optimised to observe on average 20% peptide cleavage over 2 hours with MMP-2, with selectivity observed against MMP-9 and MMP-14. The original gadolinium analogues of these compounds were then analysed by 19F NMR spectroscopy, successfully demonstrating a change in signal upon incubation with MMP-2. Development of the probes was then carried out to improve their clinical potential. Two pathways were explored; increasing compound reactivity through the introduction of spacer groups and increasing 19F sensitivity via increased local concentrations of fluorine, carried out utilising activated cell penetrating peptides (ACPPs). The latter method proved more successful, displaying similar reactivity and selectivity to 1st generation compounds. These ACPP probes are still in the early stages of development, with scope for future investigation. The transferability of 1st generation MMP-2-activated probes was also investigated via the production of a compound of analogous design, incorporating an MMP-14 selective substrate RIGFLR. Testing and development revealed this compound required incorporation of a two amino acid spacer group (SG) in order to demonstrate partial MMP-14 selectivity.