Detection of Metalloenzymes Employing Fluorescentpolymers and Liposomes

In the biological systems, proteins are important constituents. Protein-protein interactions play vital roles in physiological environments and any disruption in these interactions lead to adverse effects. However, designing artificial receptor molecules or scaffolds to imitate or replace these endogenous partners could be an avenue for better drug designing and detection tools creation. We are primarily interested in polymer and liposomal systems to detect two crucial metalloenzymes of the living world. Matrix metalloproteinases are zinc-containing endopeptidases which are required for wound healing, pregnancy and angiogenesis in normal bodily conditions. However, when overexpressed, these cause cancer, arthritis, cardiovascular disorders and fibrosis. Carbonic anhydrases (CAs) are another class of Zn2+ metalloenzymes involved in glaucoma, diabetes, epilepsy and hypertension. Sulphonamide-based inhibitors are prevalent in the market for targeting CAs, but they lack specificities in isozyme-selective inhibition or detection. Usually most of the broad spectrum inhibitors for MMPs have failed the clinical trials due to adverse side effects such as musculoskeletal pain or the inhibition of other non-targeted isozymes. Our strategy was to develop isozyme selective fluorescent water soluble polymers incorporating an active site binding inhibitor for each enzyme class and different charged and uncharged moieties for surface binding with the exposed residues of the isozymes. We have incorporated fluorophores in our polymers which acted as our detection signal generator through fluorescence. For MMPs, one of the optimized polymers was able to detect MMP-9 selectively compared to MMP-7 and -10 (discussed in Chapter 1). This polymer had shown potency in differentiating and subtyping various breast and prostate cancer cell lines from non- cancerous cell lines based on interactions with the secreted MMP-9 from these cell lines (discussed in Chapter II). Chapter III deals with the selective detection of CA II from CA VII and XII even in the complex mixture of biomacromolecules using our synthesized polymers. In Chapter IV, we investigated dye-encapsulated liposomal formulations for detection of catalytically active MMP- 7. The synthesized polymers and liposomes could serve as an alternative detection tool for detection and isozyme selective interactions of these metalloenzymes