Combinatorial Enzyme Activity and Inhibition Assays.

At the SPOCC Centre a FRET based assay using the combination of anthranillic acid and 3-nitro-tyrosine for peptide substrate libraries on biocompatible PEG-based resins was developed in the early 90’s. This assay has been used for the investigation of a large variety of enzymes of all classes. Metallo-proteases involved in osteoporosis, parasitic cysteine proteases, and serine proteases and aspartic proteases have all been investigated using the one-bead-one-compound substrate library assay. Once a good substrate has been identified for an important enzyme the substrate can be used in a second screen for highly active inhibitors in the one bead two compound assays, where the substrate is attached to library of putative inhibitors for the enzyme of interest. When the library is incubated with enzyme those resin beads containing active inhibitors are inactive with the substrate and remain dark where those which contain no inhibitors the substrate is cleaved and fluorescence is observed. This assay is very suited for quantitative high speed sorting on the Compas bead sorter developed in collaboration with Union Biometrica. In this way a range of new inhibitors have been developed towards the cysteine protease CPB 2.8 from lexmania mexicana and towards the metalloproteases involved in osteoporosis such as MMP9, MMP12, and 13.