LAUSR

Inflammatory caspases (caspase-1, −4 and −5 in humans and caspase-11 in mice) are cysteine-dependent, aspartate-specific proteases implicated in inflammatory, autoimmune and autoinflammatory disorders. To date, assays seeking to test the activity of caspases-1, −5 and −11 have all used Ac-WEHD-AMC as their fluorogenic substrate. To explore the possibility that alternative fluorogenic peptides may exhibit enhanced assay properties, we have designed, synthesized, and characterized several novel fluorogenic peptides containing coumarin derivatives. The coumarin derivatives were incorporated into peptides with various amino acid residues: WEHDA, WEHD, LEVD, LEHD as either a side chain of a non-natural amino acid, or at the C-terminus. The fluorescence quantum yields of these peptides were obtained, allowing the viability of these substrates to enhance the signal to noise ratio in caspase enzyme assays to be assessed. Biochemical assays were then carried out to determine if the signal to noise ratio indicated by the fluorescence quantum yield results correlated with caspase activity and could be applied to inhibitor screening assays.