This project seeks to find specific peptide dendrimers through combinatorial chemistry, to be used as highly specific, fluorogenic protease substrates. A general approach is formulated that should allow to discover fluorescent dendrimer probes specific of any given protease. To that extent, combinatorial synthesis of dendrimers incorporating target sequences for well-known proteases will be carried out.
Then, a suitable FRET (fluorescence resonance energy transfer) strategy for protease activity detection will be developed and optimized using dendrimers with selective cleavage sequences for model proteases. Red fluorescence will be preferable to blue fluorescence, especially when considering a potential in vivo diagnostic application.
Then, fluorogenic peptide dendrimers incorporating target sequences for proteases of diagnostic interest (cathepsins, uPA, PSA) will be synthesized and tested. Finally the optimized assay will be adapted as a microarray by covalently binding the dendrimers on a glass surface. This project takes advantage of the optimized split and mix, biased dendrimer synthesis techniques developed in the host group.
Using these technique, the combinatorial library encoding/decoding is particularly efficient. The application of combinatorial chemistry to the problem of protease substrate selectivity is novel. In fact some sequences arising from libraries have been reported to be better substrates than the 'natural' target sequence.
To the present date, specific target probes on linear peptides have been used, either on a standalone basis or grafted onto a linear structure, but not with the dendrimer being carrier and substrate itself, or using its own three-dimensional structure as a feature to modulate the interaction with different proteases and therefore, to achieve selectivity in the cleavage of target aminoacid sequences.
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