Activity-based selections and amplification of non-natural protease substrates

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Proteases, which catalyze the hydrolysis of amide bonds in peptides and proteins, play essential roles in most biological processes and are very important therapeutic and diagnostic targets for a multitude of diseases, including cancer. In this proposal, the development of efficient and systematic combinatorial tools is proposed to determine the substrate specificity profiles of proteases involved in prostate cancer. Generation of highly selective and specific substrates for these three proteases will aid in designing more selective diagnostic tools as well as have the potential to aid in designing selective inhibitors. Mentored K99 Phase: The postdoctoral phase (Specific Aims I and II) of the proposal will be carried out under the mentorship of Professor Jonathan Ellman at the University of California-Berkeley. The K99 phase goal is to develop a method for the generation of completely specific protease substrates. The substrates will be peptidic and activity-based selections using a thioester scissile bond will be performed on the DNA-bound library. DMA display technology will be utilized for encoding, routing, and amplification of the library. Jon Ellman is a leading expert in the field of combinatorial chemistry and its application to protease substrates. This period will provide essential training in the fields of combinatorial chemistry and protease chemistry. The acquired knowledge will prove invaluable for completion of Specific Aims III and IV and transition into a successful career as an independent scientist. Independent ROO Phase: Specific Aims III and IV will focus on the application of the technology developed in Specific Aims I and II. Peptidic substrates targeting proteases involved in prostate cancer will be evaluated as pro-drug therapeutics for prostate cancer (Specific Aim III). The substrates afforded from these experiments will also provide the basis for the generation of protease-activated near IR probes for the in vivo imaging of prostate tumors. Relevance: This proposal has the potential to greatly improve public health through advancement of the synthesis and evaluation (using activity-based selections) of combinatorial libraries. The proposed research develops methodology that will provide completely selective protease substrates. The substrates obtained will be used as the basis for drug delivery agents and selective imaging agents.