Intracellular cyclic reagents for validating protein function

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Functional genomic analyses are providing data sets that allow hypotheses to be formulated on the therapeutic potential of proteins. Given the large number of protein activities and interactions that are predicted to be important for any given disease, it is difficult to determine the best targets for drug development. It is also too time consuming and expensive to initiate drug development programs against all these targets. To circumvent these problems, cost-effective methods are needed to rapidly validate targets that are important in disease-associated processes and that are susceptible to inhibition by trans dominant agents such as small-molecule drugs. The goal of this proposal is to develop a comprehensive platform for isolating cyclic peptides and antibody-like single-chain variable regions (scFvs) that interact with protein targets with high affinity and specificity. The proposal builds on previous technology developed in our laboratory, whereby we designed a novel genetic selection for rapidly isolating "lariats" that function as inhibitors of protein function. A lariat consists of a cyclic peptide or "noose" region with a covalently attached "tail" that contains a transcription activation domain. Combinatorial libraries of lariats, where the noose region is randomized, can be screened for interactions with a protein target using the yeast two-hybrid assay. In this proposal, the short-term objective is to develop methods to increase the stability and solubility of lariat peptides and scFvs. This will improve our ability to isolate these reagents against protein targets and to use them effectively as inhibitors for validating the therapeutic potential of proteins. The long-term goal of this proposal is to isolate lariats and scFvs against protein targets involved in regulating autocrine IGF-1 signaling in chronic myeloid leukemia (CML) and to use these reagents to identify novel targets to develop drugs for treating CML.