Miniature Protein Ligands, Inhibitors, and Scaffolds

Yale University, New Haven CT

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Abstract

Many if not all events in biology demand highly specific interactions between macromolecular partners. Each of these events could, in theory, be targeted as part of a novel therapeutic strategy. Yet our understanding of the rules that govern interactions between macro-molecules has not advanced to the point where scientists can design molecules that predictably, potently, and selectively inhibit macromolecular complexation. In this application we build on the success of a general strategy for the design of folded, miniature proteins that bind receptors for alpha helices. This strategy, which we call protein grafting, has generated two miniature proteins based on avian pancreatic polypeptide (aPP, 36 residues) that bind DNA with exceptional affinity and specificity and one other that binds a protein with comparable properties. In this application, we explore the generality of protein recognition, by asking (Aim 1) whether miniature proteins can bind other proteins (such as Bcl-2 and Bcl-XL), even one that does not possess an identifiable "deep cleft" (such as CBP). In Aim 2 we approach the daunting problem of selective kinase inhibition, by asking whether a miniature protein can render selective an otherwise non-selective kinase inhibitor, or distinguish PKC sub-types, by recognizing the surface outside the ATP or phorbol binding pocket. Finally, we ask whether the type II polyproline (PPII) helix in aPP-based miniature proteins can be replaced functionally with a 'foldamer' to explore the virtually unexplored area of foldamer-alpha helix interactions (Aim 3) and whether miniature proteins can be assembled in an intermolecular reaction of two separate pieces (Aim 4). Construction of minimized proteins that possess appreciable biological function is an important step to-wards structure-based design of small molecules that inhibit or activate the functions regulated by protein-macromolecule complexes. The combined features of high affinity, high specificity and a compact fold implies that miniature proteins could be used to dissect, modulate, or analyze a single protein function, irrespective of the other functions the protein may regulate within the proteome.