The central objective of this competing continuation research is to use structure-based mimetics to design antagonists for IL5 and to advance mechanistic understanding of receptor recognition for IL5 specific and for 4-helix bundle growth factor proteins generally. IL5 is the major hematopoietin which stimulates the proliferation, migration and activation of eosinophils and has been implicated in the pathogenesis of diseases such as asthma. Indeed, neutralizing IL5 antibodies currently are yielding promising results in asthma clinical trials, providing a proof of principle for the potential benefit of IL5 antagonists in therapies for asthma and other allergic diseases. IL5 is a homodimeric protein, dominated by two 4-helix bindle conformational domains. It functions through a cell-surface receptor that contains an alpha chain that binds IL5 selectively and a more 'public' beta chain for signal transduction. Available data for IL5 provide an important opportunity to use recognition site mimetics to decipher basic structural principles about how the 4-helix bundle framework in IL5 displays receptor binding epitopes for alpha and beta chains and to provide mimetic strategies useful to design 4-helix bundle cytokine antagonists. This project has several specific aims: (1) advance growing understanding of activating and non-activating modes of receptor subunit recruitment and design IL5 mimetic monomer and chimera antagonists, which recruit without activation; (2) starting with key miniprotein mimetic leads based on coiled coil stem loop scaffold, design advanced miniprotein antagonists, including size-minimized coiled-coil structures and miniproteins with alternative conformational scaffolds; (3) map and compare ligand binding epitopes on receptor alpha chain for IL5 vs. protein and miniprotein mimetic antagonists through mutational studies of the receptor and complementary mutagenesis of the ligands; (4) advance the use of mimetics through protein minimization, miniprotein scaffolds and antibody CDRs using the structurally related granulocyte- macrophage colony stimulating factor system. Long term, this project will lead to a fundamental understanding of recognition epitopes within the structural architecture of IL5. It will yield models for how variations within the basic 4-helix bundle architecture, common to many growth factor protein, trigger selective receptor recognition. And it will yield antagonist strategies which could lead to molecular tools for therapeutic intervention in asthma and other allergic diseases.
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