It is proposed to determine the structural requirements for the functional activities of interleukin-8, a protein inflammatory mediator responsible for diverse pathologies. The long term objectives are to provide a hypothesis for the receptor bound form and a basis set of compounds with modified activities that will lead to the design of novel anti- inflammatory drugs.
The specific aims are 1) to determine the individual residues and structural features that are critical for its multiple biological functions, 2) design and generate more efficient antagonists of IL-8 actions, based on our low affinity antagonists, and 3) design a minimal peptide that mimics the activities of the mature protein. The experimental design will be to model and synthesize analogs, based on the 3-dimensional structure, that maintain the overall fold but result in local changes so that the structural and functional roles of individual regions/residues can be assessed. The analogs proposed include hybrids between homologous structures with no function, substitutions, covalent dimers, heterodimers, dimer destabilizing modifications, and conformationally restricted analogs. The proposed analogs will be progressively adjusted as information is acquired from the initial experiments so that profiles of allowed and disallowed substitutions at key sites can be obtained as efficiently as possible. Use will be made of coded and non-coded amino acids. The effects of the changes will be determined structurally by CD spectroscopy and where appropriate 2D-NMR spectroscopic techniques, and the functional changes by assaying the analogs for receptor binding, chemotaxis and exocytosis activities and where appropriate inhibitors of these activities, as well as intracellular calcium induction and respiratory burst activities. Analogs will be chemically synthesized by automated Merrifield methods and characterized physiochemically for authenticity and structure. The chemical synthesis approach is crucial to this study because it will allow synthesis of analogs with unambiguous structure in a facile and timely manner and allows the use of non-coded amino acids. Using this approach will be realistic to provide a detailed understanding of IL-8 structure-activity relations within the prescribed time period.
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