It is proposed that a unique combination of talents from three disciplines be brought to bear on the molecular basis for the action of cyclosporin. The central theme for these highly interdependent efforts will be the apparent major cellular receptor for cyclosporin, cyclophilin, with a focus on the structure of the cyclosporin binding domain and functionally related regions in this protein. Project 1. Rational design of semi-peptide and non- peptide structures of high stereospecificity based on analysis of the three dimensional receptor structure and available structure- activity information. Project 2. Chemical characterization of the major bovine isoform and the differences in the isoforms from bovine and other species. Chemical derivatives of cyclophilin will be prepared to aid in the 3 dimensional structural studies with an initial focus on a uniquely accessible cysteine sulphydryl group. Site directed mutagenesis and expression will also be used to examine binding kinetics and to aid Project 3 in sequential assignment of residues. Collaborative X-ray crytallography, CD studies and X-ray scattering determinations will be extended. A major future activity will be characterization of the putative natural ligand and the development of correlates between the structure of isoforms and their role in immuno-regulatory phenomena. Project 3) Development of a 3 D model of the binding site by 2D-NMR methodology with cyclosporin, its homologs as well as 13C enriched and F derivatives. Particular attention will be directed toward conformational changes in both the protein and drug consequent to ligand association to suggest new synthetic directions for Project 1.
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