Complexes formed between proteins called immunophilins (immunosuppressant binding proteins) and their immunosuppressive ligands have proved to be valuable probes of cytoplasmic signal transduction. The proposed research is aimed at understanding the biological effects and structure of immunophilin-ligand complexes and, further, to identify and study their cellular targets. In the case of cyclophilin-cyclosporin A and FKBP-FK506, research carried out over the previous funding period demonstrated that the target is the Ca2+, calmodulin-dependent phosphatase calcineurin. The proposed research aims to identify the target of the FKBP-rapamycin complex as well as possible endogenous ligands of immunophilins. Strategies for the synthesis of new ligands to the FKBP family of immunophilins will be explored-the structures of these target ligands have been formulated by analyzing the structure of FKBP12 and FKBP12-ligand complexes. The properties and structures of the immunophilins FKBP13, FKBP25, and FKBP59, as well as the complexes of FKBP12 and synthetic ligands, will be investigated. Determining the mechanisms by which cyclosporin A and FK506 interfere with Ca2+-dependent signal transduction and rapamycin interferes with Ca2+-independent signal transduction may result in the identification of new targets, in analogy to the discoveries of calcineurin and FKBP (also achieved during the past funding period), for the development of therapeutic agents. New synthetic molecules will be prepared that should modulate the activity of calcineurin by forming high affinity complexes with FKBP12. Such molecules, in analogy to FK506 and cyclosporin A (when bound to cyclophilin), are expected to interfere with T cell activation (and thus function as immunosuppressants) and mast cell activation (and thus function as antiinflammatory agents). In addition, peptides and other ligands will be sought that directly modulate the activity of calcineurin. New synthetic molecules will be prepared that should exhibit rapamycin-like properties, with the possibility of greater potency, by forming ultra-high affinity complexes with FKBP12 (and thus modulate the activity of the yet-to-be discovered FKBP-rapamycin target).
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