The objectives of this research application are to (1) synthesize photoaffinity-labeled analogs of the immunosuppressive peptide Cyclosporine A (CSA) and (2) use these probes to determine if specific T lymphocyte-restricted CSA receptors exist. Cyclosporin, a novel and highly effective immunosuppressive agent, apparently acts by suppressing the production of the lymphokine IL-2, especially for mitogen or alloantigen-stimulated virgin T cells. It is widely used in transplantation, has potential as an autoimmune agent, and has antiparasitic activity. The use of PA labeled CS probes to identify its immunologically relevant target has not been reported, although a diaziridine PA-CS derivative has been synthesized and used to study the binding of CS to the hepatic bile salt transport proteins. These studies demonstrate the feasibility of synthesizing biologically active PA- CS analogs. Synthesis of PA-CS analogs will be done in Dr. Rich's lab modifying techniques he has developed over the past three years for total synthesis of cyclosporin analogs. These will include replacing amino 3 of CSA with N-methyl D-lysine and position 8 with D-Lys, to which several types of PA probes can be attached. Conditions will be optimized for PA-CS cellular labeling, and the effect of probes on cell viability and biological activity will be determined. Labeled components will be isolated and characterized by 1- and 2-dimensional gel electrophoresis, ion exchange, gel permeation, and hydrophobic chromatographic techniques, in addition to FPLC and HPLC. Isolated products will be further characterized by tryptic digests. Elucidating the nature of CSA's cellular receptor would greatly aid in deciphering the mechanism of CSA's immunosuppressive activity.
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