The known immunosuppressant Cyclosporin A (CsA), which is effective in increasing survival and decreasing morbidity in human origin transplants, is thought to exert its biological activity when it is bound to cyclophilin, a cytoplasmic protein that possesses peptidyl prolyl cis-trans isomerase (rotamase) enzymatic activity. Work has indicated that CsA is a potent inhibitor of rotamase catalysis and that rotamase inhibition is a necessary, but insufficient, condition for immune suppression. Consequently, the rational design of new immunosuppressants with greater potency and decreased toxicity than CsA will require the identification of molecular fragments capable of rotamase inhibition. In this proposal, funds are sought to pursue experiments aimed at understanding rotamase catalysis and identifying molecular fragments capable of rotamase inhibition. To accomplish this, peptide substrates of cyclophilin which contain modified amino acids will be examined. First, a new substrate for the spectrophotometric assay for rotamase catalysis which will increase the sensitivity of the assay will be identified. Second, modified peptides will be examined to see if they can inhibit rotamase catalysis. One set of these peptides is expected to be irreversible inhibitors of rotamase activity, while the other set of peptides is expected to be reversible inhibitors. If successful, the proposed research will provide important information about the mechanism of rotamase catalysis. The research will also facilitate the design of new immunosuppressants for use in organ transplants and in the treatment of immunological disorders.
