Structure-activity studies of ground- and transition-state analogue inhibitors of cyclophilin

Peptidyl-prolyl isomerases (PPIases) are ubiquitous cellular enzymes that p lay roles in cellular signaling and protein folding. In addition, these pro teins are the receptors for the widely used immunosuppressants cyclosporin A and FK506. We report the first structure-activity studies of de novo desi gned inhibitors of cyclophilin, the cellular target of cyclosporin A. Our m echanism-based inhibitors were modeled on the ground- and transition-state structures of proline-containing peptides, the natural substrates of the en zyme. Both ground-state analogues I and transition-state analogues 2 were p repared as single enantiomers from L-proline following a "self-reproduction of chirality" procedure. The binding affinities of the analogues for the a ctive site of cyclophilin were measured by a fluorescence perturbation assa y. While the transition-state analogues 2 did not display significant avidi ty for the active site (K-d = 77 muM for 2b), several ground-state analogue s bound to the enzyme with low micromolar affinity (K-d = 1.5 muM for 1e). These results proclaim that properly designed small molecular weight molecu les can form strong complexes with cyclophilin and may find use as probes i n cell biology and as therapeutic agents.