STRUCTURE-BASED DESIGN OF CALCINEURIN INHIBITORS. Charles R. Kissinger, Hans Parge, Daniel Knighton, Laura Pelletier, Anna Tempczyk, John Tatlock and Ernest Villafranca, Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, CA 92122

The crystal structure of human calcineurin (CaN) provides the foundation for structure-based design of novel immunosuppressive agents. CaN is a calmodulin-dependent protein serine/threonine phosphatase that plays a critical role in T-cell activation. CaN is the target of the immunosuppressive drugs, cyclosporin and tacrolimus (FK506). These macrocyclic compounds inhibit the enzyme only after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We have determined the crystal structures of human CaN and of the CaN-FKBP12-FK506 complex. The structure of native CaN reveals that a calmodulin-regulated auto-inhibitory sequence binds over the di-metal active site. In the CaN-FKBP12-FK506 complex, FKBP12-FK506 binds adjacent to the CaN active site but dispaces the auto-inhibitory sequence and inhibits through a non- competitive mechanism. The binding of FKBP12-FK506 appears to mimic a natural protein-protein interaction involved in CaN regulation. The structure of the complex reveals essential features of FK506 necessary to facilitate protein-protein interaction between CaN and FKBP12. The structural findings suggest strategies for design of both FK506 analogues and direct active site inhibitors of CaN.