STRUCTURE-ACTIVITY STUDIES OF RAPAMYCIN ANALOGS - EVIDENCE THAT THE C-7 METHOXY GROUP IS PART OF THE EFFECTOR DOMAIN AND POSITIONED AT THE FKBP12-FRAP INTERFACE

Background: Rapamycin is an immunosuppressant natural product, which b locks T-cell mitogenesis and yeast proliferation. In the cytoplasm, ra pamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be re placed by a variety of different substituents. We set out to examine t he effects of such substitutions on FKBP12 binding and on biological a ctivity. Results: Rapamycin C-7-modified analogs of both R and S confi gurations were shown to have high affinities for FKBP12, yet these con geners displayed a wide range of potencies in splenocyte and yeast pro liferation assays. The X-ray crystal structures of four rapamycin anal ogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 grou p remained exposed to solvent. We then prepared a rapamycin analog wit h a photoreactive functionality as part of the C-7 substituent. This c ompound specifically labeled, in an FKBP12-dependent manner, a protein of similar to 250 kDa, which comigrates with recombinant FRAP. Conclu sions: We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.