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 blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin 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 replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity.


RESULTS
Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs 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 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP.


CONCLUSIONS
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.
Luengo
J I
JI
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Yamashita
D S
DS
Dunnington
D
D
Beck
A K
AK
Rozamus
L W
LW
Yen
H K
HK
Bossard
M J
MJ
Levy
M A
MA
Hand
A
A
Newman-Tarr
T
T
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D020104:Immunophilins; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D017853:Phosphotransferases (Alcohol Group Acceptor); D019920:Photoaffinity Labels; D011090:Polyenes / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D020123:Sirolimus; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D013329:Structure-Activity Relationship; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; D015003:Yeasts / Q000187:drug effects
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D019920:Photoaffinity Labels; D011090:Polyenes; D017853:Phosphotransferases (Alcohol Group Acceptor); C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D020123:Sirolimus