5858 J. Am. Chem. Soc., Vol. 118, No. 25, 1996
Kallen et al.
light the importance of the distinct effector domains of these
macrolides, and they have been designated as “dual domain”
inhibitors.7,13 Both macrolides bind to FKBP12 through their
common binding domain, and the resulting complexes become
the actual immunosuppressive species, which, in virtue of their
distinct exposed effector domains, interact with distinct intra-
cellular targets.7,13 The FKBP12-FK506 complex binds to and
inhibits the serine-threonine phosphatase calcineurin, thereby
disrupting the signal transduction pathway emanating from the
T-cell receptor which leads to the transcription of the gene for
the cytokine IL-2.14 Recently a protein of hitherto unknown
function, which is bound by the FKBP12-rapamycin complex,
termed FRAP15a (RAFT,15b RAPT,15c mTOR15d) has been
described by several groups. Indirect evidence indicates that
this could be the relevant target for the immunosuppressive
activity of rapamycin.15a Not much is known about the precise
structural features required for the antiproliferative action of
rapamycin. The pipecolinyl ring is deeply buried in FKBP12.
The pyranose ring, the cyclohexyl ring and the region extending
from C34 to C28 (Figure 1) are in close contact with the protein,
with the three hydroxyls at positions 10, 28, and 40 involved
in hydrogen bonds. The region from C16 to C27, including
the characteristic C17-C22 triene, is exposed5a,c and therefore
available for interaction with FRAP. The triene appears to be
critical, as demonstrated by a derivative modified in that region,
which is still a potent PPIase inhibitor, but which is not
immunosuppressive.16 We report herein an X-ray crystal-
lographic analysis of 28-O-methylrapamycin (2) bound to
FKBP12 showing, as the sole major change with respect to the
FKBP12-rapamycin complex, a dramatic shift in the orientation
of the cyclohexyl ring, correlating with a loss of immunosup-
pressive activity.
Figure 2. Final 2Fo - Fc electron density map (calculated using all
data from 8 to 2.1 Å) contoured at 1σ for 28-O-methylrapamycin bound
to FKBP12. The locations of carbon atoms C16, C27, C28, C39, and
C40 (cf. Figure 1) are nearby to indicated numbers.
toward FKBP12. This result came as a surprise, as the C28-
hydroxyl had been shown to make a hydrogen bond with the
Glu-54 main chain carbonyl.5a,c Even if this single hydrogen
bond could be anticipated to contribute relatively little to the
overall binding energy, it was nevertheless expected that, due
to the close proximity of the C28-OH to the protein backbone,
O-methylation at this position would result in severe steric
interaction with FKBP12 and thus in strongly reduced binding.
The immunosuppressive activity of 2 was assessed in Vitro in
a two-way allogeneic mixed lymphocyte reaction (MLR);20 the
IC50 of 164 nM was more than 1300-fold higher than that
observed for rapamycin in the same assay (IC50 0.12 nM). In
order to gain insight into the structural basis for these results,
28-O-methylrapamycin (2) was cocrystallized with human
recombinant FKBP12. A high-resolution X-ray analysis (details
of data collection and refinement statistics are shown in Table
1) of the resulting complex yielded a structure with an estimated
accuracy of coordinates of 0.25 Å (from a Luzzati plot). The
conformation of the ligand was clearly defined, as can be seen
from the 2Fo - Fc electron density contoured at 1σ (data 8-2.1
Å) depicted in Figure 2.
Results and Discussion
In the course of a program aimed at establishing a structure-
activity relationship for rapamycin derivatives we wanted to
probe the role of the C28-hydroxyl and prepared 28-O-methyl-
rapamycin (2).17,18 In a competitive FKBP12-binding assay 2
inhibited binding of FK506 with an IC50 of 1.8 ( 0.6 nM
(rapamycin: IC50 1.1 ( 0.4 nM),19 indicating that methylation
of the C28-hydroxyl did not significantly affect the affinity
(12) (a) Dumont, F. J.; Staruch, M. J.; Koprak, S. L.; Melino, M. R.;
Sigal, N. H. J. Immunol. 1990, 144, 251-258 (b) Dumont, F. J.; Melino,
M. R.; Staruch, M. J.; Koprak, S. L.; Fischer, P. A.; Sigal, N. H. J. Immunol.
1990, 144, 1418-1424.
(13) (a) Schreiber, S. L.; Liu, J.; Albers, M. W.; Rosen, M. K.; Standaert,
R. F.; Wandless, T. J.; Somers, P. K. Tetrahedron 1992, 48, 2545-2558.
(b) Rosen, M. K.; Schreiber, S. L. Angew. Chem., Int. Ed. Engl. 1992, 31,
384-400. (c) Schreiber, S. L. Cell 1992, 70, 365-368. (d) Schreiber, S.
L.; Albers, M. W.; Brown, E. J. Acc. Chem. Res. 1993, 26, 412-420. (e)
Belshaw, P. J.; Meyer, S. D.; Johnson, D. D.; Romo, D.; Ikeda, Y.; Andrus,
M.; Alberg, D. G.; Schultz, L. W.; Clardy, J.; Schreiber, S. L. Synlett 1994,
381-392.
(17) 28-O-Methylrapamycin (2) was prepared in three steps from
rapamycin 1: (a) selective monosilylation of C40-OH with 1.1 equiv of
TBSOTf and 2.2 equiv of 2,6-lutidine in methylene chloride at 0 °C (83%);
(b) methylation of C28-OH using 3 equiv of trimethyloxonium tetrafluo-
roborate in the presence of 5 equiv proton sponge in methylene chloride at
room temperature (60%) (ref 18); (c) desilylation in 18:1 acetonitrile-HF‚-
pyridine at 0 °C (51%).
(18) (a) Diem, M. J.; Burow, D. F.; Fry, J. L. J. Org. Chem. 1977, 42,
1801-1802. (b) Evans, D. A.; Miller, S. J.; Ennis, M. D.; Ornstein, P. L.
J. Org. Chem. 1992, 57, 1067-1069. (c) Evans, D. A.; Ratz, A. M.; Huff,
B. E.; Sheppard, G. S. Tetrahedron Lett. 1994, 35, 7171-7172.
(19) In this assay, FK506 coupled to BSA is used to coat microtiter well
plates. Biotinylated human FKBP12 is allowed to bind to the immobilized
FK506 in the absence (negative control) or presence of a test sample. Bound
biotinylated FKBP12 is assessed by incubation with a streptavidin-alkaline
phosphatase conjugate, followed by incubation with p-nitrophenyl phosphate
as a substrate and determination of the OD at 405 nM. This competitive
binding assay is conceptually similar to the ones used by other authors to
determine the affinity of FK5068 and rapamycin9 to FKBP12. The affinity
of rapamycin to FKBP12 as determined in our assay is consistent with data
reported in the literature.9
(14) (a) Liu. J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.; Weissman,
I.; Schreiber, S. L. Cell 1991, 66, 807-815. (b) Liu, J.; Albers, M. W.;
Wandless, T. J.; Luan, S.; Alberg, D. G.; Belshaw, P. J.; Cohen, P.;
MacKintosh, C.; Klee, C. B.; Schreiber, S. L. Biochemistry 1992, 31, 3896-
3901. (c) Fruman, D. A.; Klee, C. B.; Bierer, B. E.; Burakoff, S. J. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 3686-3690. (d) Clipstone, N. A.; Crabtree,
G. R. Nature 1992, 357, 695-697. (e) O’Keefe, S. J.; Tamura, J.; Kincaid,
R. L.; Tocci, M. J.; O’Neill, E. A. Nature 1992 357, 692-694.
(15) (a) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith,
C. T.; Lane, W. S.; Schreiber, S. L. Nature 1994, 369, 756-758. (b)
Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S.
H. Cell 1994, 78, 35-43. (c) Chiu, M. I.; Katz, H.; Berlin, V. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 12574-12578. (d) Sabers, C. J.; Martin, M.
M.; Brunn, G. J.; Williams, J. M.; Dumont, F. J.; Wiederrecht, G.; Abraham,
R. T. J. Biol. Chem. 1995, 270, 815-822. (e) Chen, Y.; Chen, H.; Rhoad,
A. E.; Warner, L.; Caggiano, T. J.; Failli, A.; Zhang, H.; Hsiao, C.-L.;
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(20) Spleen cells from two genetically different donors are mixed and
cultivated for 4 days in the absence or presence of a serially diluted test
sample and rapamycin as a standard. 3H-thymidine is then added, the cells
are harvested after another 16 h incubation period, and 3H-thymidine-
incorporation, a measure for cell proliferation, is determined.
(16) Ocain, T. D.; Longhi, D.; Steffan, R. J.; Caccese, R. G.; Sehgal, S.
N. Biochem. Biophys. Res. Commun. 1993, 192, 1340-1346.