Creating diverse target-binding surfaces on FKBP12: Synthesis and evaluation of a rapamycin analogue library

Article abstract of DOI:10.1021/co200057n

FK506 and rapamycin are immunosuppressive drugs with a unique mode of action. Prior to binding to their protein targets, these drugs form a complex with an endogenous chaperone FK506-binding protein 12 (FKBP12). The resulting composite FK506-FKBP and rapamycin-FKBP binding surfaces recognize the relatively flat target surfaces of calcineurin and mTOR, respectively, with high affinity and specificity. To test whether this mode of action may be generalized to inhibit other protein targets, especially those that are challenging to inhibit by conventional small molecules, we have developed a parallel synthesis method to generate a 200-member library of bifunctional cyclic peptides as FK506 and rapamycin analogues, which were referred to as "rapalogs". Each rapalog consists of a common FKBP-binding moiety and a variable effector domain. The rapalogs were tested for binding to FKBP12 by a fluorescence polarization competition assay. Our results show that FKBP12 binds to most of the rapalogs with high affinity (K_I values in the nanomolar to low micromolar range), creating a large repertoire of composite surfaces for potential recognition of macromolecular targets such as proteins.

Full text of DOI:10.1021/co200057n

RESEARCH ARTICLE
pubs.acs.org/acscombsci
Creating Diverse Target-Binding Surfaces on FKBP12: Synthesis and
Evaluation of a Rapamycin Analogue Library
Xianghong Wu,† Lisheng Wang,‡ Yaohua Han,X Nicholas Regan,|| Pui-Kai Li,||
Miguel A. Villalona,§,^ Xiche Hu,X Roger Briesewitz,*,‡,^ and Dehua Pei*,†,^
†Department of Chemistry, ‡Department of Pharmacology, §Division of Medical Oncology, Department of Internal Medicine,
Division of Medicinal Chemistry, College of Pharmacy, and ^The Ohio State Comprehensive Cancer Center,
The Ohio State University, Columbus, Ohio 43210, United States
XDepartment of Chemistry, University of Toledo, Toledo, Ohio 43606, United States
S Supporting Information
b
ABSTRACT: FK506 and rapamycin are immunosuppressive
drugs with a unique mode of action. Prior to binding to their
protein targets, these drugs form a complex with an endogenous
chaperone FK506-binding protein 12 (FKBP12). The resulting
composite FK506-FKBP and rapamycin-FKBP binding surfaces
recognize the relatively flat target surfaces of calcineurin and
mTOR, respectively, with high affinity and specificity. To test
whether this mode of action may be generalized to inhibit other
protein targets, especially those that are challenging to inhibit by
conventional small molecules, we have developed a parallel
synthesis method to generate a 200-member library of bifunc-
tional cyclic peptides as FK506 and rapamycin analogues, which were referred to as “rapalogs”. Each rapalog consists of a common
FKBP-binding moiety and a variable effector domain. The rapalogs were tested for binding to FKBP12 by a fluorescence polarization
competition assay. Our results show that FKBP12 binds to most of the rapalogs with high affinity (KI values in the nanomolar to low
micromolar range), creating a large repertoire of composite surfaces for potential recognition of macromolecular targets such as proteins.
KEYWORDS: cyclic peptides, FKBP, FK506, rapamycin, structure-activity relationship
’ INTRODUCTION
part (the effector domain) interacts with the target protein and
provides target specificity. The cocrystal structures of FK506-
FKBP12-calcineurin and rapamycin-FKBP12-mTOR reveal that
the composite FKBP12-drug binding surfaces are large and
bind to relatively flat surfaces on their target proteins.9ꢀ11 The
two-dimensional binding character of the composite FKBP-drug
binding surface is in stark contrast to that of most small-molecule
drugs, which typically bind in deep binding pockets that provide
three-dimensional binding spaces. A deep binding pocket in a
target protein allows for the establishment of many molecular
interactions between a small molecule and the protein so that
high affinity binding can occur. Proteins with distinct binding
pockets are usually enzymes, ion channels, and receptors. These
classes of proteins make up the majority of all drug targets that
have already been exploited.12 On the other hand, many potential
drug targets exert their biological activities through proteinꢀ
protein interactions. Proteinꢀprotein interactions often take
place via large and flat surfaces that are considered “undruggable”
because small molecules cannot establish sufficient molecular
FK506 and rapamycin are natural products that are produced
by soil microorganisms. Both molecules are effective immuno-
suppressive drugs that are used for organ transplantation.1,2 In
addition, rapamycin has shown activity as an anticancer agent.3
The molecular target of FK506 is the protein phosphatase cal-
cineurin whereas that of rapamycin is a protein kinase, mamma-
lian target of rapamycin (mTOR). Unlike most small-molecules
drugs, FK506andrapamycin bythemselves show only verymodest
affinity for their respective targets. To be efficacious, FK506 and
rapamycin must first form a binary complex with the endogenous
FK506-binding protein 12 (FKBP12), a peptidyl-prolyl isomer-
ase that is widely expressed in human cells. The resulting FK506-
FKBP12 and rapamycin-FKBP12 complexes bind with high affinity
and specificity and inhibit the enzymatic activities of calcineurin and
mTOR, respectively.4ꢀ7 Compared to the drugs alone, the drug-
FKBP12 binary complexes show increased binding affinities be-
cause the FKBP12 protein surface also makes favorable molecular
interactions with the target protein surface.8 Thus, by binding to
FKBP12, FK506 and rapamycin create composite binding surfaces
that allow both drug-target and FKBP12-target interactions.
FK506 and rapamycin are bifunctional molecules (Figure 1).
One part of the molecules binds to FKBP12, while the second
Received: March 24, 2011
Revised:
June 17, 2011
Published: July 19, 2011
r
2011 American Chemical Society
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RESEARCH ARTICLE
Figure 1. Structures of FK506, rapamycin, meridamycin, and rapalogs SLF, 1 and 2.
interactions with these flat target surfaces to bind with high
affinity and selectivity.
calcineurin/mTOR-binding motifs of FK506/rapamycin (com-
pound 2, Figure 1). When bound to FKBP12, these rapalogs
should create diverse composite binding surfaces on FKBP12,
which may be screened for binding to otherwise intractable
protein targets.
Through the formation of composite drug-FKBP binding
surfaces, Nature has found a solution to successfully target flat
protein surfaces with small molecules and has repeatedly ex-
ploited this approach. In addition to FK506 and rapamycin,
meridamycin, a macrolide produced by the soil bacterium Strep-
tomyces hygroscopicus, is also a high-affinity FKBP ligand that ant-
agonizes the immunosuppressive activity of FK506.13 The mo-
lecular target of the meridamycin-FKBP complex is currently
unknown. Likewise, antascomicins A-E bind to FKBP12 with
about the same nanomolar affinity as FK506 and rapamycin and
the antascomycin-FKBP complexes inhibits the function of a yet
unknown cellular target(s).14 Nature has also used other proteins
to form composite binding surfaces. One such example is the
peptidyl-prolyl isomerase cyclophilin A (CypA). The natural
products that bind to cyclophilin and form drug-chaperon com-
plexes include cyclosporin A (CsA) and sanglifehrin A. CsA is a
cyclic undecapeptide, and the CsA-CypA complex creates a com-
posite surface that binds and inhibits calcineurin.15 Both CsA and
CypA make molecular contacts with calcineurin and contribute
to the overall affinity and specificity.16 Sanglifehrin A has shown
’ RESULTS AND DISCUSSION
Our objective is to synthesize a large library of rapalogs that
retain the ability to bind to FKBP12, but each have a different
effector domain to create a diverse array of composite surfaces for
screening against a target protein. To achieve this goal, we must
first devise a minimal structural unit that is capable of binding to
FKBP12 with high affinity and specificity. Next, we need to re-
place the effector domain of rapamycin with structurally diverse
moieties. In the present work, we chose peptides as the effector
domains, because peptides of diverse structures can be generated
from a small set of amino-acid building blocks and are synthe-
tically accessible, although other types of structures may also
be used.
Identification of a Minimal FKBP12-Binding Motif. Many
FK506 and rapamycin analogues have previously been synthe-
sized and tested, resulting in ample SAR data with respect to
FKBP12 binding. For example, the work of Holt and co-
workers18 established that a macrocycle containing a 3,3-dimeth-
yl-2-ketobutyryl-L-pipecolinate (Dkb-Pip) core (compound 1a,
Figure 1) retains much of the FKBP-binding activity of rapamy-
cin (apparent Ki of 30 nM). Addition of an (R)-1-(phenylethyl)-
benzyl group (1b) to the core further increases the binding
affinity by ∼30-fold. To facilitate later library synthesis, we elected
to replace the (R)-1-(phenylethyl)benzyl moiety with a simpler,
more readily available building block such as an amino acid (R1 in
2, Figure 1). In addition, we felt that the residue immediately
N-terminal to the Dkb-Pip core (R2 in 2, Figure 1) might also
affect FKBP12 binding. To identify a competent, minimal
FKBP12-binding motif, we designed 15 cyclic peptides that
featured 10 different R1 residues including L-alanine, D-alanine,
exceptionally high affinity for CypA in a cell free assay (IC50
=
∼7 nM).17 The Sanglifehrin-CypA complex has immunosup-
pressive activity, although the target of the complex remains to be
identified.
Despite the different effector domains of FK506, rapamycin,
meridamycin, and antascomycins, which allow them to bind to
different target proteins, the four compounds possess a common
FKBP-binding moiety, which consists of a triketo pipecolyl core
(Figure 1). We hypothesize that it may be possible to generalize
the mechanism-of-action of FK506 and rapamycin to target
otherwise intractable protein sites. To test this hypothesis,
we have developed a synthetic route that allows the rapid syn-
thesis of large libraries of cyclic bifunctional molecules. These
synthetic molecules (or rapalogs) consist of a common FKBP12-
binding moiety but different effector domains which replace the
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RESEARCH ARTICLE
Table 1. SAR of Rapalogs 2aꢀya
Scheme 1. Synthetic Route for Key Building Blocks of
Rapalogs Librarya
building blocks
R2 R3 R4
compound
R1
R5
R6 IC50 (μM)
2a
L-Ala
D-Ala
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
240 ( 7
298 ( 80
303 ( 27
310 ( 2
13 ( 4
2b
2c
D-Abu
a Reagents and conditions: (a) 5 equiv of allyl bromide, 2 equiv of
Cs2CO3, acetone, 2 h; (b) 20% piperidine in DCM, 20 min; (c) 1.5 equiv
of dihydro-4,4-dimethyl-2,3-furandione, 10% DMAP, Ar gas, toluene,
reflux, overnight; (d) DIC, Fmoc- amino acids (R2), 5% DMAP,1 h;
(e) 5% Pd(Ph3P)4, 3 equiv of N-methylaniline, THF.
2d
D-β-Glu
2e
D-β-homoPhe D-Ala L-Ala L-Ala
2f
D-homoPhe
L-β-Ile
L-Ile
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
D-Ala L-Ala L-Ala
147 ( 23
165 ( 42
316 ( 46
266 ( 46
26 ( 3
2g
2h
Synthesis of cyclic peptides 2aꢀn began with the preparation
of a key building block 3 (Scheme 1). Starting from the com-
mercially available N-Fmoc-L-pipecolinic acid, its carboxyl group
was protected as an allyl ester by treatment with allyl bromide
under basic conditions. Removal of the Fmoc group with pipe-
ridinegave amine 4, whichwasacylated with dihydro-4,4-dimethyl-
2,3-furandione to give alcohol 5.18 Coupling of alcohol 5 with
three different N-Fmoc amino acids followed by allyl removal
with Pd(PPh3)4 afforded the building block 3aꢀc in good yields
(∼45% overall). Next, the 15 cyclic peptides were synthesized in
parallel on Rink amide resin (Scheme 2). N-Fmoc-Glu-R-allyl ester
was coupled to the amino group of Rink resin using O-benzotriazole-
N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU)
as the coupling agent. After removal of the Fmoc group, the N-
terminal amine was acylated with the 10 different N-Fmoc amino
acids (R1) described above. Subsequent addition of building
blocks 3aꢀc and L-Ala-L-Ala were carried out using standard
peptide chemistry. It should be noted that following the coupling
of R1 and R3 residues, removal of the Fmoc groups with pipe-
ridine should be carried out quickly and the resulting amines
should be immediately acylated with the next amino acid to avoid
undesired cyclization at the ester moieties. Prior to peptide cycli-
zation, the C-terminal allyl group was removed by treatment with
a catalytic amount of Pd(PPh3)4 in the presence of N-methylani-
line, and the N-terminal Fmoc group was removed by piperidine.
Peptide cyclization was achieved by using benzotriazole-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBop)
as the coupling reagent. Finally, treatment with 50% trifluoroa-
cetic acid (TFA) in dichloromethane released the peptides from
the resin and deprotected the amino acid side chains. The resulting
crude peptides were quickly passed through a silica gel column to
remove the salts and used directly in activity assays.
2i
D-Ile
2j
L-β-homoPhe L-Phe L-Ala L-Ala
D-β-homoPhe L-Phe L-Ala L-Ala
2k
18 ( 2
2l
L-β-Ile
L-Phe L-Ala L-Ala
L-Phe L-Ala L-Ala
250 ( 57
386 ( 43
4 ( 1
2m
2n
D-β-Glu
D-β-homoPhe L-Thr L-Ala L-Ala
2o
D-β-homoPhe D-Ala L-Ala D-Ala L-Ala
D-β-homoPhe D-Ala L-Ala L-Ala L-Ala
D-β-homoPhe D-Ala D-Ala D-Ala D-Ala
D-β-homoPhe L-Thr L-Ala L-Ala L-Ala
D-β-homoPhe L-Phe L-Ala D-Ala L-Ala
D-β-homoPhe L-Phe L-Ala L-Ala L-Ala
D-β-homoPhe L-Thr
20 ( 6
2p
6 ( 2
2q
13 ( 2
2r
9 ( 3
2s
31 ( 6
2t
10 ( 1
2u
37 ( 10
18 ( 6
2v
D-β-homoPhe D-Ala
2w
2x
D-β-homoPhe D-Ala L-Ala D-Ala L-Ala L-Ala
D-β-homoPhe D-Ala L-Ala L-Ala L-Ala L-Ala
D-β-homoPhe L-Phe L-Ala D-Ala L-Ala L-Ala
13 ( 2
5 ( 1
2y
8 ( 2
FK506
Rapamycin
SLF
0.22 ( 0.08
0.057 ( 0.02
2.6 ( 1.5
a The IC50 values reported were mean ( SD from multiple independent
titration experiments (n) (rapalogs, n = 2ꢀ4; FK506, n = 5; rapamycin,
n = 3; SLF, n = 18).
(S)-2-aminobutyric acid (D-Abu), (R)-3-aminoadipic acid (D-β-
Glu), (R)-3-amino-5-phenylpentanoic acid (D-β-homoPhe), (S)-3-
amino-5-phenylpentanoic acid (L-β-homoPhe), D-homopheny-
lalanine (D-homoPhe), L-β-isoleucine (L-β-Ile), L-isoleucine, and
D-isoleucine and three R2 residues (D-Ala, L-Thr, and L-Phe)
(Table 1, compounds 2aꢀn). To facilitate solid-phase synthesis,
an invariant glutamine was added to the C-terminal side of the R1
residue, for the purpose of backbone peptide cyclization and
providing an anchor for attachment to the solid support. For this
set of compounds, the dipeptide Ala-Ala was used as the effector
domain (Figure 1).
Peptides 2aꢀn were tested for binding to FKBP12 by a fluor-
escence polarization competition assay.19,20 A previously reported
synthetic ligand of FKBP1221 (SLF in Figure 1), was labeled with
the fluorescent dye fluorescein.20 Binding of the labeled ligand to
FKBP12 increases its fluorescence anisotropy (FA) value. Addi-
tion of an unlabeled rapalog to the reaction inhibits the binding of
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Scheme 2. Solid-Phase Synthesis of Rapalogs 2aꢀna
a Reagents and conditions: (a) N-Fmoc-Glu-R-allyl ester, HOBT, HBTU, NMM, 2 h; (b) 20% piperidine in DMF; (c) N-Fmoc-amino acids (R1),
HOBt, HBTU, NMM, 2 h; (d) building block 3, HOBt, HBTU, NMM, 2 h; (e) standard Fmoc/HBTU chemistry; (f) 20% Pd(Ph3P)4, 9 eq
N-methylaniline, THF; (g) PyBop, HOBT, NMM, overnight; (h) 50% TFA in DCM.
(Table 1). This is not surprising, since D-β-homoPhe is structu-
rally similar to the 2-cyclohexylethyl-β-hydroxyketone moiety of
rapamycin and the (R)-1-(phenylethyl)benzyl moiety in SLF and
compound 1b (Figure 1). Among the three R2 residues examined,
the smaller residues (L-Thr and D-Ala) were somewhat more ef-
fective than L-Phe (Table 1 compare compounds 2e, 2k, and 2n).
Therefore, we chose the tetrapeptide L-Thr (or D-Ala)-Dkb-Pip-D-
β-homoPhe as the minimal structural unit for binding to FKBP12.
Effect of Ring Size and Building Blocks in the Effector
Domain. For our approach to be successful, FKBP12 must be
able to tolerate structurally diverse effector domains of different
ring sizes and building blocks. As an initial test of the feasibility,
Figure 2. Representative titration curves showing the competition of
we synthesized compounds 2oꢀy, which all contain L-Thr (or D-
FK506 or rapalogs 2oꢀ2y (1ꢀ200 μM) with SLF-fluorescein (100 nM)
Ala, L-Phe)-Dkb-Pip-D-β-homoPhe as the FKBP12-binding do-
for binding to FKBP12 (200 nM) as monitored by fluorescence
main but have zero to four L- or D-Ala residues as the effector
anisotropy (FA). The percentage of FA was plotted against the rapalog
domain (Table 1). All of the compounds bound to FKBP12 with
high affinity with IC50 values in the range of 5ꢀ37 μM. In com-
parison, rapamycin, FK506, and SLF had IC50 values of 0.057,
0.22, and 2.6 μM, respectively, under the same conditions. These
results suggest that FKBP12 can tolerate a wide variety of effector
domains including different ring sizes and both L- and D-amino
acids as building blocks.
Synthesis and Evaluation of a Rapalog Library. To gain
further insight into the SAR with respect to the influence of the
effector domain structure on FKBP12-binding activity, we syn-
thesized a 200-member rapalog library on Rink amide resin in
concentration (in logarithmic scale).
SLF to FKBP12 and decreases the FA value (Figure 2). By using
this competition assay in the presence of increasing concentra-
tions of the rapalogs 2aꢀn, we determined the IC50 values
(concentrations of rapalogs at which the FA value of SLF is
reduced by 50%) for the peptides (Table 1). The results show
that a D-β-homoPhe at the R1 position resulted in the best binding
affinity for FKBP12 (IC50 = 4ꢀ18 μM) and L-β-homoPhe was
slightly less effective (IC50 = 26 μM), whereas the other eight
building blocks were much less effective (IC50 g 147 μM)
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Table 2. Binding Affinities (IC50, μM) of Rapalog Library Members to FKBP12a
R3
R4
D-Thr
Gly
Ala
D-Val
Pro
Lys
Trp
Asp
D-Phe
Nle
D-Thr
11
12
11
12
11
12
11
12
11
12
11
12
11
12
11
12
11
12
11
12
17 ( 1
5 ( 1
27 ( 2
14 ( 4
25 ( 7
26 ( 7
28 ( 13
14 ( 5
5 ( 1
15 ( 1
4 ( 1
ND
18 ( 5
ND
19 ( 1
>100
>100
37 ( 10
8 ( 3
18 ( 4
9 ( 1
9 ( 3
ND
12 ( 1
25 ( 5
24 ( 9
10 ( 1
17 ( 6
9 ( 3
10 ( 1
13 ( 7
21 ( 2
3 ( 1
22 ( 4
4 ( 1
8 ( 2
15 ( 4
19 ( 4
2 ( 1
4 ( 1
4 ( 1
ND
65 ( 4
28 ( 2
21 ( 6
95 ( 25
ND
>100
80 ( 35
30 ( 7
5 ( 2
57 ( 18
32 ( 6
38 ( 11
28 ( 9
71 ( 17
48 ( 8
36 ( 17
71 ( 15
26 ( 7
67 ( 10
>100
Gly
13 ( 5
16 ( 2
23 ( 4
ND
25 ( 6
70 ( 16
14 ( 4
41 ( 4
>100
17 ( 1
49 ( 12
>100
5 ( 2
ND
Ala
63 ( 11
>100
6 ( 1
62 ( 2
35 ( 6
33 ( 10
21 ( 3
>100
57 ( 6
12 ( 5
>100
8 ( 4
D-Val
Pro
55 ( 5
89 ( 16
41 ( 8
38 ( 11
62 ( 8
>100
12 ( 4
52 ( 25
36 ( 8
29 ( 5
>100
9 ( 3
8 ( 2
21 ( 6
ND
>100
2 ( 1
>100
>100
ND
64 ( 10
43 ( 1
80 ( 28
ND
27 ( 6
ND
46 ( 2
87 ( 5
47 ( 10
45 ( 12
ND
6 ( 3
Lys
93 ( 31
22 ( 4
12 ( 6
14 ( 1
7 ( 2
58 ( 10
15 ( 5
41 ( 9
66 ( 2
13 ( 1
60 ( 4
71 ( 23
>100
12 ( 1
ND
>100
37 ( 6
53 ( 15
ND
Trp
Asp
D-Phe
Nle
75 ( 16
89 ( 12
13 ( 2
18 ( 5
50 ( 16
81 ( 16
72 ( 19
>100
16 ( 2
6 ( 1
13 ( 4
5 ( 2
84 ( 24
ND
34 ( 10
15 ( 3
11 ( 2
50 ( 17
38 ( 14
50 ( 11
68 ( 12
23 ( 13
21 ( 4
42 ( 10
4 ( 1
4 ( 2
69 ( 6
6 ( 3
14 ( 1
11 ( 3
13 ( 2
44 ( 6
5 ( 1
22 ( 1
15 ( 1
21 ( 6
34 ( 5
35 ( 1
33 ( 5
ND
10 ( 3
7 ( 1
12 ( 2
40 ( 15
38 ( 6
24 ( 5
24 ( 6
10 ( 2
ND
>100
>100
ND
31 ( 9
35 ( 11
13 ( 1
79 ( 2
>100
28 ( 6
17 ( 2
40 ( 11
8 ( 1
a The IC50 values reported were mean ( SD from 2 to 4 independent titration experiments.
parallel by following a procedure similar to that described in
Scheme 2. All of the compounds contained the tetrapeptide D-
Ala-Dkb-Pip-D-β-homoPhe as the FKBP12-binding domain. For
half of the library members (compound 11), a dipeptide of
random sequence (R3 and R4) was employed as the effector
domain, while a tripeptide (R3-R4-L-Ala) was used as the effector
domain for the other half (compound 12) (Table 2). At each
random position (R3 and R4), 10 different amino acids (D-Thr,
Gly, Ala, D-Val, Pro, Lys, Trp, Asp, D-Phe, and Nle) were used.
After synthesis, side-chain deprotection, and cleavage from the
resin, the rapalogs were analyzed by matrix-assisted laser desorp-
tion ionization-time-of-flight mass spectrometry (MALDI-TOF
MS) and each showed a single species with the expected m/z
value (Supporting Information, Figures S3 and S4).
Table 3. Comparison of the Binding Affinities of Crude vs
Purified Rapalogs to FKBP12
crude sample
purified sample
Rapalog (R3, R4) IC50 (μM)a KI (μM) IC50 (μM)a KI (μM)
11a (Asp, D-Phe)
15 ( 1
0.48 ( 0.04 6.3 ( 1
0.20 ( 0.01
12a (2p) (Ala, Ala) 6.0 ( 2.0 0.19 ( 0.03 4.9 ( 1.4 0.15 ( 0.04
12b (Asp, Ala)
12c (Gly, Ala)
5.0 ( 2.0 0.16 ( 0.06 7.0 ( 2.1 0.22 ( 0.10
4.7 ( 2.1 0.16 ( 0.06 6.3 ( 2.8 0.20 ( 0.09
a The IC50 values reported were mean ( SD from 2 to 4 independent
titration experiments.
The 200 compounds were individually assayed for their binding
affinities to FKBP12 by the FA competition assay and their IC50
values are listed in Table 2. Evaluation of the 200 compounds
revealed several important trends. First, out of the 182 com-
pounds whose IC50 values were reliably determined, the great
majority of them (163 compounds) bound to FKBP12 with
excellent to respectable affinities (IC50 of 2ꢀ93 μM). Some of
the compounds were more potent than SLF and only ∼10-fold
less potent than FK506 (IC50 = 0.22 μM). Second, although for a
particular peptide sequence the two different ring sizes may result
in as much as 10-fold difference in the IC50 values, in general, the
compounds from the two sublibraries of different ring sizes have
similar potencies, consistent with our earlier observations. Third,
at the R3 position, an Asp resulted in the most potent ligands
against FKBP12 (IC50 = 2.0ꢀ60 μM), whereas Lys generally
gave poorer activities. Finally, the R4 residue had minimal effect
on the binding affinity and all 10 amino acids were well tolerated.
The only exception was Asp, which gave high activities for most
compounds, even when the R3 residue was not optimal (e.g.,
when R3 was Lys).
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Figure 3. Top (a) and side view (b) of the overall FKBP12-rapalog 11b complex. FKBP12 is rendered as a surface with a probe radius of 1.4 Å, and the
rapalog is in a licorice representation with hydrogen atoms omitted for clarity. C, O, and N atoms are colored in cyan, red, and blue, respectively.
(c) Stereo view of the complex between FKBP12 (shown in silver color except for the O and N atoms involved in binding) and rapalog 11c [color
scheme: C (cyan), O (red), and N (blue)]. Dashed lines indicate intermolecular hydrogen bonding, electrostatic, and CH-π interactions between the
rapalog and the active-site residues of FKBP12.
To confirm the library screening results, which were carried
out with crude samples, we chose four of the rapalogs that had
relatively high activities to FKBP12 and purified them to homo-
geneity by HPLC [compounds 11a, 12a (or 2p), 12b, and 12c in
Table 3). The pure samples were assayed for binding to FKBP12
under the same conditions to give IC50 values of 6.3, 4.9, 7.0, and
6.3 μM, respectively (Table 3). These values were e2-fold dif-
ferent from those derived from the crude samples; these differ-
ences were well within the margin of error for the competition
assay method.
Molecular Modeling of the FKBP-Rapalog Complexes. To
gain some structural insight into the above observed SAR,
molecular modeling was carried out for the binding of FKBP12
to rapalogs 11b (R3 = Ala, R4 = Asp) and 11c (R3 = Asp, R4 =
Ala) using a two stage protocol as described under Experimental
Section. In the model, the D-Ala-Dkb-Pip-D-β-homoPhe FKBP-
binding motif is mostly buried in the active site, whereas the ef-
fector domain is largely exposed to the solvent (Figure 3a and b).
A key FKBP-rapalog binding interaction occurs in the form of
CH-π interactions between the side chain of Pip and the hydro-
phobic pocket formed by the side chains of Tyr-26, Phe-46, Trp-
59, and Phe-99 of FKBP12 (Figure 3c). The carbonyl groups of
Dkb and Pip also make two key hydrogen bonds with the side
chain of Tyr-82 and the mainchain ꢀNH of Ile-56, respectively.
The pro-(S) methyl group of Dkb makes hydrophobic contact
with Phe-36 side chain, while its pro-(R) methyl group interacts
with the side chain of His-87. In addition, the phenyl ring of
D-β-homoPhe contacts the side chains of Tyr-82 and His-87. The
side chain of the D-Ala (R2) points away from the protein, con-
sistent with the tolerance of different side chains at this position
(e.g., D-Ala, Thr, and Phe). Some of the effector domain residues
also contribute to the overall binding affinity. The side chain of
Asp atposition R3 is physically proximal toand engages in chargeꢀ
charge interaction with the guanidinium group of FKBP Arg-42
(Figure 3c). A similar electrostatic interaction also occurs when
an Asp or Glu is placed at the R4 position (Figure 3a and b). This
is in excellent agreement with our experimental observations
(Table 2). Finally, the side chain amide of the anchoring Gln is
3.82 Å from the side chain of Glu-54, suggesting that they may be
involved in electrostatic interactions as well. Overall, the mod-
eled binding mode explains why FKBP12 is capable of binding to
the wide variety of rapalogs in this work.
Comparison with Previous Approaches. The unique mode-
of-action of FK506, rapamycin, and other natural products has
inspired other investigators as well as us to develop several appro-
aches to modulate the biological activity of small molecules. For
example, we demonstrated that the affinity of a linear peptide for
the Fyn SH2 domain can be enhanced when the peptide is
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ACS Combinatorial Science
RESEARCH ARTICLE
coupled to an FKBP ligand and bound to FKBP.22 This affinity
enhancement is likely due to the establishment of favorable
FKBP-SH2 interactions. In another application it was shown that
conjugation of an FKBP ligand to Congo Red, a dye molecule
that binds to beta-amyloid peptide, improved the ability of the
dye molecule to disrupt amyloid plaque formation.23 Presum-
ably, binding of FKBP to the molecule creates a steric block
FKBP that hinders the beta-amyloid peptide aggregation. In a
third experiment, it was shown that linking a HIV protease
inhibitor to an FKBP ligand increased the half-life of the drug in
mice.24 It was thought that the association of the bifunctional
molecule with FKBP in mammalian cells may have slowed the
molecule’s metabolism and excretion. These previous studies
required a pre-existing ligand with high affinity and specificity to
the target of interest; however, for many high-value targets such
as the flat surfaces involved in proteinꢀprotein interactions, no
such ligand is available (or possible). To recognize these flat
surfaces, which are generally considered as “undruggable” by the
conventional small-molecule approaches, we are exploring an
alternative approach to recapitulate the mode of action of rap-
amycin and other natural products. Our strategy is to generate a
large library of bifunctional cyclic peptides, which contain a
common FKBP-binding motif on one side and diverse effector
domains on the other side. These cyclic peptides should bind to
FKBP12 via their shared FKBP-binding motif to form a library of
FKBP-cyclic peptide complexes, each of which displays a unique
and relatively flat surface formed by both the FKBP protein and
the variable face of the cyclic peptide. Screening of the library of
composite surfaces against a target surface may identify com-
plexes that can inhibit proteinꢀprotein interactions. In the cur-
rent study, we have shown that the tetrapeptide D-Ala-Dkb-Pip-
D-β-homoPhe acts as an effective minimal structural motif that
binds to FKBP12 with high affinity and specificity. When the
motif was incorporated into ∼200 cyclic peptides of different
ring sizes and amino acid building blocks, the vast majority of
them (177 out of the 196 peptides tested) were capable of
binding to FKBP12 with IC50 values of 2ꢀ95 μM as determined
by the FKBP competition assay, corresponding to KI values of
60ꢀ3000 nM (Table 1 and 2). By using the same assay, a KI value
of 7.0 nM was obtained for FK506, in reasonable agreement with
the KI value of 1ꢀ2 nM previously reported for inhibition of
FKBP rotamase activity by FK506.18,25 Thus, the binding affini-
ties of our rapalogs for FKBP12 are generally one to 3 orders of
magnitude lower than that of FK506. Most importantly, our
results as well as the previously reported examples18,26,27 demon-
strate that essentially any cyclic peptide or other types of
macrocycles should be able to bind to FKBP12, as long as they
contain a FKBP-binding motif such as the tetrapeptide identified
in this work in a proper conformation. The solid-phase synthesis
method we have developed may be easily adapted for the
combinatorial synthesis of one-bead-one-compound (OBOC)
libraries. In fact, the combinatorial synthesis and screening of
large OBOC libraries of rapalogs are currently underway in our
laboratories and will be reported in due course.
molecule drugs, proteinꢀprotein interaction often involves large,
flat surfaces, which are challenging targets for traditional small
molecules. As has been repeatedly demonstrated in nature, the
drugꢀprotein composite surfaces are flat and sufficiently large in
areas and should be able to bind to the flat surfaces involved in
proteinꢀprotein interactions. Second, the rapalog-FKBP com-
posite surfaces may be utilized for isoform-specific inhibition of
proteins and enzymes that belong to large families of structurally
similar proteins such as protein kinases and protein tyrosine
phosphatases (PTPs). The human genome encodes 500 protein
kinases and ∼100 PTPs.29,30 Each enzyme family has a highly
conserved active site, making it difficult to develop specific in-
hibitors against a particular kinase or PTP. Indeed, most of the
kinase inhibitors so far designed target the ATP-binding site. But
because the ATP-binding site is conserved, few of these inhibi-
tors are truly selective for the intended kinase target. For the
same reason, few selective PTP inhibitors are currently available.
On the other hand, for both kinases and PTPs, the protein sur-
faces outside the active site are highly divergent. It is conceivable
to design or screen for a rapalog-FKBP composite surface that
recognizes a unique surface area outside (but near) the kinase/
PTP active site. While binding to such a site by a conventional
small molecule may not have significant effect on the biological
activity of the enzymes, association with a rapalog-FKBP com-
plex would create a steric block to the active site, preventing
the access of kinase/PTP substrates, which are large protein
molecules. This is precisely the mechanism by which FK506
and cyclosporin A inhibit the serine/threonine phosphatase
calcineurin.9
’ EXPERIMENTAL SECTION
Materials. N-Fmoc amino acids were purchased from Ad-
vanced ChemTech (Louisville, KY), Peptides International
(Louisville, KY), or NovaBiochem (La Jolla, CA). HBTU and
1-hydroxybenzotriazole hydrate (HOBt) were from Peptides
International. TFA was purchased from Sigma-Aldrich. Dihy-
dro-4,4-dimethyl-2,3-furandione, tetrakis(triphenylphosphine)-
palladium, allyl bromide, cesium carbonate, and solvents were
purchased from Aldrich, Fisher Scientific (Pittsburgh, PA), or
VWR (West Chester, PA). Rink resin (0.20 mmol/g, 100ꢀ
200 μm) was purchased from Advanced ChemTech. 1H and 13
C
NMR spectra were recorded on a 400 MHz spectrometer
(operated at 400 and 100 MHz, respectively). Chemical shifts
are reported as δ values (ppm). NMR data were collected by
using DMSO-d6 or CDCl3 as solvent. Reaction progress in
solution phase was monitored by thin-layer chromatography
(TLC), using 0.25 mm silica gel plates with visualization by
irradiation with a UV lamp. Reaction progress in solid phase was
monitored by ninhydrin test, whenever possible. HRMS data
were collected with electrospray ionization mass spectrometry or
direct probe ionization. MALDI-TOF mass analysis was per-
formed on a Bruker III MALDI-TOF instrument in an auto-
mated manner at Campus Chemical Instrument Center of The
Ohio State University. The data obtained were analyzed by either
Moverz software (Proteometrics LLC, Winnipeg, Canada) or
Bruker Daltonics flexAnalysis 2.4 (Bruker Daltonic GmbH,
Germany). Flash column chromatography was carried out on
silica gel 40.
Potential Applications. We envision at least two important
applications for the above rapalog libraries. First, the rapalog-
FKBP composite surfaces may be screened for inhibition of
proteinꢀprotein interactions. Proteinꢀprotein interaction is
ubiquitous in biology and provides an exciting class of largely
unexploited drug targets.28 Unlike the conventional small-
molecule drug targets, which typically contain deep pockets or
clefts to make three-dimensional interactions with the small-
Allyl Nr-Fluorenyloxycarbonyl-L-Pipecolate. To a solution
of 1.0 equiv of Fmoc-L-pipecolinic acid (0.5 mmol, 176 mg),
dissolved in 10 mL of acetone (saturated by K2CO3), 2 equiv of
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RESEARCH ARTICLE
Cs2CO3 (1 mmol, 326 mg) and 5 equiv of allyl bromide
(2.5 mmol, 302 mg) were added. The solution was stirred for
4 h at room temperature. The crude product was purified by
using flash column chromatography on a silica gel column with
hexane/ethyl acetate (2:1) as eluent to give a clear oil (yield
99%): 1H NMR (400 MHz CDCl3) δ1.30ꢀ1.74 (m, 6H), 2.30
(t, 1H), 3.10 (dt, 1H), 4.12 (t, 1H), 4.33 (m, 3H), 4.60 (dd, 2H),
5.30 (dd, 2H), 5.95 (m, 1H), 7.28ꢀ7.81(m, 8H). 13C NMR (100
MHz, CDCl3) δ 171.3, 156.5, 144.1, 141.3, 131.8, 127.7, 127.1,
125.1, 120.0, 118.6, 67.7, 65.7, 54.5, 47.3, 41.8, 26.9, 24.7, 20.7.
HRMS (ESI): calcd for C24H25NO4Na (M + Na+) 414.1682,
found 414.1697.
4.16ꢀ446 (m, 6H), 7.10ꢀ7.78 (m, 13H). 13C NMR (100 MHz,
CDCl3) δ 205.0, 204.0, 174.1, 172.0, 170.4, 166.3, 158.0, 156.8,
143.7, 141.3, 137.8, 128.6, 127.8, 127.2, 125.1, 120.0, 118.3, 74.1,
70.0, 67.8, 67.1, 47.1, 46.6, 42.4, 40.6, 38.9, 30.9, 19.3. HRMS
(ESI) calcd for C36H38N2O8Na (M + Na+) 649.2526, found
649.2529.
3c. 1H NMR (400 MHz CDCl3) δ 1.14 (s, 9H), 1.17 (s, 3H),
1.47 (s, 6H), 1.23ꢀ1.79 (m, 6H), 3.10 (m, 1H), 3.30 (m, 1H),
3.51 (m,1H), 4.01ꢀ4.59 (m, 7H), 5.30 (bs, 1H), 7.19ꢀ7.80 (m,
8H). 13C NMR (100 MHz, CDCl3) δ 205.2, 204.4, 174.0, 172.4,
170.4, 166.3, 158.0, 156.8, 143.7, 141.3, 137.8, 128.6, 127.8,
127.2, 125.1, 120.0, 118.3, 74.1, 70.0, 67.8, 59.0, 56.2, 47.1, 43.3,
33.3, 28.3, 27.3, 24.0, 20.0. HRMS (ESI) calcd for C35H44N2O9-
Na (M + Na+) 659.2944 found 659.2941.
Allyl-L-pipecolinate (4). Allyl NR-fluorenyloxycarbonyl-L-pi-
pecolate was added to 20% piperidine in dichloromethane
(DCM) solution. The solution is stirred for 20ꢀ25 min at room
temperature and monitored by TLC. After evaporation of sol-
vent, the crude product was purified by flash chromatography on
a silica gel column eluted with hexane/ethyl acetate/EtOH/
diisopropylethylamine (40:40:19:1) (80% yield). 1H NMR (400
MHz CDCl3) δ 1.42ꢀ2.01 (m, 6H), 2.64 (t, 1H), 3.05 (d, 1H),
3.45 (dd, 1H), 4.59 (dd, 2H), 5.25 (dd, 2H), 5.89 (m, 1H). 13C
NMR (100 MHz, CDCl3) δ 171.2, 132.0, 118.4, 65.2, 58.6, 45.7,
29.2, 25.6, 24.1. HRMS: (ESI): calcd for C9H15NO2 (M + H+)
170.1181, found 170.1171.
Synthesis of Rapalog Library. The library was synthesized on
0.50 g of Rink resin (0.20 mmol/g, 100ꢀ200 μm). Standard
Fmoc/HBTU peptide chemistry was employed for all of the
synthesis steps unless otherwise noted. The coupling reactions
typically employed 2 equiv of Fmoc-amino acids, 2 equiv of
HBTU, 2 equiv of HOBt, and 4 equiv of NMM for 2 h and were
monitored by Ninhydrin tests. The Fmoc group was removed by
treatment with 20% piperidine in dimethylformamide (DMF)
for 5ꢀ25 min. After each step, the beads were exhaustively
washed with DMF and DCM. Starting from the Fmoc-protected
rink resin, the Fmoc group was removed with piperidine, and
the exposed amine was acylated with N-Fmoc-Glu-R-allyl ester
(2 equiv), followed by the coupling of Fmoc-D-β-homoPhe
(2 equiv). The resin was treated with 20% piperidine for 5 min,
exhaustively washed with DMF, and immediately coupled to
building block 3a (1.5 equiv). The resulting resin was split into
10 equal aliquots, and each aliquot was coupled to a different
Fmoc-amino acid (D-Thr, Gly, Ala, D-Val, Pro, Lys, Trp, Asp,
D-Phe, and Nle). After the coupling reaction was complete, each
aliquot was further split into 10 equal portions to give 100 indivi-
dual samples. Each portion (5 mg) was individually treated with
20% piperidine (5 min) to remove the Fmoc group, washed
exhaustively, and immediately coupled to one of the 10 Fmoc-
amino acids described above. At this point, each of the 100 sam-
ples was split into two equal portions. The first half was washed and
stored for later cyclization to produce the sublibrary 11, while the
second half was subjected to another round of coupling reaction (to
Fmoc-L-Ala) to expand the ring size of the cyclic peptides
(sublibrary 12). Prior to cyclization, the C-terminal allyl group
was removed by treatment with Pd(PPh3)4 (0.2 equiv) and N-
methylaniline (9.0 equiv) in anhydrous THF (45 min). The resin
was washed sequentially with THF, DMF, and DCM, and treated
with 20% piperidine to remove the N-terminal Fmoc group.
For peptide cyclization, the resin was suspended in a solu-
tion of PyBOP/HOBt/NMM (5, 5, and 10 equiv, respectively)
in DMF, and the mixture was incubated on a carousel shaker for
17ꢀ20 h. The cyclization reaction was terminated when Ninhydrin
tests showed negative results. The resulting resin was washed with
DMF and DCM, dried under vacuum, and stored at 4 ꢀC. Cleavage
of the peptides from the resin and side-chain deprotection was
achieved by treating the resin with 50% trifluoroacetic acid in DCM
for 1.5 h. The solvents were evaporated under vacuum, and the
crude peptides were dissolved in DCM containing 10% diethyl-
propylamine. The solution was quickly passed through a silica
gel column to remove the salts, evaporated to dryness, and stored
at 4 ꢀC until use. Compounds 2aꢀy were prepared in a similar
manner.
Allyl Nr-(3,3-dimethyl-4-hydroxy-2-ketobutyryl)-L-pipe-
colinate (5). To a solution of amine 4 (0.33 mmol, 55 mg)
dissolved in 1.5 mL of toluene was added dihydro-4,4-dimethyl-
2,3-furandione (0.49 mmol, 62 mg) and 4-dimethylaminopyr-
idine (DMAP) (0.033 mmol, 4 mg). The solution was stirred for
17ꢀ20 h at reflux temperature under argon atmosphere. After
removal of solvent, the crude product was purified by flash
chromatography on a silica gel column with hexane/ethyl acetate
(2:1) as eluent (81% yield). 1H NMR (400 MHz CDCl3) δ 1.50
(s, 6H), 1.23ꢀ1.80 (m, 6H), 3.22 (dt. 1H), 3.50 (d, 1H), 3.56 (q,
1H), 4.44 (s, 2H), 4.66 (dd, 2H), 5.30 (dd, 2H), 5.90 (m, 1H).
13C NMR (100 MHz, CDCl3): δ 205.9, 170.1, 168.1, 131.4,
119.3, 69.3, 66.2, 51.6, 49.5, 44.2, 26.3, 24.8, 21.3, 20.9. HRMS
(ESI) calcd for C15H23NO5Na (M + Na+) 320.1474 found
320.1476.
Building Block 3. To a solution of allyl ester 5 (1.0 mmol) in
freshly distilled DCM (3 mL) was added the proper Fmoc-amino
acid (1.05 mmol), N,N0-diisopropyl carbodiimide (2.0 mmol),
and DMAP (0.05 mmol). The resulting mixture was stirred for
1 h at room temperature. The crude allyl ester products were
purified by silica gel column chromatography using hexane/ethyl
acetate (3:1) as eluent (90ꢀ95% yield). Next, the allyl ester
(0.9 mmol) was dissolved in 4 mL of distilled tetrahydrofuran
(THF), and Pd(Ph3P)4 (0.045 mmol) and N-methylaniline
(2.7 mmol) were added. The solution was stirred for 40 min at
room temperature under argon atmosphere, and the color of the
solution changed from light yellow to brown. The crude products
were purified by flash silica gel column chromatography using
hexane/ethyl acetate/AcOH (66:33:1) as eluent (70ꢀ80% yields).
3a. 1H NMR (400 MHz CDCl3) δ 1.27 (s, 6H), 1.32 (d, 3H),
1.24ꢀ1.59 (m, 6H), 2.97 (m, 1H), 3.26 (m, 1H), 3.37 (m, 1H),
4.22ꢀ4.39 (m, 6H), 7.28ꢀ7.65 (m, 8H). 13C NMR (100 MHz,
CDCl3) δ 206.1, 203.5, 178.3, 172.5, 170.0, 163.3 157.0, 143.8,
141.3, 135.1, 132.2, 128.6, 127.8, 127.4, 125.1, 120.0, 70.0, 67.8,
67.1, 47.1, 46.6, 42.4, 41.6, 38.9, 30.9, 19.2. HRMS (ESI) calcd
for C30H34N2O8Na (M + Na+) 573.2213, found 573.2216.
3b. 1H NMR (400 MHz CDCl3) δ 1.16ꢀ1.65 (m, 6H), 1.34
(s, 6H), 2.90 (s, 2H), 3.00 (m, 1H), 3.30 (m, 1H), 3.36 (m, 1H),
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Evaluation of the Rapalog Library. To check the quality of
library synthesis, each of the compounds from above (2aꢀy,
sublibrary 11, and sublibrary 12) was dissolved in 0.1% TFA
(in water) and analyzed by ESI or MALDI-TOF MS. Most of the
compounds showed single species of the expected m/z ratios
(Supporting Information, Figure S2ꢀS4). Four of the com-
pounds were selected for further purification by HPLC on a
semipreparative C-18 column, which was eluted by a linear gra-
dient of 0ꢀ100% CH3CN in water (containing 0.05% trifluor-
oacetic acid) over 20 min. The pure samples were assayed against
FKBP12 and compared to the results obtained with the crude
samples (Table 3). Because of the small sample quantities and
lack of suitable optical activity, the sample concentrations were
determined as follows. The amount of crude compound (0.25 mg
for a compound of the molecular mass of 1000) was estimated on
the basis of the resin loading capacity (0.20 mmol/g) and a 50%
overall synthesis (isolated) yield. For the purified compounds, it
was assumed that HPLC purification had 70% sample recovery.
Sample partition was carried out by dissolving the crude com-
pound in 1 mL of DCM and withdrawing appropriate volumes of
the stock solution for different experiments (e.g., activity assay
and HPLC purification). For activity assay, the samples were
evaporated to dryness and redissolved in appropriate volumes
of DMSO.
FKBP Binding Assay. The fluorescence polarization based
competition assay was performed in a 384-well plate. Each re-
action (20 μL total volume) contained 137 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4, 200 nM
recombinant FKBP12, 100 nM fluorescent probe FLU-SLF
(kindly provided by I. Graef of Stanford University), and varying
concentrations of rapalogs (0ꢀ5000 μM). After the addition of
the rapalogs as the last component, the binding reactions were
incubated for 1 h at room temperature to reach binding equili-
brium. Anisotropy values were measured on a FlexStation 3 plate
reader (Molecular Devices, Sunnyvale, CA). KI values were
calculated from the corresponding IC50 values using the method
by ChengꢀPrusoff31 [KI = IC50/(1 + D/KDF)], where D is the
concentration of FLU-SLF (100 nM) and KDF is the binding
affinity of FLU-SLF for FKBP (3.3 nM).20
Molecular Modeling. At the first stage, three-dimensional
structures were generated for rapaplogs 11a and 11b by geome-
try optimization with the SPARTAN 02 program. Since the
rapalogs have similar FKBP-binding motif to compound 13 of
Holt et al.,18 the coordinates for atoms in the binding motif were
modeled using the coordinates of compound 13 in a 2.2 Å resolu-
tion X-ray crystal structure of its complex with FKBP12. The
binding motif was then fused with the cyclic peptide sequences of
rapalogs 11a and 11b at both ends to generate an initial geo-
metry. The latter was optimized at the HF/6-31+G* level with
the geometry of the binding motif fixed. The optimized structure
appears to be quite rigid, because of the formation of multiple
hydrogen bonds between backbone amide -NH and carbonyl
oxygens, as in antiparallel β-strands. During the second stage, the
optimized rapalog structure was docked with FKBP12 by means
of quantum mechanics/molecular mechanics (QM/MM) opti-
mization to form the FKBP12-rapalog complex. Initially, the
rapalog was placed into the binding site of FKBP12 utilizing
the crystal structure of a previously reported FKBP12-rapalog
complex18 (PDB access number 1FKI) as a template. The QM
layer contained the rapalog and the protein residues that directly
interact with the rapalog, and was treated at the PM3 level. The
MM layer included the rest of the FKBP12 protein and was
described by the AMBER force field. The QM/MM optimization
was performed using the Gaussian 03 package.32 The QM/MM
two-layer partitioning was performed as previously described.33
’ ASSOCIATED CONTENT
S
Supporting Information. HPLC, NMR, MS, data of tar-
b
get peptides. This material is available free of charge via the Inter-
net at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 614-688-4068 (D.P.), 614-688-4395 (R.B.). Fax: 614-
292-1532 (D.P.), 614-292-7232 (R.B.). E-mail: pei.3@osu.edu
(D.P.), roger.briesewitz@osumc.edu. (R.B.).
Funding Sources
This work was supported by the National Institutes of Health
(GM062820 and CA132855), the FORE Cancer Research
Foundation, and the Jack Roth Fund for Lung Cancer.
’ ABBREVIATIONS
CypA, peptidyl-prolyl isomerase cyclophilin A; CsA, cyclosporin
A; SAR, structureꢀactivity relationship; D-Abu, (S)-2-aminobu-
tyric acid; D-β-homoPhe, (R)-3-amino-5-phenylpentanoic acid;
L-β-homoPhe, (S)-3-amino-5-phenylpentanoic acid; D-homo-
Phe, D-homophenylalanine; L-β-Ile, L-β-isoleucine; HBTU, O-benzo-
triazole-N,N,N,N-tetramethyluronium hexafluorophosphate; NMM,
N-methylmorpholine; PyBop, benzotriazole-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate; DMAP, 4-(dimethylamino)
pyridine; FA, fluorescence anisotropy; OBOC, one-bead-one-com-
pound; HOBt, 1-hydroxybenzotriazole hydrate
’ REFERENCES
(1) Vezina, C.; Kudelski, A.; Sehgal, S. N. Rapamycin (AY-22,989), a
new antifungal antibiotic. I. Taxonomy of the producing streptomycete
and isolation of the active principle. J. Antibiot. 1975, 28, 721–726.
(2) Tanaka, H.; Kuroda, A.; Marusawa, H.; Hatanaka, H.; Kino, T.;
et al. Structure of FK506, a novel immunosuppressant isolated from
Streptomyces. J. Am. Chem. Soc. 1987, 109, 5031–5033.
(3) Yuan, R.; Kay, A.; Berg, W. J.; Lebwohl, D. Targeting tumor-
igenesis: development and use of mTOR inhibitors in cancer therapy.
J. Hematol. Oncology 2009, 2, 45.
(4) Liu, J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.; Weissman, I.;
et al. Calcineurin is a common target of cyclophilin-cyclosporin A and
FKBP-FK506 complexes. Cell 1991, 66, 807–815.
(5) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith,
C. T.; et al. A mammalian protein targeted by G1-arresting rapamycin-
receptor complex. Nature 1994, 369, 756–758.
(6) Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.;
Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a
rapamycin-dependent fashion and is homologous to yeast TORs. Cell
1994, 78, 35–43.
(7) Chiu, M. I.; Katz, H.; Berlin, V. RAPT1, a mammalian homolog
of yeast Tor, interacts with the FKBP12/rapamycin complex. Proc. Natl.
Acad. Sci. U. S. A. 1994, 91, 12574–12578.
(8) Futer, O.; DeCenzo, M. T.; Aldape, R. A.; Livingston, D. J.
FK506 binding protein mutational analysis. Defining the surface residue
contributions to stability of the calcineurin co-complex. J. Biol. Chem.
1995, 270, 18935–18940.
(9) Griffith, J. P.; Kim, J. L.; Kim, E. E.; Sintchak, M. D.; Thomson,
J. A.; et al. X-ray structure of calcineurin inhibited by the immunophilin-
immunosuppressant FKBP12-FK506 complex. Cell 1995, 82, 507–522.
494
dx.doi.org/10.1021/co200057n |ACS Comb. Sci. 2011, 13, 486–495

ACS Combinatorial Science
RESEARCH ARTICLE
(10) Kissinger, C. R.; Parge, H. E.; Knighton, D. R.; Lewis, C. T.;
Pelletier, L. A.; et al. Crystal structures of human calcineurin and
the human FKBP12-FK506-calcineurin complex. Nature 1995, 378,
641–644.
(11) Choi, J.; Chen, J.; Schreiber, S. L.; Clardy, J. Structure of the
FKBP12-rapamycin complex interacting with the binding domain of
human FRAP. Science 1996, 273, 239–242.
(30) Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg, I.;
Osterman, A.; Godzik, A.; Hunter, T.; Dixon, J.; Mustellin, T. Protein
tyrosine phosphatases in the human genome. Cell 2004, 117, 699–711.
(31) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes 50%
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22, 3099–3108.
(12) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. How many
drug targets are there? Nat. Rev. Drug Discovery 2006, 5, 993–996.
(13) Salituro, G. M.; Zink, D. L.; Dahl, A.; Nielsen, J.; Wu, E.; Huang,
L.; Kastner, C.; Dumont, F. J. Meridamycin: A novel nonimmunosup-
pressive FKBP12 ligand from Streptomyces Hygroscopicus. Tetrahedron
Lett. 1995, 36, 997–1000.
(14) Fehr, T. S., J-J.; Schuler, W.; Gschwind, L.; Ponelle, M.;
Schilling, W.; Wioland, C. Antascomicins A, B, C, D and E. Novel
FKBP12 binding compounds from a Micromonospora strain. J. Antibiot.
1996, 49, 230–233.
(15) Jin, L.; Harrison, S. C. Crystal structure of human calcineurin
complexed with cyclosporin A and human cyclophilin. Proc. Natl. Acad.
Sci. U. S. A. 2002, 99, 13522–13526.
(16) Etzkorn, F. A. C., Z.Y.; Stolz, L. A.; Walsh, C. T. Cyclophilin
residues that affect noncompetitive inhibition of the protein serine
phosphatase activity of calcineurin by the cyclophilin-cyclosporin A
complex. Biochemistry 1994, 33, 2380–2388.
(32) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J., J. A.; Vreven, T.; Kudin,
K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.;
Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.;
Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.;
Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, Revision C.03; Gaussian, Inc.: Wallingford, CT, 2004.
(17) Zenke, G.;Strittmatter, U.;Fuchs, S.;Quesniaux, V. F.;Brinkmann,
V.; et al. Sanglifehrin A, a novel cyclophilin-binding compound showing
immunosuppressive activity with a new mechanism of action. J. Immunol.
2001, 166, 7165–7171.
(33) Liu, Y. M.; Hu, X. H. Molecular determinants for binding of
ammonium ion in the ammonia transporter AmtB - A quantum chemical
analysis. J. Phys. Chem. A 2006, 110, 1375–1381.
(18) Holt, D. A.; Luengo, J. L.; Yamashita, D.; Oh, H.-J.; Konilian,
A. L.; Yen, H.-K.; Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy,
M. A.; Eggleston, D.; Liang, J.; Schults, L. W.; Stout, T.; Clardy, J.
Design, Synthesis, and Kinetic Evaluation of High-Affinity FKBP
Ligands and the X-ray Crystal Structures of Their Complexes with
FKBP12. J. Am. Chem. Soc. 1993, 115, 9925–9938.
(19) Dubowchik, G. M.; Ditta, J. L.; Herbst, J. J.; Bollini, S.; Vinitsky,
A. Fluoresceinated FKBP12 ligands for a high-throughput fluorescence
polarization assay. Bioorg. Med. Chem. Lett. 2000, 10, 559–562.
(20) Braun, P. D.; Wandless, T. J. Quantitative analyses of bifunc-
tional molecules. Biochemistry 2004, 43, 5406–5413.
(21) Maynard-Smith, L. A.; Chen, L. C.; Banaszynski, L. A.; Ooi,
A. G.; Wandless, T. J. A directed approach for engineering conditional
protein stability using biologically silent small molecules. J. Biol. Chem.
2007, 282, 24866–24872.
(22) Briesewitz, R.; Ray, G. T.; Wandless, T. J.; Crabtree, G. R.
Affinity modulation of small-molecule ligands by borrowing endogenous
protein surfaces. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1953–1958.
(23) Gestwicki, J. E.; Crabtree, G. R.; Graef, I. A. Harnessing
chaperones to generate small-molecule inhibitors of amyloid beta
aggregation. Science 2004, 306, 865–869.
(24) Marinec, P. S.; Chen, L.; Barr, K. J.; Mutz, M. W.; Crabtree,
G. R.; et al. FK506-binding protein (FKBP) partitions a modified HIV
protease inhibitor into blood cells and prolongs its lifetime in vivo. Proc.
Natl. Acad. Sci. U. S. A. 2009, 106, 1336–1341.
(25) Harrison, R. K.; Stein, R. L. Substrate specificities of the
peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506
binding protein: evidence for the existence of a family of distinct
enzymes. Biochemistry 1990, 29, 3813–3816.
(26) Somers, P. K.; Wandless, T. J.; Schreiber, S. L. Synthesis and
analysis of 506BD, a high-affinity ligand for the immunophilin FKBP.
J. Am. Chem. Soc. 1991, 113, 8045–8056.
(27) Kawai, M.; Lane, B. C.; Hsieh, G. C.; Mollison, K. W.; Carter,
G. W.; Luly, J. R. Structure-activity profiles of macrolactam immuno-
suppresant FK506 analogues. FEBS Lett. 1993, 316, 107–113.
(28) Wells, J. A.; McClendon, C. L. Reaching for high-hanging
fruit in drug discovery at protein-protein interfaces. Nature 2007, 450,
1001–1009.
(29) Johnson, S. A.; Hunter, T. Kinomics: methods for deciphering
the kinome. Nat. Methods 2005, 2, 17–25.
495
dx.doi.org/10.1021/co200057n |ACS Comb. Sci. 2011, 13, 486–495