Retention of immunosuppressant activity in an ascomycin analogue lacking a hydrogen-bonding interaction with FKBP12

Article abstract of DOI:10.1021/jm980252z

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Δ^23,24-dehydroascomycin, was likewise evaluated. De

Full text of DOI:10.1021/jm980252z

4456
J . Med. Chem. 1999, 42, 4456-4461
Brief Articles
Reten tion of Im m u n osu p p r essa n t Activity in a n Ascom ycin An a logu e La ck in g a
Hyd r ogen -Bon d in g In ter a ction w ith F KBP 12
Paul E. Wiedeman,* Stephen W. Fesik, Andrew M. Petros, David G. Nettesheim, Karl W. Mollison,
Benjamin C. Lane, Yat Sun Or, and J ay R. Luly
Abbott Laboratories, Pharmaceutical Products Division, 200 Abbott Park Road, Department 47N, Building AP-52N,
Abbott Park, Illinois 60064-6217
Received April 27, 1998
C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its
immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic
pathway, ∆23,24-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-
bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyasco-
mycin was found to be equipotent to the parent compound both in its immunosuppressive
potency and in its interaction with the immunophilin, FKBP12. Conversely, ∆23,24-dehydro-
ascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency.
NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to
understand this phenomenon at the molecular level. The NMR structures of the complexes
formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting
that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex
formation.
In tr od u ction
ecule, respectively. The location of the C24 hydroxyl
group is of particular interest in that it is found at, or
near, the border between the sites that interact with
FKBP12 and calcineurin.7 In addition, there is recent
speculation that this hydroxyl group is a hydrogen bond
donor that mimics the NH group of the peptide sub-
strate.8
As part of a continuing effort to probe the structure-
activity relationships of these macrolactam immuno-
suppressants,9 the importance of the hydrogen bonds
involving the C24 hydroxyl group for the formation of
the FKBP12/ascomycin complex was examined by com-
parison with its C24-deoxy analogue and the synthetic
precursor to the deoxy analogue, ∆23,24-dehydroascomy-
cin. In this paper, the synthesis, biological activity, and
NMR studies of C24-deoxyascomycin are reported.
Through the development and use of the cyclic peptide
cyclosporin A, immunosuppressive therapy has been
effective in the prevention of acute organ rejection
following transplantation procedures and shows the
potential to control autoimmune and inflammatory
diseases.1 In addition, the discovery of FK506, found
while screening fermentation broths for interleukin 2
production inhibitors, heightened interest in this thera-
peutic arena by initiating entry into a novel second
structural class of compounds having a 10-100-fold
enhancement of immunosuppressive potency.2,3 A nec-
essary first step in FK506 action is binding to a cis-
trans peptidyl-prolyl isomerase, the FK506 binding
protein (FKBP12). The FK506/FKBP12 complex inhibits
a serine/threonine phosphatase, calcineurin, the inhibi-
tion of which ultimately prevents transcriptional proc-
esses leading to interleukin 2 production from occurring
within the T cell nucleus.4 Three-dimensional structures
of FKBP12 alone and when complexed to FK506 and
FK506 analogues have been determined by X-ray crys-
tallography and NMR spectroscopy.5 Ascomycin, the
analogue of interest for this report with in vitro potency
comparable to that of FK506,6 differs from FK506 only
at the C21 substituent in which an ethyl group replaces
an allyl side chain. The FKBP12/FK506 complex is
stabilized by a series of hydrophobic interactions and
intermolecular hydrogen bonds. Among the intermo-
lecular hydrogen bonds found in all FKBP12/ligand
complexes are those involving the C24 hydroxyl moiety.
Glutamic acid 54 and glutamine 53 hydrogen bond with
this moiety directly or through a bridging water mol-
Resu lts a n d Discu ssion
Syn t h esis of C24-Deoxya scom ycin . Ascomycin
was obtained as a fermentation product from Strepto-
myces hygroscopicus var. ascomyceticus by methods
similar to those described in the literature.10 The
synthesis of C24-deoxyascomycin was accomplished
from ascomcyin by a variation of a two-step process
(Scheme 1).11 Transformation to the ∆23,24-enone was
selectively and efficiently achieved by using p-toluene-
sulfonic acid in toluene at 75-80 °C. Evidence for
formation of the dehydration product, which like asco-
mycin and FK506 exists as a mixture of amide rotamers,
was obtained from the 13C NMR spectrum of the product
in which new signals at 147.8/146.2 (C24) and 129.0/
127.6 (C23) ppm corresponding to the R and â carbons
of the enone system were observed. Reduction of the
10.1021/jm980252z CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/01/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4457
Sch em e 1a
dehydroascomycin, reflective of its poor interaction with
FKBP12, was approximately 2 orders of magnitude less
potent than the other compounds (Table 1). In the
second assay in which the PLN hyperplasia response
to alloimmune challenge with histoincompatible sple-
nocytes was measured, the results were similar to those
found for the in vitro experiment. Ascomycin, FK506,
and C24-deoxyascomycin exhibited similar potencies,
whereas ∆23,24-dehydroascomycin did not give a signifi-
cant response (Table 1).
NMR Stu d ies of th e F KBP 12/C24-Deoxya scom y-
cin Com p lex. The tight binding affinity and high
immunosuppressive potency of C24-deoxyascomycin was
surprising since two of the five hydrogen bonds in the
FKBP12/ascomycin complex cannot be formed in the
FKBP complex with the C24 deoxy analogue. In an
attempt to understand this phenomenon at the molec-
ular level, NMR studies were conducted on the FKBP12/
C24-deoxyascomycin complex.
a
Reagents: (a) p-TsOH‚H2O, toluene, reflux; (b) H2, Pd-C,
MeOH.
As a first step, the conformation of C24-deoxyasco-
mycin when bound to FKBP12 was determined from
isotope-filtered NMR experiments. In an isotope-filtered
2D TOCSY spectrum, the scalar-coupled protons were
readily traced. This information coupled with an analy-
sis of the isotope-filtered 2D NOE spectrum allowed the
1H NMR signals corresponding to C24-deoxyascomycin
when bound to FKBP12 to be assigned. To determine
the conformation of C24-deoxyascomycin when bound
to FKBP12, 126 intraligand NOEs were assigned from
the isotope-filtered 2D NOE spectrum of the complex.
Proton-proton distance restraints were generated from
the NOE data, and 200 structures were generated that
were consistent with the distance restraints using a
dynamical simulated annealing protocol.14 A total of 62
low-energy structures were chosen from this set that
best satisfied the NOE restraints. The structure of the
ligand when bound to FKBP was very well-defined by
the NMR data and deviated only slightly from idealized
geometry. The average rms deviation of all heavy atoms
from a calculated average structure was 0.28 ( 0.11 Å,
and no NOE violations were found to be greater than
0.1 Å (see Supporting Information).
Ta ble 1. Biological Evaluation of Ascomycin and Ascomycin
Analogues
IC50 (nM)
FKBP12
binding potency
MLR
PLN potency ED50
(mg/kg/day IP)
compound
FK506
ascomycin
∆
2.3
2.1
0.23
0.26
22
0.15
0.15
0.32
39%a
0.65
23,24-dehydroascomycin 529
C24-deoxyascomycin
0.77
a
Percent response of ∆23,24-dehydroascomycin at 3 mg/kg/day
ip.
∆
23,24-enone was then accomplished by exposure to
hydrogen gas in the presence of palladium on activated
carbon at room temperature. The 13C and 1H NMR
spectra obtained were consistent with the reduction of
the enone moiety and retention of the two trisubstituted
olefins.
P h a r m a cology. To examine the binding affinity of
the FK506 analogues to FKBP12, a binding assay was
utilized in which the analogue competes with labeled
ascomycin for binding to an FKBP12-CMP-KDO syn-
thetase fusion protein.12 As shown in Table 1, FK506,
ascomycin, and C24-deoxyascomycin all bind compara-
bly. The lone standout in this assay is ∆23,24-dehydro-
ascomycin which is several hundred times less potent.
Although this may be explained in part by the loss of
the hydrogen bonds involving the C24 hydroxyl moiety,
the decreased affinity may also be due to conformational
changes caused by the introduction of two new sp2
hybridized centers. Surprisingly, reintroduction of sp3
hybridization, without the hydrogen bonding, results in
total restoration of the interaction with FKBP12.
The immunosuppressive activities of these compounds
were examined in an in vitro model of allogeneic T
lymphocyte activation (MLR)3 as well as in an in vivo
model of lympho-proliferation, the popliteal lymph node
(PLN)13 assay. In the MLR assay, human peripheral
blood mononuclear leukocytes (PBL) were exposed to a
pool of mitomycin C-treated PBL. Four days hence, the
incorporation of [3H]thymidine into cellular DNA was
measured by liquid scintillation spectrometry. Complete
inhibition of this response was observed for FK506,
As shown in Figure 1, the bound conformation of C24-
deoxyascomycin was found to be very similar to that of
ascomycin when bound to FKBP12.5d Superposition of
the common heavy atoms of the C24-deoxyascomycin
analogue and ascomycin gives an rms deviation of 0.35
Å. This is consistent with the similar chemical shifts
observed for ascomycin and C24-deoxyascomycin. Ex-
cept in the immediate vicinity of C24, the chemical shifts
of the analogue were very similar to ascomycin, includ-
ing the characteristic upfield proton resonances of the
3, 4, and 5 positions of the piperidine ring.15 Thus based
on chemical shifts, the overall structure of the complex
must be similar despite the lack of the two hydrogen
bonds involving the C24-OH in ascomycin.
Although the conformations of ascomycin and the C24
deoxy analogue are similar, it is possible that the
ligands interact with the protein in a different manner.
Thus, the backbone chemical shifts of FKBP were
compared while bound to the two ligands. As shown in
Figure 2, the substitution of a proton for the OH
functional group of ascomycin results in significant
changes in chemical shift of the protein all along the
ascomycin, and C24-deoxyascomycin. By contrast, ∆23,24
-

4458 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
F igu r e 1. Surperposition of the average, minimized structure of C24-deoxyascomycin bound to FKBP (gray) to the average,
minimized structure of ascomycin bound to FKBP (black).
F igu r e 2. Analysis of chemical shift differences observed between the two FKBP12 complexes. The shifts were obtained by
1
subtracting the chemical shifts of the HR, 1HN, and 15N resonances of [U-15N,13C]FKBP12 ligated to unlabeled C24-deoxyascomycin
1
from the corresponding resonances of [U-15N,13C]FKBP12 ligated to unlabeled ascomycin. The scale of the H shift differences is
not the same as that of the 15N differences.
loop from Gly-51 to Ile-56, the region of the protein
closest to the site of modification on the ligand. Most of
these changes are relatively small and may simply be
related to the presence or absence of the oxygen at C24.
However, an unusually large 15N chemical shift differ-
ence of 2.2 ppm was observed for the Val-55. A likely
cause of this large chemical shift difference is the direct
effect of the hydrogen bond to the Glu-54 carbonyl. In
the FKBP12/ascomycin complex, the hydrogen bond
between the C24-OH of ascomycin and the Glu-54
carbonyl of FKBP12 stabilizes the amide bond reso-
nance structure in which there is increased double-bond
character between nitrogen and carbon16 resulting in a
decrease of the electron density on the nitrogen atom
of Val-55 and a downfield shift relative to the C24 deoxy
analogue.
in the two complexes (see Supporting Information).
Thus the NOE data indicates that the complexes formed
between FKBP12 and ascomycin and the C24 deoxy
analogue are structurally very similar.
Con clu sion s
In the three-dimensional structure of the FKBP12/
FK506 complex, two of the five intermolecular hydrogen
bonds involve the C24 hydroxyl group. One hydrogen
bond is formed directly between the Glu-54 carbonyl and
the C24 hydroxyl group, and the second, mediated by a
water molecule, involves Gln-53. Despite the absence
of these hydrogen bonds between the ligand and the
protein in the FKBP12/C24-deoxyascomycin complex,
the C24 deoxy analogue interacts as strongly to FKBP12
as does ascomycin in a competitive binding assay. In
addition, both molecules display similar immunosup-
pressant activities. The conformations of the bound
ligands are similar as are the overall structures of the
FKBP/ligand complexes. These observations suggest
that the hydrogen bonds involving the C24 hydroxyl
group do not significantly enhance the binding of
ascomycin to the protein. One possible explanation is
that upon hydrogen bond formation, a loss of entropy
is obtained by decreasing the motion of an otherwise
mobile loop of FKBP12 and confining the location of a
water molecule to mediate one of the hydrogen bonds.
To further investigate possible differences in the
conformation of FKBP12 when bound to the two ligands,
a 4D [13C,1H,15N,1H] NOESY and a 4D [13C,1H,13C,1H]
NOESY was recorded on both complexes. Two 2D ω1,
ω2 slices from the 4D [13C,1H,15N,1H] NOESY spectra
1
at the same 15N (ω3) and H (ω4) amide frequencies of
Gln-53 and Glu-54 in both complexes were compared.
These amides are located near the two hydrogen bonds
observed in the FKBP/ligand complex. The NOEs are
nearly identical in the spectra of the two complexes.
Similarly, the 4D [13C,1H,13C,1H] NOE data is the same

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4459
(0.35 g, 35%): 13C NMR (125 MHz, CDCl3) δ 212.2, 210.8,
196.3, 193.4, 169.4, 169.3, 166.1, 165.1, 138.9, 138.3, 132.1,
131.6, 131.1, 124.4, 124.1, 98.4, 97.4, 84.2, 82.3, 82.2, 76.8, 75.5,
73.8, 73.5, 73.5, 73.1, 72.4, 57.2, 57.1, 56.5, 56.3, 56.2, 55.4,
54.7, 52.6, 49.3, 48.4, 43.8, 39.2, 39.1, 38.3, 35.3, 34.9, 34.9,
34.7, 34.6, 34.3, 33.3, 32.7, 31.2, 30.6, 28.6, 27.7, 26.5, 26.3,
26.1, 24.5, 24.3, 24.0, 20.9, 20.7, 19.9, 19.3, 16.1, 16.0, 15.7,
15.5, 14.9, 14.4, 13.7, 13.5, 11.7; MS (FAB + KI) m/z 814 (M
+ K+); IR (KBr) cm-1 3440, 2940, 1742, 1715, 1650, 1455, 1382,
1198, 1175, 1100. Anal. (C43H69NO11) C, H, N.
Biologica l Meth od s. F KBP 12 bin d in g a ssa y: Human
FKBP12 was cloned as a fusion partner with CMP-KDO
synthetase (CKS) and purified as described by Edalji et al.12
An ascomycin conjugate of alkaline phosphatase was prepared
by active ester coupling of an ascomycin C22 derivative
(carboxymethyloxime) with alkaline phosphatase (ascomycin-
AP; Abbott Laboratories, Abbott Park, IL). In the assay,
FKBP12-CKS fusion protein was dissolved in 10-35 µg/mL
in 20 mM sodium phosphate buffer, pH 7.4, and adsorbed to
the wells of an Immuno Plate Maxisorp (Nunc, Naperville, IL)
by incubation at ambient temperature for 2 h. A solution of
phosphate-buffered saline (PBS), pH 7.4, containing 2% bovine
serum albumin (BSA) and 0.2% Tween 20 was added to the
wells. After rinsing the wells with 0.2% Tween 20 in PBS, the
test compound in the PBS/BSA/Tween 20 buffer, or buffer
alone, was added to the wells. An equal volume of ascomycin-
AP ligand at 1 µg/mL in PBS/BSA/Tween 20 was added to the
wells and incubated 2 h at ambient temperature. After rinsing
with 0.2% Tween 20 in PBS, p-nitrophenyl phosphate at 1 mg/
mL in 0.1 M aminomethylpropanol was added to the wells and
the temporal change in 405-nm absorbance recorded.
Both effects may lead to an unfavorable entropic process
that compensates to a large degree any favorable
enthalpic reaction that might be gained upon forming
hydrogen bonds. The major driving force for complex
formation may be hydrophobic in nature. Indeed, FK506
binds in a hydrophobic pocket lined with nonpolar
groups. The ligands themselves contain many nonpolar
groups and are quite insoluble in water. The loss of two
hydrogen bonds appears not to be as significant to the
complex formation as was originally assumed.
Exp er im en ta l Section
Ascomycin and FK506 were produced by fermentation using
procedures similar to those that have been previously de-
scribed in the literature.10 Unless otherwise indicated, reagents
were purchased from Sigma Chemical Co. (St. Louis, MO).
Penicillin-streptomycin was purchased from Gibco Labora-
tories (Grand Island, NY). Complete RPMI 1640 contained 10%
heat-inactivated fetal bovine serum, 50 µM 2-mercaptoethanol,
2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin. FK506 binding protein (FKBP12) and the fusion
protein composed of FKBP12 and CMP-KDO synthetase
(FKBP12/CKS) were prepared at Abbott Laboratories (Abbott
Park, IL) as previously described.12 PMBCs were isolated by
Ficoll-Hypaque density gradient centrifugation of heparinized
blood from healthy donors. Stimulator PBMCs were treated
with 25 µg/mL mitomycin C for 30 min and washed before
being used.
Ch em istr y. Gen er a l Meth od s. See Su p p or tin g In for -
m a tion . Syn th esis of ∆23,24-Deh yd r oa scom ycin . Ascomycin
(6.00 g, 7.58 mmol) was dissolved in toluene (120 mL) and the
solution heated to 75-80 °C. p-Toluenesulfonic acid monohy-
drate (300 mg) was introduced in one portion. After 1 h, the
reaction was incomplete and additional p-toluenesulfonic acid
monohydrate (300 mg) was added. Thirty minutes later, the
reaction was complete, and the mixture was cooled to room
temperature. Without concentration, the reaction solution was
passed through a plug of silica gel (75 g) eluting with ether
(1000 mL). Concentration of the eluent in vacuo provided 5.47
g of crude product as a yellow solid. Purification by flash
chromatography (silica gel, elution with 35% acetone in
hexane) furnished the title compound as a colorless solid (3.16
g, 54%): mp 122-124 °C; 13C NMR (125 MHz, CDCl3) δ 201.2
and 199.6 (C22), 196.0 and 191.4 (C9), 169.2 and168.8 (C1),
165.9 and 165.1 (C8), 147.8 and 146.2 (C24), 139.1 and 138.0
(C19), 133.2 and 131.7 (C27), 131.1 and 130.0 (C28), 129.0 and
127.6 (C23), 123.9 and 123.4 (C20), 98.7 and 97.9 (C10), 84.1
(C31), 80.4 and 79.6 (C26), 76.0 and 75.8 (C15), 74.1 and 73.8
(C32), 73.5 and 73.5 (C13-OMe), 73.4 and 71.6 (C14), 57.5
and 56.2 (C15-OMe), 56.8 (C2), 56.6 (C13-OMe), 56.4 (C31-
OMe), 53.6 and 53.3 (C21), 49.1 and 48.8 (C18), 39.8 and 39.1
(C25), 38.4 and 33.7 (C6), 35.7 and 35.0 (C29), 34.9 and 34.7
(C11), 34.6 and 34.4 (C30), 33.0 and 32.7 (C16), 32.4 (C12),
31.2 (C33), 30.6 and 30.5 (C34), 27.9 and 27.4 (C3), 26.5 and
26.4 (C17), 25.5 and 25.4 (C35), 24.7 and 24.2 (C5), 21.0 (C17-
Me), 20.3 and 18.9 (C4), 16.1 and 15.9 (C11-Me), 14.5 and 14.2
(C27-Me), 12.9 and 12.5 (C36), 11.8 and 11.7 (C25-Me); MS
(CI) m/z 791 (M + NH4+); IR (KBr) cm-1 3440, 2930, 1745,
1690, 1645, 1625, 1450, 1390, 1242, 1190, 1170, 1100. Anal.
(C43H67NO11) C, H, N.
Hu m a n m ixed leu k ocyte r esp on se: One-way allogeneic
mixed leukocyte response assays were performed as described
by Kino et al. with slight modification.3 Briefly, 1 × 105
responder PBMCs were mixed with 4 × 105 stimulator PBMCs,
composed of 1 × 105 PBMCs from each of four different donors,
in 0.2 mL of complete RPMI 1640 and cultured for 96 h at 37
°C. During the final 6 h, the cells were labeled with 0.5 µCi/
well of tritiated thymidine ([3H]TdR; DuPont NEN Research
Products, Boston, MA). The cells were harvested by vacuum
filtration onto glass fiber filters, and the radioactivity was
measured by liquid scintillation spectrometry. Test com-
pounds, dissolved at 10 µM in dimethyl sulfoxide, were diluted
in complete RPMI 1640 and added to the responder PBMCs
before addition of the stimulator PBMCs.
P LN h yp er p la sia a ssa y: Inbred male rats (125-150 g)
were purchased from Harlan Sprague-Dawley, Inc. (Indian-
apolis, IN) and housed for 1 week before use. Spleen cells from
Brown Norway rats (RT-1n) were subjected to ammonium
chloride to lyse red cells, washed in phosphate-buffered saline
(PBS), X-irradiated (2000 rads), washed again, and injected
subcutaneously with a 27-gauge needle in the right hind foot
of Lewis strain rats (RT-11) using 5 × 106/100 µL. Test
compound was dissolved in a vehicle consisting of 10% ethanol,
40% propylene glycol, and 2% cremophore with the balance
being sterile 5% dextrose in water (Abbott Laboratories, Abbott
Park, IL); 2 mL/kg of the test compound solution was given ip
to groups of 8 animals once daily, on days 0-3. On day 4, the
PLN were removed and weighed on a microbalance. The mean
weight of nodes from a group of uninjected control animals
was subtracted to determine the net increase, and percent
inhibition in reference to a vehicle control group was calcu-
lated.
Syn th esis of C24-Deoxya scom ycin . ∆23,24-Dehydroasco-
mycin (1.00 g, 1.29 mmol) was added to a suspension of 10%
palladium on carbon (100 mg) in methanol. The reaction vessel
was evacuated and filled with nitrogen several times before
being placed under a hydrogen atmosphere (balloon). The
reaction mixture was vigorously stirred with a magnetic stirrer
for 1.5 h. The catalyst was removed by filtration through Celite
and rinsed with methanol. The filtrate was concentrated in
vacuo to supply 0.93 g (93%) of the desired product as a
colorless foam. Analytically pure material was obtained by
flash column chromatography (silica gel, elution with 25%
acetone in hexane) although a loss in product was encountered
Sta tistica l a n a lysis: Unless otherwise indicated, all data
are presented as the mean ( SEM and were analyzed using
Student’s t-test of unpaired samples.
NMR sa m p le p r ep a r a tion for F KBP /liga n d com p lexes:
Recombinant human FKBP was cloned from a J urkat T-cell
cDNA library and expressed at high levels in Escherichia coli
using translational coupling to the 5′ end of the E. coli kdsB
gene. [U-13C,15N]FKBP were purified from cells grown on
minimal medium containing [15N]ammonium chloride and
[U-13C]acetate17 using ion-exchange and size-exclusion chro-
matography.12 The protein was concentrated to about 3 mM

4460 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
and exchanged into either an H2O or D2O solution (pH 6.5)
containing potassium phosphate (50 mM), sodium chloride
(100 mM), and dithiothreitol-d10 (5 mM). FKBP12/ligand
complexes (1/1) were prepared by incubating labeled FKBP12
with an excess amount of either unlabeled ascomycin or
unlabeled C24-deoxyascomycin for 24-48 h at room temper-
ature.
Krause, P. Brettheim, and J . Andrews for assistance in
collecting biological data.
Su p p or tin g In for m a tion Ava ila ble: Isotope-filtered 2D
TOCSY and NOESY spectra and structural statistics for C24-
deoxyascomycin bound to [U-13C,15N]FKBP; 13C(ω1), 1H(ω2)
planes from the 4D [13C,1H,15N,1H] NOESY spectrum of
[U-15N,13C]FKBP12 complexed with ascomycin or 24-deoxy-
ascomycin extracted at the 1HN,15N chemical shifts of the
individual residues Glu-54 and Gln-53, respectively; 13C(ω1),
1H(ω2) planes from the 4D [13C,1H,13C,1H] NOESY spectrum
of ligated [U-15N,13C]FKBP12 extracted at the 1H,13C chemical
shifts of Arg-57 Hδ2 and Val-55 Hγ1, respectively; chemistry
general methods. This information is available free of charge
via the Internet at http://pubs.acs.org.
NMR exp er im en ts: All NMR spectra of the FKBP/ligand
complexes were recorded on either a Bruker AMX500 (500
MHz) or an AMX600 (600 MHz) spectrometer. NMR spectra
were processed and analyzed using in-house written software
on Silicon Graphics computers. 2D isotope-filtered NOESY and
TOCSY spectra of the unlabeled ligand while bound to
[U-13C,15N]FKBP were recorded at 40 °C using pulse sequences
described earlier.18 Mixing times of 80 and 15 ms were used
for the NOESY and TOCSY experiments, respectively.
4D NOESY spectra of [U-13C,15N]FKBP while complexed
with unlabeled ligand were recorded at 30 °C. The 4D
[13C,1H,15N,1H]NOESY spectrum19 was acquired with eight
scans per experiment with 12 × 80 × 10 × 512 complex points
using sweep widths of 3289, 7463, 2128, and 10 000 Hz in
ω1(13C), ω2(1H), ω3(13C), and ω4(1H), respectively. Water sup-
pression was accomplished20 using two spin-lock pulses of 0.5-
and 2.5-ms duration. The 13C carrier frequency was set at 38
ppm. The regions from 70-49 and 27-5.5 ppm were folded
once. The 4D [13C,1H,13C,1H] NOESY spectrum21 was acquired
with eight scans per experiment with 12 × 64 × 12 × 512
complex points using sweep widths of 3289, 5208, 3289, and
10 000 Hz in ω1(13C), ω2(1H), ω3 (13C), and ω4 (1H), respectively.
The 13C spectral widths were folded in the same manner as in
the 4D [13C,1H,15N,1H] NOESY experiment. The 1H carrier was
centered at 3.7 ppm. In both 4D experiments, a 10-ms
homospoil was applied during the 50-ms NOESY mixing time
to suppress unwanted magnetization, and a series of randomly
spaced 90° pulses were used to saturate the 13C spins at the
beginning of the pulse sequence.21b
NOE-d er ived d ista n ce r estr a in ts a n d str u ctu r e ca l-
cu la tion s: The proton-proton distances used as restraints for
the structure calculations of the C24 deoxy derivative of
ascomycin were obtained by counting contours in the isotope-
filtered NOESY data set collected with a mixing time of 80
ms. NOEs were classified as either strong (1.8-2.8 Å), medium
(1.8-3.4 Å), or weak (1.8-4.4 Å). For distances involving
methyl groups, 1.0 Å was added to the upper bound to correct
for pseudoatom.22 A total of 126 NOE restraints were used
along with 5 lower bound restraints that were determined on
the basis of the lack of observable NOEs.
3D structures were calculated using a hybrid distance
geometry/dynamical simulated annealing protocol.14 Using the
XPLOR23 program and the NOE-derived distance restraints,
200 initial structures were generated and subjected to 200
steps of Powell restrained energy minimization to remove bad
van der Waals contacts. During this minimization, and
throughout the entire simulated annealing protocol, the NOE
force constant was maintained at 50 kcal‚mol-1‚Å-2, and
electrostatic terms were excluded. The minimization step was
followed by 7.5 ps of molecular dynamics (time-step of 3 fs) at
2000 K during which the van der Waals force constant was
decreased from its initial value of 20 kcal‚mol-1‚Å-2 to a value
of 0.003 kcal‚mol-1‚Å-2 while increasing all other force con-
stants (bond, angle, etc.). The structures were then cooled from
2000 to 100 K in steps of 50 K. Each step of the cooling process
consisted of 1.25 ps of restrained molecular dynamics (time-
step of 5 fs). The van der Waals force constant was increased
at each step by multiplying the previous value by 1.28 until a
final value of 4.0 kcal‚mol-1‚Å-2 was obtained. The van der
Waals radius was decreased stepwise to a final value of 0.8
times the value used in CHARM for Frepel.24 In the last stage
of the refinement, the structures were subjected to 1000 steps
of Powell restrained energy minimization.
Refer en ces
(1) (a) Bach, J . F. Cyclosporine in autoimmune diseases. Transplant.
Proc. 1989, 21, 97-113. (b) Feutren, G. Clinical experience with
sandimmune (cyclosporine) in autoimmune disease. Transplant.
Proc. 1992, 24, 55-60. (c) Kino, T.; Goto, T. Discovery of FK-
506 and update. Ann. N. Y. Acad. Sci. 1993, 685, 13-21. (d)
Klintmalm, G. A review of FK506: A new immunosuppressant
agent for the prevention and rescue of graft rejection. Trans-
plant. Rev. 1994, 8, 53-63. (e) Thomson, A. W.; Carroll, P. B.;
McCauley, J .; Woo, J .; Abu-Elmagd, K.; Starzl, T. E.; Van Thiel,
D. H. FK506: A novel immunosuppressant for treatment of
autoimmune disease. Rationale and preliminary clinical experi-
ence at the University of Pittsburgh. Springer Semin. Immu-
nopathol. 1993, 14, 323-344. (f) Thomson, A. W.; Starzl, T. E.
FK506 and autoimmune disease: perspective and prospects.
Autoimmunity 1992, 12, 303-313. (g) Chaudhuri, K.; Torley, H.;
Madhok, R. Cyclosporin. Br. J . Rheumatol. 1997, 36, 1016-1021.
(2) (a) Kino, T.; Hatanaka, H.; Hashimoto, M.; Nishiyama, M.; Goto,
T.; Okuhara, M.; Kohsaka, M.; Aoki, H.; Imanaka, H. FK-506, a
novel immunosuppressant isolated from
a Streptomyces I.
Fermentation, isolation, and physico-chemical and biological
characteristics. J . Antibiot. 1987, 40, 1249-1255. (b) Kino, T.;
Hatanaka, H.; Miyata, S.; Inamura, N.; Nishiyama, M.; Yajima,
T.; Goto, T.; Okuhara, M.; Kohsaka, M.; Aoki, H.; Ochiai, T.
FK506, a novel immunosuppressant isolated from a Streptomy-
ces II. Immunosuppressive effect of FK-506 in vitro. J . Antibiot.
1987, 40, 1256-1265.
(3) Kino, T.; Inamura, N.; Sakai, F.; Nakahara, K.; Goto, T.;
Okuhara, M.; Kohsaka, M.; Aoki, H.; Ochiai, T. Effect of FK-
506 on human mixed lymphocyte reaction in vitro. Transplant.
Proc. 1987, 19 (Suppl. 6), 36-39.
(4) (a) Schreiber, S. L.; Crabtree, G. R. The mechanism of action of
cyclosporin A and FK506. Immunol. Today 1992, 13, 136-142.
(b) Liu, J .; Farmer, J .; Lane, W. S.; Freidman, J .; Weissman, I.;
Schreiber, S. L. Calcineurin is a common target of cyclophilin-
cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66, 807-
815. (c) Friedman, J .; Weissman, I. Two cytoplasmic candidates
for immunophilin action are revealed by affinity for a new
cyclophilin: One in the presence and one in the absence of CsA.
Cell 1991, 66, 799. (d) 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. Inhibition of T cell signaling by
immunophilin-ligand complexes correlates with loss of cal-
cineurin phosphatase activity. Biochemistry 1992, 31, 3896-
3901.
(5) (a) Van Duyne, G. D.; Standaert, R. F.; Karplus, P. A.; Schreiber,
S. L.; Clardy, J . Atomic structure of FKBP-FK506, an immu-
nophilin-immunosuppressant complex. Science 1991, 252, 839-
842. (b) Michnick, S. W.; Rosen, M. K.; Wandless, T. J .; Karplus,
M.; Schreiber, S. L. Solution structure of FKBP, a rotamase
enzyme and receptor for FK506 and rapamycin. Science 1991,
252, 836-839. (c) Moore, J . M.; Peattie, D. A.; Fitzgibbon, M.
J .; Thomson, J . A. Solution structure of the major binding protein
for the immunosuppressant FK506. Nature 1991, 351, 248-250.
(d) Petros, A. M.; Gampe, R. T., J r.; Gemmecker, G.; Neri, P.;
Holzman, T. F.; Edalji, R.; Hochlowski, J.; Jackson, M.; McAlpine,
J .; Luly, J . R.; Pilot-Matias, T.; Pratt, S.; Fesik, S. W. NMR
studies of an FK-506 analogue, [U-13C]Ascomycin, bound to
FKBP: conformation and regions of ascomycin involved in
binding. J . Med. Chem. 1991, 34, 2925-2928. (e) Meadows, R.
P.; Nettesheim, D. G.; Xu, R. X.; Olejniczak, E. T.; Petros, A.
M.; Holzman, T. F.; Severin, J .; Gubbins, E.; Smith H.; Fesik,
S. W. Three-dimensional structure of the FK506 binding protein/
ascomycin complex in solution by heteronuclear three- and four-
dimensional NMR. Biochemistry 1993, 32, 754-765. (f) Xu, R.
X.; Meadows, R. P.; Fesik, S. W. Heteronuclear 3D NMR Studies
of water bound to an FK506 binding protein/immunosuppressant
complex. Biochemistry 1993, 32, 2473-2480.
Ack n ow led gm en t. We thank the Analytical Re-
search Department of Abbott Laboratories for spectral
measurements. We also thank L. Miller, T. Fey, R.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4461
(6) Hatanaka, H.; Kino, T.; Miyata, S.; Inamura, N.; Kuroda, A.;
Goto, T.; Tanaka, H.; Okuhara, M. FR-900520 and FR-900523,
novel immunosuppressants isolated from a Streptomyces II.
Fermentation, isolation and physico-chemical and biological
characteristics. J . Antibiot. 1988, 41, 1592-1601.
McAlpine, J .; Luly, J . R.; Pilot-Matias, T.; Pratt, S.; Fesik, S.
W. NMR studies of an FK-506 analogue, [U-13C]ascomycin,
bound to FKBP: conformation and regions of ascomycin involved
in binding. J . Med. Chem. 1991, 34, 2925-2928. (c) Wandless,
T. J .; Michnick, S. W.; Rosen, M. K.; Karplus, M.; Schreiber, S.
L. FK506 and rapamycin binding to FKBP: common elements
in immunophilin-ligand complexation. J . Am. Chem. Soc. 1991,
113, 2339-2341.
(7) Schreiber, S. L. Chemistry and biology of the immunophilins
and their immunosuppressive ligands. Science 1991, 251, 283-
287.
(8) Ikeda, Y.; Schultz, L. W.; Clardy, J .; Schreiber, S. L. Structural
basis for peptidomimicry by a natural product. J . Am. Chem.
Soc. 1994, 116, 4143-4144.
(9) Kawai, M. K.; Lane, B. C.; Hsieh, G. C.; Mollison, K. W.; Carter,
G. W.; Luly, J . R. Structure-activity profiles of macrolactam
immunosuppressant FK-506 analogues. FEBS Lett. 1993, 316,
107-113.
(10) (a) Arai, T.; Koyama, Y.; Suenaga, T.; Honda, H. Ascomycin, an
antifungal antibiotic. J . Antibiot. 1962, 15, 231-232. (b) Morisa-
ki, M.; Arai, T. Identity of immunosuppressant FR-900520 with
ascomycin. J . Antibiot. 1992, 45, 126-128. (c) Or, Y. S.; Clark,
R. F.; Xie, Q.; McAlpine, J .; Whittern, D. N.; Henry, R. H.; Luly,
J . R. The chemistry of ascomycin: Structure determination and
synthesis of pyrazole analogues. Tetrahedron 1993, 49, 8771-
8786. (d) Hatanaka, H.; Kino, T.; Miyata, S.; Inamura, N.;
Kuroda, A.; Goto, T.; Tanaka, H.; Okuhara, M. FR-900520 and
FR-900523, novel immunosuppressants isolated from a Strep-
tomyces II. Fermentation, isolation and physicochemical and
biological characteristics. J . Antibiot. 1988, 41, 1592-1601.
(11) Cooper, M. E.; Donald, D. K.; Hardern, D. N. World Patent WO
89 05304, 1989.
(12) Edalji, R.; Pilot-Matias, T. J .; Pratt, S. D.; Egan, D. A.; Severin,
J . M.; Gubbins, E. G.; Petros, A. M.; Fesik, S. W.; Burres, N. S.;
Holzman, T. F. High level expression of recombinant human FK-
binding protein from a fusion precursor. J . Protein Chem. 1992,
11, 213-223.
(13) Adapted for rats from: Mollison, K. W.; Fey, T. A.; Krause, R.
A.; Thomas, V. A.; Mehta, A. P.; Luly, J . R. Comparison of FK-
506, rapamycin, ascomycin, and cyclosporine in mouse models
of host-versus-graft disease and heterotopic heart transplanta-
tion. Ann. N. Y. Acad. Sci. 1993, 685, 55-57.
(14) Nilges, M.; Clore, G. M.; Gronenborn, A. M. Determination of
three-dimensional structures of proteins from interproton dis-
tance data by dynamical simulated annealing from a random
array of atoms. FEBS Lett. 1988, 239, 129-136.
(15) (a) Petros, A. M.; Gemmecker, G.; Neri, P.; Olejniczak, E. T.;
Nettesheim, D. Xu, R. X.; Gubbins, E. G.; Smith, H.; Fesik, S.
W. NMR studies of an FK-506 analogue, [U-13C]ascomycin,
bound to FK-506-binding protein. J . Med. Chem. 1992, 35, 2467-
2473. (b) Petros, A. M.; Gampe, R. T., J r.; Gemmecker, G.; Neri,
P.; Holzman, T. F.; Edalji, R.; Hochlowski, J .; J ackson, M.;
(16) Saito, H.; Tanaka, Y.; Nukada, K. The hydrogen bond studied
by nitrogen-14 nuclear magnetic resonances. III. A study of
electron redistribution associated with hydrogen bonding and
metal-complex formation of formamide and N-methylacetamide.
J . Am. Chem. Soc. 1971, 93, 1077-1081.
(17) Venters, R. A.; Calderone, T. L.; Spicer, L. D.; Fierke, C. A.
Uniform 13C isotope labeling of proteins with sodium acetate for
NMR studies: application to human carbonic anhydrase II.
Biochemistry 1991, 30, 4491-4494.
(18) Petros, A. M.; Kawai, M.; Luly, J . R.; Fesik, S. W. Conformation
of two nonimmunosuppressive FK506 analogues when bound to
FKBP by isotope-filtered NMR. FEBS Lett. 1992, 308, 309-314.
(19) Kay, L. E.; Clore, G. M.; Bax, A.; Gronenborn, A. M. Four-
dimensional heteronuclear triple-resonance NMR spectroscopy
of interleukin-1â in solution. Science 1990, 249, 411-414.
(20) Messerle, B. A.; Wider, G.; Otting, G.; Weber, C.; Wu¨thrich, K.
Solvent suppression using a spin lock in 2D and 3D NMR
spectroscopy with H2O solutions. J . Magn. Reson. 1989, 85, 608-
613.
(21) (a) Zuiderweg, E. R. P.; Petros, A. M.; Fesik, S. W.; Olejniczak,
E. T. Four-dimensional [13C,1H,13C,1H] HMQC-NOE-HMQC
NMR spectroscopy: Resolving tertiary NOE distance constraints
in the spectra of larger proteins. J . Am. Chem. Soc. 1991, 113,
370-372. (b) Clore, G. M.; Kay, L. E.; Bax, A.; Gronenborn, A.
M. Four-dimensional 13C/13C-edited nuclear overhauser enhance-
ment spectroscopy of a protein in solution: application to
interleukin 1â. Biochemistry 1991, 30, 12-18.
(22) Wu¨thrich, K.; Billeter, M.; Braun, W. Pseudo-structures for the
20 common amino acids for use in studies of protein conforma-
tions by measurements of intramolecular proton-proton dis-
tance constraints with nuclear magnetic resonance. J . Mol. Biol.
1983, 169, 949-961.
(23) Kuszewski, J .; Nilges, M.; Bru¨nger, A. T. Sampling and efficiency
of metric matrix distance geometry: A novel partial metrization
algorithm. J . Biomol. NMR 1992, 2, 33-56.
(24) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J .;
Swaminathan, S.; Karplus, M. CHARMM: A program for
macromolecular energy, minimization, and dynamics calcula-
tions. J . Comput. Chem. 1983, 4, 187-217.
J M980252Z