Cyclophilin Inhibition by a (Z)-Alkene cis-Proline Mimic

Full text of DOI:10.1021/jo990409a

2998
J . Org. Chem. 1999, 64, 2998-2999
Cyclop h ilin In h ibition by a (Z)-Alk en e
cis-P r olin e Mim ic
Scott A. Hart and Felicia A. Etzkorn*,†
Department of Chemistry, University of Virginia,
McCormick Road, Charlottesville, Virginia 22901
F igu r e 1. cis/ trans-Pro equilibrium and (Z)-alkene Ala-cis-Pro
mimic 1.
Received March 8, 1999
The peptidyl-prolyl isomerases (PPIases) cyclophilin (CyP)
and FK506 binding protein (FKBP) catalyze the cis-trans
isomerization of Xaa-Pro amide bonds. Several compounds
that bind these enzymes, including cyclosporin and FK506,
are potent immunosuppressants,1,2 although the immuno-
suppressant activity is independent of PPIase activity.3-7
Understanding the mechanism by which these enzymes
catalyze cis-trans amide isomerization will help elucidate
the native roles of these ubiquitous enzymes in cellular
processes.8 We present here a (Z)-alkene-based substrate
mimic that inhibits the PPIase activity of human cyclophilin
A (hCyPA).
Proline is unique among the 20 natural amino acids due
to its dialkylated amine, which causes amide bonds preced-
ing proline to adopt a significantly higher percentage of cis
configuration than other amino acids. Xaa-cis-Pro amides
appear in 10-30% of short prolyl peptides9 (Figure 1) and
approximately 6% of Xaa-Pro amides in proteins of known
structure.10 This relatively high occurrence of cis amides
complicates protein folding when a particular prolyl amide
must isomerize before a protein can reach its native folded
structure. On the time scale of protein folding, thermal cis-
trans amide isomerization is slow, leading to slow steps in
the folding of proline-containing proteins.9,11-13 PPIases
facilitate this process in nature by catalyzing prolyl cis-
trans amide isomerization. A twisted amide mechanism was
proposed on the basis of both the observation that the keto-
carbonyl of the FK506 R-keto amide was orthogonal to the
amide plane in the bound conformation14-16 and secondary
kinetic isotope effects.17-19 More recently, it has been sug-
gested that these enzymes bind the Xaa-Pro substrate and
distort the amide bond by pyramidalizing the prolyl nitrogen
through hydrogen bonding.20-22 It was proposed that the
hydrogen bond in FKBP is donated intramolecularly from
the prolyl C-terminal amide in the substrate,20-22 while the
active site Arg-55 is proposed to act as the hydrogen donor
in hCyPA, since the orientation of the substrate in the
hCyPA active site does not allow a similar intramolecular
hydrogen bond.8 Indeed, the Arg55Ala mutant was catalyti-
cally inactive yet retained the ability to bind cyclosporin.3,8
While the immunosuppression exhibited by cyclosporin
and FK506 is independent of PPIase activity, it has been
shown these drugs do bind in the isomerase active site23,24
and are competitive inhibitors of PPIase activity.25 A better
mechanistic understanding may lead to other drugs divorced
of immunosuppression activity, as well as insights into other
PPIase dependent processes such as protein folding13 and
related chaperone activity,26 voltage gating in ion channels,27
maturation and infectivity of HIV-1,28-30 selectivity of Xaa-
Pro amides by HIV-1 protease,31 molecular switching in the
HIV-1 capsid protein,32 and regulation of mitosis by the
newly discovered PPIase Pin1.33-35
Structural studies have demonstrated that CyP is highly
selective for cis substrates. The tetrapeptide Ac-Ala-Ala-Pro-
Ala-amidomethylcoumarin was shown bound in the hCyPA
active site with a cis conformation about the central Ala-
Pro amide.36 Crystallography has also demonstrated that the
Ala-Pro dipeptide binds to hCyPA in the cis conformation.37
Additionally, the tripeptide succinyl-Ala-Pro-Ala-p-nitro-
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† Tel: (804)-924-3135. Fax: (804)-924-3567. email: etzkorn@virginia.edu.
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10.1021/jo990409a CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/14/1999

Communications
J . Org. Chem., Vol. 64, No. 9, 1999 2999
Sch em e 1
F igu r e 2. % inhibition vs concentration of 4 (µM). Calculated
value of IC50 ) 6.5 ( 0.5 µM was from a logarithmic fit by
Kaleidagraph.
and chymotrypsin, over the course of approximately 1 h at
room temperature, the amount of pNA released corre-
sponded to the total amount of inhibitor present, confirming
that chymotrypsin was not simply cleaving a small amount
of (E)-alkene impurity. The presence of hCyPA had no affect
on the rate of pNA release from 4, eliminating the mecha-
nistically intriguing possibility of alkene isomerization by
hCyPA.
anilide crystallized in the active site of Escherichia coli CyP
was found in the cis conformation.38 These observations take
on greater significance considering that 70-90% of small
prolyl peptides in solution are trans.9
We previously reported the stereoselective synthesis of 1,
a (Z)-alkene cis-Pro mimic of the Ala-Pro dipeptide (Figure
1),39 by a route similar to the stereoselective synthesis of
(E)-alkene non-proline dipeptide isosteres.40 The (Z)-alkene
cis-Pro mimic overlays the analogous dipeptide on the two
vectors shown in Figure 1 with an RMS deviation of 0.17 Å,
indicating that it is an ideal conformationally locked mimic
of the bound substrate.39 Succinyl-Ala-Ala-Pro-Phe-p-nitro-
anilide (succ-AAPF-pNA) is an excellent short peptide
substrate for hCyPA, with a Km(cis) of 870 µM.25 We designed
mimic 4 based directly on this substrate by replacing the
central Ala-Pro core with (Z)-alkene cis-Pro mimic 1.
The peptide mimic was synthesized as shown in Scheme
1. Amides were coupled in solution using HBTU, and the
succinyl N-terminus was installed with succinic anhydride.
The sterically demanding coupling of acid 1 proceeded in
good yield (73%). Concerns about â,γ-unsaturated acid or
amide isomerization to the R,â-unsaturated compound proved
unfounded, as no migration of the alkene was observed in
any of the reactions.39
The chymotrypsin coupled assay was used to evaluate
inhibition.3,41,42 Final substrate (succ-AAPF-pNA) concentra-
tion was 100 µM (10 µM cis),25 hCyPA concentration was
20 nM, and the concentration of inhibitor 4 was varied from
0.5 to 240 µM. Progress of the assays was monitored by
release of p-nitroaniline (pNA), since it is known that chymo-
trypsin hydrolyzes only trans substrates.41 However, we
observed that chymotrypsin recognized peptide mimic 4 and
slowly released pNA in the absence of hCyPA, indicating
that chymotrypsin may recognize cis substrates, although
poorly. The amount of pNA released from 4 during a typical
assay was approximately 0.6% of the total inhibitor concen-
tration. In control experiments containing only inhibitor 4
Due to p-nitroaniline insolubility, steady-state data to
determine competitive inhibition could not be obtained by
this assay.25 The IC50 was determined to be 6.5 ( 0.5 µM
from a plot of percent inhibition vs inhibitor concentration
(Figure 2). A bicyclic lactam cis-Pro mimic has been found
to bind hCyPA with a Kd of 5 µM by fluorescence satura-
tion.43-45 An (E)-alkene trans-Pro mimic included in an
FKBP substrate sequence was shown to inhibit the PPIase
activity of FKBP with a Ki of 8.6 µM.46 Since cis substrates
have been found several times in the active site of CyP, it is
interesting that a trans substrate mimic inhibited FKBP
PPIase activity with a similar magnitude. FKBP is known
to bind R-keto amides with the ketone orthogonal to a cis
amide,15 which led to the hypothesis of a twisted amide tran-
sition state.14,16 Unlike CyP, no structures of FKBPs have
been reported with peptide substrates bound, so the prefer-
ence for the cis or trans conformation is unknown, and these
two inhibitor/PPIase complexes cannot be compared directly.
We have designed and synthesized an inhibitor of cyclo-
philin based on a (Z)-alkene amide bond isostere. The central
Ala-cis-Pro core of the substrate succ-AAPF-pNA was re-
placed by a (Z)-alkene isostere. Substrate mimic 4 inhibits
the PPIase activity of hCyPA with an IC50 of 6.5 ( 0.5 µM.
Ack n ow led gm en t. This work was supported by J ef-
fress Foundation Grant J -355 and NIH Grant GM52516-
01. We thank Professor Mark Lipton and J ennifer Kowalski
of Purdue University for generously providing purified
hCyPA protein.
Su p p or tin g In for m a tion Ava ila ble: Experimental proce-
dures for assays, synthesis, and characterization of compounds 2,
3, and 4. This material is available free of charge via the Internet
at http://pubs.acs.org.
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