Oligopeptide cyclophilin inhibitors: A reassessment

Article abstract of DOI:10.1016/j.ejmech.2011.09.019

Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory interaction of linear proline-containing peptides with the active site of CypA can be increased through a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA, a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the context of the CsA framework a net charge of -7 clustered at the amino acid side chain of position 1 resulted in slightly improved CypA inhibition.

Full text of DOI:10.1016/j.ejmech.2011.09.019

European Journal of Medicinal Chemistry 46 (2011) 5556e5561
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Oligopeptide cyclophilin inhibitors: A reassessment
*
Michael Schumann, Günther Jahreis, Viktoria Kahlert, Christian Lücke, Gunter Fischer
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives
have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans
isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate
peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory
interaction of linear proline-containing peptides with the active site of CypA can be increased through
a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported
for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory
CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA,
a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit
CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the
context of the CsA framework a net charge of e7 clustered at the amino acid side chain of position 1
resulted in slightly improved CypA inhibition.
Received 1 September 2011
Received in revised form
12 September 2011
Accepted 14 September 2011
Available online 21 September 2011
Keywords:
Cyclophilin
Cyclosporin A
Prolyl bond cis/trans isomerization
Inhibition
cis/trans ratio
Isothermal titration calorimetry
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
direction of the contacting sites in their respective complexes
topologies are defined by the superposition of the proline ring of
Cyclophilins represent a subfamily of peptidyl prolyl cis/trans
isomerases (PPIases, EC. 5.2.1.8) that can bind and catalytically
interconvert prolyl cis/trans isomers in all folding states of
a proline-containing polypeptide chain. The functional importance
of the prototypic enzyme cyclophilin A (CypA) for a variety of
biological processes such as viral infections [1e4], chronic inflam-
mation [5e8] and malignancies [9e13] has stimulated a consider-
able interest in the development of CypA inhibitors as mechanistic
tools and potential drugs for various diseases.
The immunosuppressive cyclic undecapeptide cyclosporin A
(CsA) and non-immunosuppressive CsA derivatives represent low-
nanomolar inhibitors of the PPIase activity of CypA [14,15]. Beside
inhibition of CypA catalytic activity, CypAeCsA complex formation
also precedes calcineurin inhibition and subsequent suppression of
IL-2 expression which may explain how CsA mediated immuno-
suppression can arise [16]. Non-peptidic small molecule inhibitors
have been designed but require considerable improvement to
exhibit CsA-like inhibitory potencies [17]. While the active cleft of
CypA orients CsA and oligopeptide substrates in the opposite
the substrate and the MeVal-11 side chain of CsA [18]. The marked
increase of about >104 fold in the CypA affinity for CsA when
compared to a linear oligopeptide substrate can be rationalized by
the transition state-like structural constraints imposed by the
macrocycle [19]. In an extensive search of heptapeptide sequences
derived from the HIV-1 Gag polyprotein capsid domain containing
-Xaa-Gly-Pro- moieties only very weak CypA binders could be
identified [20]. On the other hand, the internally positioned -Gly-
Pro- sequence motif was found as an essential feature for CypA
binders identified by phage display screening and by planar peptide
arrays [21]. Consequently, the question arises whether linear
peptide inhibitors of low molecular masses, whose particular
amino acid sequences and internal charge distributions allow for
a transition state complementary geometry, might exist. Recently,
based on the identification of critical residues of CypA for the
CypAeCsA interaction by calculating the electronic structure of
both molecules [22], a virtual oligopeptide library database has
been ranked for CypA affinity by using a molecular docking algo-
rithm [23]. Interestingly, the calculations have resulted in a few
charged oligopeptides that exhibited interaction energies superior
to those of the CypAeCsA interaction. Based on these calculations
the authors have carried out an experimental approach with PPIase
assays, surface plasmon resonance analyses and viral replication
data. The inhibitory tripeptide Trp-Gly-Pro eventually emerged,
exhibiting a CypA binding and inhibiting affinity nearly as strong as
Abbreviations: CypA, cyclophilin A; CsA, cyclosporin A; PPIase, peptidyl prolyl
cis/trans isomerase; ITC, isothermal titration calorimetry.
* Corresponding author. Tel.: þ49 345 5522800; fax: þ49 345 5511972.
E-mail address: fischer@enzyme-halle.mpg.de (G. Fischer).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.019

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561
5557
those found for CsA. Even more, marked antiviral effects in a HIV-1
replication assay have been noted [23]. From the cellular viewpoint,
these findings would be particularly intriguing since they implicate
protein degradation products, especially those resulting from
catalysis by proline-specific proteases, in the regulation of endog-
enous CypA activity.
Structurally, the major differences found between previously
tested peptides and Trp-Gly-Pro were found in the charge distri-
bution next to the critical proline and the presence of a Trp residue.
To explain the possible origin for the said CsA-like biochemical
effects, the interplay between a negative charge at the backbone
and a potentially Trp-mediated increase of the cis/trans ratio was
considered to play a dominating role.
Here we investigate anionic peptides in conjunction with an
NMR-based analysis of prolyl cis/trans isomers to evaluate their
CypA inhibitory potency. We show below that various PPIase assays
and isothermal titration calorimetry revealed a drastic loss of CypA-
peptide complex stability for peptides bearing a negative charge
next to proline on the backbone. Taken together, our results do not
only contradict CypA inhibition but furthermore indicate a lack of
affinity of short linear oligopeptides, such as Trp-Gly-Pro, for CypA.
utilized to analyze CypA inhibitor interactions [24]. Our ITC
experiments indicated an exothermally formed CypAeCsA complex
(
D
H ¼ 10.6 kcal molꢀ1) with a Kd value of 7.6 nM together with
a complex stoichiometry of 1.02 in good agreement with published
data [24,25]. However, under the same conditions, titration of Trp-
Gly-Pro with CypA did not indicate any binding up to 25
in the titration cell (Fig. 1).
mM peptide
It can be hypothesized that the reportedly high affinity of Trp-
Gly-Pro resulted from the combination of a high cis prolyl isomer
content and a negatively charged proline. Indeed, 1H NMR inves-
tigations on linear oligopeptide substrates revealed that the KM
value of a cis isomer is significantly lower than that of the respective
trans isomer [26,27]. It is also known that proline-containing linear
peptides undergo relatively slow cis/trans equilibration after
dissolution in aqueous buffer and that this might lead to a lag and
latency issue for the cis isomer content. It follows that very slow
isomerization kinetics might be able to disturb our ITC experiment
[28]. Thus, we characterized the prolyl isomer composition of Trp-
Gly-Pro in buffer solution at pH 7.5 by NMR spectroscopy.
2.2. Isomer-specific NMR signals
2. Results and discussion
To identify isomer-specific 1H and 13C resonances of Trp-Gly-Pro
the signals were completely assigned by means of homo- and
heteronuclear 2D NMR spectra (Supplementary Tables S1 and S2).
The trans and cis prolyl bond isomers were identified based on the
2.1. Isothermal titration calorimetry
Direct comparison of surface plasmon resonance, which has
been used as an activity independent binding assay by Pang et al.
[23], and isothermal titration calorimetry (ITC) has been already
C
b
and C
sequential ROEs between Gly2 H
strong resonance overlap, the cis/trans ratio was obtained from the
g
chemical shift values [29], and additionally confirmed by
a
and either Pro3 H or H . Due to
a
d
Fig. 1. Representative isothermal titration calorimetry of CsA (A) and Trp-Gly-Pro (B) with CypA in 10 mM HEPES buffer pH 7.5, 100 mM NaCl at 20 ꢁC. For the CsA experiment each
peak corresponds to the injection of 15 l of 100 M cyclophilin into the titration cell containing 10 M CsA. For the tripeptide the titration cell contained 10 M CypA which was
titrated with 100 M peptide solution. Capacity of the titration cell was 1.4 ml. Reference titration of buffer versus buffer, CypA versus buffer and peptide versus buffer were
m
m
m
m
m
separately obtained and subtracted from the thermogram of the sample titrations. Cumulative heat of reaction is displayed as a function of the mole ratio of inhibitor/CypA (lower
part). The solid line of the CsA titration represents the least square fit of the compiled data points. (A) CsA binding to CypA gave an association constant Ka ¼ (1.3 ꢂ 0.6) ꢃ 108 M,
a complex stoichiometry N ¼ 1.023 ꢂ 0.08 and a reaction enthalpy
D
H ¼ 10.6 ꢂ 0.1 kcal molꢀ1 (B) CypA did not show any affinity for the tripeptide in the isothermal titration
calorimetry.

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M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561
2D NMR data of Trp-Gly-Pro. Interestingly, under the solvent
conditions used in the PPIase assays, this tripeptide showed a high
cis content of 41% as based on integration of the CaH signals in the
1H/13C-HSQC spectrum (Fig. 2). This finding opens up the possibility
of assuming the cis isomers of linear peptides bearing a negative
charge as highly inhibitory toward CypA. We note that in the NMR
experiments the cis/trans isomerization rate was shown to be fast
enough to equilibrate the isomers within 30 min after dissolution
indicating that the ITC experiment was not corrupted by the
isomerization kinetics.
2.3. Inhibition of PPIase activity
We have carried out CypA inhibition experiments where Trp-
Gly-Pro was allowed to preequilibrate in HEPES buffer pH 7.5,
100 mM NaCl prior to testing.
Surprisingly, no inhibition of CypA could be seen in the
chymotrypsin-coupled standard PPIase assay even at high concen-
trations of Trp-Gly-Pro. According to the reported IC50 value of
Fig. 3. Inhibition of the PPIase activity of cyclophilin A (CypA) by Trp-Gly-Pro (trian-
gles), Succinyl-Ala-Ala-Pro-Phe (squares), VK-053 (diamonds), and cyclosporin A (CsA)
(circles) in the protease-coupled assay. The PPIase activity of 0.5 nM CypA was
measured in 10 mM HEPES buffer pH 7.5, 100 mM NaCl at 10 ꢁC using Succinyl-Ala-Ala-
Pro-Phe-4-nitroanilide as substrate. Each data point represents the average of three
measurements. The error bars represent standard deviation. PPIase activity of CypA
was inhibited by VK-053 and CsA. Drawn lines were calculated from the hyperbolic
equation using an IC50 value of 4.9 ꢂ 0.4 nM (VK-053) and 9.3 ꢂ 1.5 nM, (CsA),
respectively.
33.1 nM [23], a negligible residual activity should result at 100 mM
Trp-Gly-Pro in this assay contrasting the almost uninhibited isom-
erization catalysis depicted in Fig. 3. However, under similar
conditions our experiments revealed an IC50 value of 9.5 ꢂ 1.5 nM
(Fig. 3) for CsAwhich is within the range of inhibition data published
elsewhere [25].
We then examined the effects of increasing concentrations of
another anionic peptide, Succinyl-Ala-Ala-Pro-Phe on the PPIase
activity of CypA. This peptide is an intrinsic constituent in the
chymotrypsin-coupled standard PPIase assay [24] because it is
increasingly present in the assay mixture bycleavage of Succinyl-Ala-
Ala-Pro-Phe-4-nitroanilide as the isomerization reaction proceeds.
While tetrapeptide 4-nitroanilides bind CypA with KM values in the
high micromolar range [26,27], their peptidic fragment of proteolysis
failed to compete in the catalyzed substrate isomerization up to
In the light of the above findings it is important to analyze
whether the net negative charge of an inhibitor is generally
repulsive for its interaction with CypA. Given that the very low
CypA affinity of linear oligopeptides makes them unsuitable for
detecting small effects, the CsA framework would provide a sensi-
tive probe of binding energy changes.
The CsA derivative VK-053 has been synthesized and charac-
terized in a PPIase assay by comparison with CsA. Seven carboxylate
residues covalently added to the side chain of MeBmt amino acid at
the 1-position of CsA (Supplementary Scheme S1) suffice to
100 mM concentrations (Fig. 3). Lack of active site binding affinity of
the anionic peptide is in accord with the strict first-order kinetics
observed in the time courses of protease-coupled PPIase assays [31].
This result is reminiscent to the PPIase Pin1 which is mechanistically
related to CypA. Here, prevention of a negatively charged peptide
backbone along with chain elongation increased binding to the active
site of Pin1 more than ten-fold [32].
implement
a moderate increase in the inhibitory potency
(4.9 ꢂ 0.4 nM) when compared to CsA (Fig. 3). We note that even
clustering the negative charge adjacent to the binding site of
a transition state-like CypA inhibitor did not impair its intrinsically
high affinity.
Fig. 4. Inhibition of the PPIase activity of cyclophilin A (CypA) by Trp-Gly-Pro in the
protease-free assay [29]. The PPIase activity of 0.5 nM CypA was measured in 10 mM
HEPES buffer (pH 7.5), 100 mM NaCl at 10 ꢁC using Succinyl-Ala-Ala-Pro-Phe-4-
nitroanilide as substrate.
Fig. 2. Section from the 1H/13C-HSQC spectrum of Trp-Gly-Pro (in 50 mM phosphate
buffer, pH 7.5, 25 ꢁC) displaying only the C
a
H signals of the trans and cis prolyl bond
conformers. The 1D 1H trace is shown on the top.

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561
5559
To confirm that the chymotrypsin content in the protease-
coupled PPIase assay did not deteriorate the inhibitory nature of
Trp-Gly-Pro, a protease-free PPIase assay was utilized [30]. How-
ever, the pattern of the doseeactivity curve was found to be similar
to that of the protease-coupled assay, confirming the inert nature of
Trp-Gly-Pro in the CypA activity assays (Fig. 4).
Succinyl-Ala-Ala-Pro-Phe-OH, Mmcalc 504.5, Mmfound 505.0,
Rt ¼ 9.9 min; Trp-Gly-Pro-OH, Mmcalc 358.4, Mmfound 359.0,
Rt ¼ 9.7 min.
4.2. VK-053 (MeBmt-SCH2CH2CO-NH(D
-Glu)6-Gly-OH]1-CsA)
VK-053 was synthesized from CsA as depicted in Supplementary
Scheme S1. CsA was purchased from LC Laboratories with >99%
purity. Silica and RP18 columns for flash chromatography were
used from Teledyne Isco. All other reagents and chemicals were
received from commercial sources in analytical grade and were
used without further purification.
Analytical HPLC runs were executed on DIONEX equipment or
SYKAM equipment. MALDI-TOF mass spectra were measured at
Ultraflex II TOF/TOF, BrukereDaltonik GmbH, and electrospray
ionization (ESI) mass spectra were measured at ESI-Iontrap
ESQUIRE-LC, BrukereDaltonik GmbH.
3. Conclusions
We have presented a detailed study of the active site directed
interaction of CypA with linear peptides and cyclosporins with the
aim of elucidating the effects of negative charges and cis isomer
content on CypA inhibition. Our results were shown to be in
complete disagreement with the recent predictions of molecular
docking simulations and subsequent experimental findings that
a charged linear tripeptide realizes a CsA-like inhibitory efficiency
toward CypA. In contrast, we have shown that short proline-
containing peptides behave inert in CypA-catalyzed prolyl isom-
erizations up to high micromolar concentrations. Furthermore, we
have examined the question of the charge dependence of CypA
inhibition using a 1-position substituted CsA derivative containing
a clustered net charge of ꢀ7. A small improvement of the inhibitory
potency could be achieved indicating that the active site of CypA
tolerates anionic residues in the transition state-like inhibitors.
Finally, our findings suggest that endogenous CypA will maintain its
full enzymatic activity in the presence of most short oligopeptides
resulting from protein degradation in the cell. Together, these
findings indicate that CypA-oligopeptide interaction is opposed by
electrostatic repulsion between a negative charge close to the
reactive site of a substrate-like inhibitor and CypA and that a similar
charge, if located more distant to the active site, appears to
augment inhibition in the context of a transition state-like peptide
inhibitor conformation. Obviously, this conformation cannot be
realized by Trp-Gly-Pro. These results also demand reassessment of
previous findings that the protective effect of Trp-Gly-Pro against
HIV-1IIIB replication is due to cyclophilin inhibition.
4.2.1. [O-Acetyl]1-CsA
CsA (2 g, 1.664 mmol) and 4-(dimethylamino)pyridine
(331.4 mg, 2.71 mmol) were added to acetic anhydride (20 ml) and
stirred at room temperature overnight. The solvent was removed in
vacuo and the residue was purified by flash chromatography
(solvent A dichloromethane, solvent B methanol, silica column
24 g). Method: 20 ꢁC, flow 30 ml/min, 5 min isocratic 0% B in 7 min
to 4.8% B followed by isocratic 9 min with 4.8% B. Yield, 96%, light
yellow solid. Analytical HPLC, solvent A 0.1% TFA/H2O, solvent B
0.1% TFA/acetonitrile, RP C8, 250.00 ꢃ 4.600 mm. Method: 65 ꢁC,
50e100% B in 30 min, flow 1 ml/min.
[O-Acetyl]1-CsA, Mmcalc 1244.9, Mmfound 1245.0, Rt ¼ 16.5 min.
4.2.2. [O-Acetyl]1-CsA bromide
Azobisisobutyronitril (10 mg) was added to a solution of [O-
Acetyl]1-CsA (500 mg, 0.402 mmol) and N-bromosuccinimide
(1.25 eq., 90 mg, 0.5024 mmol) in carbon tetrachloride (75 ml) and
the mixture was heated to reflux for 3 h. The solvent was removed
under reduced pressure. Residue material was taken up in ethyl
acetate (150 ml), washed twice with brine, dried over MgSO4 and
filtered. The solvent was removed under reduced pressure. The
crude product was used for further reactions.
4. Experimental protocols
4.1. Peptide synthesis
Analytical HPLC: solvent A 0.1% TFA/H2O, solvent B 0.1% TFA/
acetonitrile, column RP C8, 250.00 ꢃ 4.600 mm. Method: 65 ꢁC,
50e100% B in 30 min, flow 1 ml/min.
Peptides were synthesized by solid-phase peptide synthesis
with the robot SYRO II (MultiSynTech, Witten, Germany) using
0.15 mmol of preloaded Fmoc-phenylalanine- or Fmoc-proline-2-
chlorotrityl resins (Novabiochem, Merck-Chemicals, Darmstadt,
Germany). The syntheses were performed by Fmoc strategy and
standard protocol (PyBOP/N-methyl-morpholine in DMF). Piperi-
dine (20%) in DMF was the standard cleavage cocktail used for Fmoc
detachment.
Succinylation was done with succinic anhydride, N-ethyl-
diisopropylamine and 4-(dimethylamino)-pyridine (Merck-Chem-
icals, Darmstadt, Germany) in DMF.
After synthesis, peptides were cleaved from the resin with TFA/
TIS/water (95:3:2, v/v/v) for 2 h and purified with a preparative
HPLC (Abimed, Langenfeld, Germany) on an Interchrom Modulo-
Cart Strategy 5, C18-2, 250 ꢃ 10 column (Interchim, Montlucon,
France) using a water/ACN gradient containing 0.1% TFA in the
solvents. Peptides were detected at 220 nm.
[O-Acetyl]1-CsA bromide, Mmcalc 1322.8, Mmfound 1344.8
[M þ Na]þ, Rt ¼ 16.9 min.
4.2.3. [MeBmt-SCH2CH2COOH]1-CsA
Methyl 3-mercaptopropionate (1.5 eq., 72.6
ml, 0.67 mmol)
and Cs2CO3 (1 eq., 130 mg, 0.402 mmol) were added to a solution
of the crude [O-Acetyl]1-CsA bromide (0.402 mmol) in acetoni-
trile (50 ml) were added (1 eq., 130 mg, 0.402 mmol dissolved in
20 ml water). The solution was stirred at room temperature
overnight.
The solvent was removed under reduced pressure and replaced
by methanol (30 ml). After the addition of 0.2 M aqueous LiOH
(30 ml) the mixture was stirred at room temperature for 3 h. The
reaction was acidified with diluted HCl and the solvent was evap-
orated. The product was purified by flash chromatography. Solvent
A 0.1% AcOH/H2O, Solvent B 0.1% AcOH/acetonitrile, RP C18, 130 g.
Method: 20 ꢁC, flow 75 ml/min, 5 min isocratic 65% B in
5 mine100% B, 7 min isocratic 100% B.
The purified peptides were lyophilized and their purity was veri-
fied by analytical HPLC using a LiChroCARTÒ column (LiChrospherÒ
100, RP8, 5
m
m; 125 ꢃ 4 mm) (Merck, Darmstadt, Germany) with the
following conditions: gradient of 5e100% ACN in water (with 0.1%
TFA) at a flow rate of 1 ml/min over 30 min; and UV-detection at
220 nm. The molecular masses of the peptides were confirmed by ESI
mass spectrometry.
Yield, 34% (relative to [O-Acetyl]1-CsA); white solid. Analytical
HPLC, solvent A 0.1% TFA/H2O, solvent B 0.1% TFA/acetonitrile, RP
C8, 125.00 ꢃ 4.600 mm. Method: 20 ꢁC, 50e100% B in 30 min, flow
1 ml/min.

5560
M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561
[MeBmt-SCH2CH2COOH]1-CsA, Mmcalc 1306.8, Mmfound 1306.9,
sequence. 1H and 13C chemical shifts were referenced to external
Rt ¼ 8.7 min.
2,2-dimethyl-2-silapentane-5-sulfonate.
4.2.4. [MeBmt-SCH2CH2CO-NH(
D
-Glu)6-Gly-OH]1-CsA
l, 0.0345 mmol) and 2-(1H-7-
4.5. Isothermal titration calorimetry
N-ethyldiisopropylamine (5.9
m
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoropho-
sphate (HATU) (4.8 mg, 0.01265 mmol) were added to a solution of
[MeBmt-SCH2CH2COOH]1-CsA (15 mg, 0.0115 mmol) in dry DMF
(1 ml) and stirred for 5 min at room temperature. Then, a solution
Titration experiments were performed in 10 mM HEPES buffer
pH 7.5 (AppliChem A1069, Darmstadt, Germany), 100 mM NaCl
using a MicroCal VP-ITC at 20 ꢁC. Protein samples were dialyzed
against the batch of buffer used for titration. A peptide stock
solution was prepared, and prior to the experiment, it was diluted
into the same batch of buffer used for dialysis. All solutions used
were degassed before filling the sample cell and syringe. The ITC
stirring speed was set to 310 rpm. For titration of CsA with CypA
of H-(
D-Glu)6-Gly-OH (14.7 mg, 0.01725 mmol) and N-ethyl-
diisopropylamine (5.9
ml, 0.0345 mmol) in dry DMF (1 ml) was
added and the mixture was stirred at room temperature overnight.
The reaction was acidified with diluted HCl and the solvent was
evaporated. Preparative HPLC, solvent A 0.05% TFA/H2O, solvent B
0.05% TFA/acetonitrile, RP C8 250 ꢃ 21 mm. Method: 20 ꢁC, 17 ml/
min, gradient 30e80% B in 60 min. Yield: 73%; white solid.
Analytical HPLC, solvent A 0.05% TFA/H2O, solvent B 0.05% TFA/
acetonitrile, RP C8, 125.00 ꢃ 4.600 mm. Method: 20 ꢁC, 5e100% B in
30 min, flow 1 ml/min.
300
in 15
a 100
m
l of 100
l steps. For titration of Trp-Gly-Pro with CypA 300
M peptide solution were added to 1.4 ml of 10 M CypA in
m
M CypA were added to 1.4 ml of a 10
m
M CsA solution
m
m
ml of
m
15
ml steps. Since the initial injection generally delivers inaccurate
data, only 2
ml were injected in this step. For ITC of CsA with CypA,
the recorded data of this first step were discarded. Reference
titration of buffer versus buffer, CypA versus buffer and peptide
versus buffer were separately obtained and subtracted from the
thermogram of the sample titrations. Binding isotherms were fitted
according to a one-site model of binding. Errors correspond to the
standard deviations of the non-linear least-squares fit of the data
points of the titration curve.
[MeBmt-SCH2CH2CO-NH(D
-Glu)6-Gly-OH]1-CsA, Mmcalc 2138.1,
Mmfound 2138.2, Rt ¼ 16.4 min.
4.3. PPIase assays
4.3.1. Protease-coupled assay
Human recombinant CypA was prepared as described elsewhere
[25]. This assay was done according to a published procedure [15]
at 10.0 ꢁC in 10 mM HEPES buffer pH 7.5 (AppliChem A1069,
Darmstadt, Germany), 100 mM NaCl, 2 nM bovine serum albumin
(SigmaeAldrich A1900, Steinheim, Germany).
Acknowledgments
This study was supported by the DFG SFB 610 and the BMBF
Project ProNet3. We are grateful to Dr. Angelika Schierhorn and
Suzanne Roß for experimental support.
4.3.2. Protease-free assay
Succinyl-Ala-Ala-Pro-Phe-4-nitroanilide (18.7 mg, Bachem L-
Appendix. Supplementary material
1400, Bubendorf, Switzerland) was dissolved in 1 ml anhydrous
0.55 M LiCl/TFE solvent mixture and stored at 4 ꢁC. Solvent jumps
were initiated by addition of an aliquot of stock solution of peptide
in 0.55 M LiCl/TFE into 10 mM HEPES buffer pH 7.5 (AppliChem
A1069, Darmstadt, Germany), 0.5 nM human recombinant CypA,
2.0 nM bovine serum albumin (SigmaeAldrich A1900, Steinheim,
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.09.019.
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