Facile synthesis of a fluorescent cyclosporin A analogue to study cyclophilin 40 and cyclophilin 18 ligands

Article abstract of DOI:10.1021/ml1001272

There are strong indications for the involvement of cyclophilin 40 in diseases caused by misregulation of steroid hormone receptors, like prostate or breast cancer. To identify novel inhibitors for this immunophilin, we developed a simplified fluorescence

Full text of DOI:10.1021/ml1001272

TECHNOLOGY NOTE
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Facile Synthesis of a Fluorescent Cyclosporin A Analogue
To Study Cyclophilin 40 and Cyclophilin 18 Ligands
Steffen Gaali,† Christian Kozany,† Bastiaan Hoogeland, Marielle Klein, and Felix Hausch*
€
AG Chemical Genomics, Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munchen, Germany
ABSTRACT There are strong indications for the involvement of cyclophilin 40 in
diseases caused by misregulation of steroid hormone receptors, like prostate or
breast cancer. To identify novel inhibitors for this immunophilin, we developed a
simplified fluorescence polarization assay based on the synthesis of a fluorescein-
labeled tracer. This tracer was produced by a facile four-step synthesis involving
Grubbs metathesis and standard amide bond coupling, to label cyclosporin A with
fluorescein. We show the binding of this tracer to Cyp40 and Cyp18 with KD values
of 106 ( 13 or 12 ( 1 nM, respectively, by analyzing the anisotropy change and
demonstrate its competition with cyclosporin A. Binding data obtained by fluores-
cence polarization were corroborated by an enzymatic activity assay. The de-
scribed tracer allows for a robust assay in a high-throughput format to support the
development of novel Cyp40 ligands.
KEYWORDS Cyclophilin, Cyp18, Cyp40, cyclosporin A, CsA, fluorescent analogue,
fluorescence polarization assay
yclophilin 40 (Cyp40) was first identified as an inter-
action partner of the estrogen receptor.1 Later, it was
found to be involved in the assembly of numerous
knockdown studies, which blocked AR responsiveness and
CsA sensitivity in prostate cancer cells and reduced their
proliferation.15
C
steroid hormone receptors (SHR), like the glucocorticoid
receptor (GR),2 the progesterone receptor (PR),3 the aryl
hydrocarbon receptor (AhR),4 or the androgen receptor
(AR).5 The efficient hormone binding of these SHRs is
regulated by their interaction with the chaperone Hsp90.6 In
addition to Cyp40, there are several other cochaperones
known to be part of these Hsp90-SHR complexes, which are
able to modulate the activity of SHRs, including the FK506-
binding proteins 51 and 52 (FKBP51 and FKBP52),7,8 the
protein phosphatase 5 (PP5),8 and Xap2/Ara9.9 These co-
chaperones compete for the binding to Hsp90 via their
tetratricopeptide repeat (TPR) motif.6,8 Cyp40, FKBP51,
and FKBP52 have an additional peptidyl-prolyl-isomerase
(PPIase) domain that is also the binding site for the immuno-
suppressants cyclosporin A (CsA) and FK506 or rapamycin,
respectively.1,10,11
As a modulator of SHRs, Cyp40 might be clinically relevant
for endocrine disorders. For instance, Cyp40 was found to be
overexpressed in breast12 and prostate cancer cells.5 Altera-
tions of the normal balance of immunophilins could lead to
the progression of ER-positive breast cancers to an estrogen-
independent state.13 Moreover, Cyp40 may influence the
response to cancer treatment, as it was found to be up-
regulated in nonresponders of radiotherapy in rectal
cancer.14 Cyp40 was further linked to prostate cancer by
the observation that the Cyp40 ligand CsAwas able to inhibit
growth of several prostate cancer cell lines. Recently, the
specific role of Cyp40 was confirmed and further defined by
The human genome encodes at least 16 unique cyclophi-
lins, all containing a highly conserved Cyp18-homology
domain.17 Many of them bind thightly to the pan-specific
cyclophilin ligands CsA, sanglifehrin A,18 or the CsA analo-
gue NIM811 (in clinical trial for hepatitis C therapy).16,19 To
further define the pharmacology of cyclophilin ligands and
the therapeutical potential of Cyp40, more selective inhibi-
tors are needed. For the development of such ligands,
fluorescence polarization assays can be applied using tra-
cers targeting the PPIase domain of isolated, recombinant
Cyp40. The selectivity of inhibitors could be examined by
similar in vitro assays with purified Cyp18 or potentially
other cyclophilins. A similar assay has been described for
Cyp18, albeit using tracers that had to be laboriously synthe-
sized in 11 steps20 or requiring publically unavailable natural
products.21 Here, we describe a straightforward four-step
synthesis of a fluorescent-labeled CsA analogue starting
from commercially available CsA. This tracer can be used
in fluorescence polarization assays targeting Cyp40.
CsA (Scheme 1) is a cyclic undecapeptide that is clinically
used as an immunosuppressive agent.22 It binds unspecifi-
cally to various cyclophilins in the nanomolar range, making
it a promising starting point for the development of a
fluorescent tracer.16
Received Date: June 2, 2010
Accepted Date: August 3, 2010
Published on Web Date: August 25, 2010
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DOI: 10.1021/ml1001272 ACS Med. Chem. Lett. 2010, 1, 536–539
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TECHNOLOGY NOTE
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Figure 1. (a) Binding of CsA-Fl (10 nM) to Cyp18 (dashed squares) and Cyp40 (continuous triangles) measured by fluorescence polarization.
(b) Competition of CsA with CsA-Fl (10 nM) for the binding to Cyp18 (10 nM) and Cyp40 (100 nM) measured by fluorescence polarization.
Scheme 1a
a Reagents and conditions: (a) Boc2O, TEA, MeOH, room temperature. (b) Grubbs Cat. II. Gen, DCM, reflux. (c) 10% TFA, DCM, 0 °C. (d) NHS-
fluorescein, TEA, DCM/THF 2:1, room temperature.
The cocrystal structure of Cyp18/CsA shows that the
terminal trans-alkene methyl substituent of the unnatural
amino acid butenyl-methyl-L-threonine (position 1 of CsA in
Scheme 1) sticks out of the binding pocket and is sol-
vent accessible.23 Because the binding pocket of Cyp40 is
highly conserved and structurally superimposable to that of
Cyp18,24 we decided to use this accessible alkene moiety
for the introduction of a fluorescent label. Cross-metathesis
using a second generation Grubbs catalyst was chosen, as it
has proven its applicability to CsA and numerous different
types of complex substrates throughout the literature.25 We
decided to attach an amine-containing linker to CsA that
could be easily coupled to carboxy-containing fluorophores.
Because metathesis catalysts are known to be sensitive to
primary amines,26 we first tried to couple Boc-allylamine or
allylammoniumchloride to CsA, which resulted in poor
yields probably because of the close distance between the
amine and the double bond. We thought to overcome this
problem by using a longer linker. Therefore, we first pro-
tected commercially availalable para-vinylaminobenzene 1
with Boc2O to obtain 2 with quantitative yields. This was
then coupled to CsAusing second generation Grubbs catalyst
to give 3 in good yields. The primary amine was liberated
using 10% TFA to produce 4, which was reacted with 5/6-
carboxyfluorescein N-hydroxysuccinimide (NHS-fluorescein)
to give the final compound CsA-Fl.
We then characterized the binding of CsA-Fl to Cyp40 and
Cyp18 in a miniaturized microtiter plate-based fluorescence
polarization assay.27 N-Terminal His-tagged Cyp40 and
Cyp18 were recombinantly expressed and purified by Ni-NTA
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DOI: 10.1021/ml1001272 ACS Med. Chem. Lett. 2010, 1, 536–539
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Figure 2. (a) Inhibition of the PPIase activity of Cyp40 (100 nM) by CsA (dashed, ꢀ) and CsA-Fl (continuous, O). (b) Inhibition of the PPIase
activity of Cyp18 (10 nM) by CsA (dashed, ꢀ) and CsA-Fl (continuous, O).
Table 1. Binding and Inhibition Constants (nM) Measured by
can be used for screening and subsequent profiling of
inhibitors of Cyp40 to identify structures for the develop-
ment of potential new drugs against breast and prostate
cancer.
Fluorescence Polarization or Enzymatic PPIase Assay
cyclophilin
FP assay (Ki/KD)
PPIase assay (Ki)
CsA
Cyp40
Cyp18
227 ( 22
34 ( 6
231 ( 55
7 ( 1
SUPPORTING INFORMATION AVAILABLE Synthetic pro-
cedures and characterization data, expression and purification of
cyclophilins, inhibition of PPIase activity, and fluorescence polar-
ization assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
CsA-Fl
Cyp40
Cyp18
106 ( 13
12 ( 1
101 ( 24
12 ( 4
affinity chromatography. The functionality of the purified
proteins was verified by a coupled enzymatic assay measur-
ing their PPIase activity. We determined Kd values of 106 (
13 nM for Cyp40 and 12 ( 2 nM for Cyp18 (Figure 1a and
Table 1). The absolute change in anisotropywas substantially
larger for Cyp40 as compared to Cyp18, likely reflecting the
bigger size of the former. To verify the binding affinity of
tracer CsA-Fl to Cyp40 and Cyp18, a coupled enzymatic
PPIase assay was performed (Figure 2).28 The measured
values of 101 ( 24 nM for Cyp40 and 12 ( 4 nM for Cyp18
match very well with the FP assay results shown above.
To demonstrate the use of the tracer CsA-Fl for the
characterization of unlabeled ligands, we tested its perfor-
mance in FP competition assays (Figure 1b). The prototypic
ligand CsA could efficiently compete with CsA-Fl for the
binding to Cyp40 and Cyp18. The measured Kd values were
again corroborated with a PPIase assay (Figure 2 and
Table 1). For Cyp40, there was an excellent match between
FPand PPIase results, while for Cyp18, a slightly lower Ki was
observed in the PPIase assay.
In general, CsA-Fl binds slightly more potently to both
Cyp18 and Cyp40 than CsA likely due to additional contacts
of the conjugated fluorescein. The affinities measured in this
work are consistent with the literature values for Cyp40 as
well as with the majority of reports for Cyp 18. For Cyp18,
substantial discrepancies in CsA affinities have been re-
ported. The consensus values, however, match very well
with the results reported in this work.29
In summary, we describe the facile synthesis of the
fluorescein-labeled tracer CsA-Fl, which shows high affinity
binding to Cyp40 and Cyp18. CsA is able to compete with the
tracer for the binding to Cyp40 and Cyp18. Therefore, CsA-Fl
enables a simplified assay in high-throughput format, which
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: (þ49)89-30622610. E-mail: hausch@mpipsykl.
mpg.de.
Author Contributions: † These authors contributed equally.
Funding Sources: This work was supported by the CIPSM. We
thank Professor Florian Holsboer for generous financial support.
ACKNOWLEDGMENT We thank Claudia Dubler (LMU, Munich,
Germany) and Elisabeth Weyher (MPI of Biochemistry, Martinsried,
Germany) for NMR spectroscopy and MS measurements.
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