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. 2010 Aug 25;1(9):536–539. doi: 10.1021/ml1001272

Facile Synthesis of a Fluorescent Cyclosporin A Analogue To Study Cyclophilin 40 and Cyclophilin 18 Ligands

Steffen Gaali 1,†, Christian Kozany 1,†, Bastiaan Hoogeland 1, Marielle Klein 1, Felix Hausch 1,*
PMCID: PMC4007835  PMID: 24900244

Abstract

graphic file with name ml-2010-001272_0005.jpg

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 fluorescence polarization were corroborated by an enzymatic activity assay. The described 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

Cyclophilin 40 (Cyp40) was first identified as an interaction partner of the estrogen receptor.1 Later, it was found to be involved in the assembly of numerous 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 cochaperones 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 immunosuppressants 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 Alterations 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 CsA was able to inhibit growth of several prostate cancer cell lines. Recently, the specific role of Cyp40 was confirmed and further defined by knockdown studies, which blocked AR responsiveness and CsA sensitivity in prostate cancer cells and reduced their proliferation.15

The human genome encodes at least 16 unique cyclophilins, 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 analogue 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 inhibitors are needed. For the development of such ligands, fluorescence polarization assays can be applied using tracers 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 synthesized 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 unspecifically to various cyclophilins in the nanomolar range, making it a promising starting point for the development of a fluorescent tracer.16

Scheme 1.

Scheme 1

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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 solvent 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 protected commercially availalable para-vinylaminobenzene 1 with Boc2O to obtain 2 with quantitative yields. This was then coupled to CsA using 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 affinity chromatography. The functionality of the purified proteins was verified by a coupled enzymatic assay measuring 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 anisotropy was 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.

Figure 1.

Figure 1

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(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.

Table 1. Binding and Inhibition Constants (nM) Measured by Fluorescence Polarization or Enzymatic PPIase Assay.

  cyclophilin FP assay (Ki/KD) PPIase assay (Ki)
CsA Cyp40 227 ± 22 231 ± 55
Cyp18 34 ± 6 7 ± 1
CsA-Fl Cyp40 106 ± 13 101 ± 24
Cyp18 12 ± 1 12 ± 4
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Figure 2.

Figure 2

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(a) Inhibition of the PPIase activity of Cyp40 (100 nM) by CsA (dashed, ×) and CsA-Fl (continuous, ○). (b) Inhibition of the PPIase activity of Cyp18 (10 nM) by CsA (dashed, ×) and CsA-Fl (continuous, ○).

To demonstrate the use of the tracer CsA-Fl for the characterization of unlabeled ligands, we tested its performance 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 FP and 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 reported. 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 can be used for screening and subsequent profiling of inhibitors of Cyp40 to identify structures for the development of potential new drugs against breast and prostate cancer.

Acknowledgments

We thank Claudia Dubler (LMU, Munich, Germany) and Elisabeth Weyher (MPI of Biochemistry, Martinsried, Germany) for NMR spectroscopy and MS measurements.

Supporting Information Available

Synthetic procedures and characterization data, expression and purification of cyclophilins, inhibition of PPIase activity, and fluorescence polarization assay. This material is available free of charge via the Internet at http://pubs.acs.org.

†

These authors contributed equally.

This work was supported by the CIPSM. We thank Professor Florian Holsboer for generous financial support.

Supplementary Material

ml1001272_si_001.pdf (106.5KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml1001272_si_001.pdf (106.5KB, pdf)

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