This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

A fluorescent polarization-based assay for the identification of disruptors of theRCAN1–calcineurin A protein complex

M. Carme Mulero a,1, Mar Orzáez b,1, Joaquim Messeguer c, Ángel Messeguer c, Enrique Pérez-Payá b,d,Mercè Pérez-Riba a,*

a Molecular Genetics Laboratory, Institut d’Investigació Biomèdica de Bellvitge, L’Hospitalet de Llobregat, 08907 Barcelona, Spainb Department of Medicinal Chemistry, Centro de Investigación Príncipe Felipe, 46013 Valencia, Spainc Department of Chemical and Biomolecular Nanotechnology, IQAC, CSIC, 08034 Barcelona, Spaind Instituto de Biomedicina de Valencia, CSIC, 46010 Valencia, Spain

a r t i c l e i n f o

Article history:Received 10 August 2009Received in revised form 29 October 2009Accepted 30 October 2009Available online 3 November 2009

Keywords:RCAN1Calcineurin ANFATImmunosuppressionFluorescence polarization assayMillipolarization units

a b s t r a c t

Calcineurin is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase involved in manybiological processes and developmental programs, including immune response. One of the most studiedsubstrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsiblefor T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most com-monly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action,completely blocking the calcineurin phosphatase activity while also requiring continuous administration,bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has beendescribed and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region,spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin–NFAT signaling pathwayin vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calci-neurin–NFAT interaction without affecting the general calcineurin phosphatase activity. Here we reportthe development of an optimized in vitro high-throughput fluorescence polarization assay based on thedisruption of the RCAN1198–218–CnA interaction for identifying molecules with immunosuppressantpotential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover,three small molecules with a potential immunosuppressive effect were also identified.

� 2009 Elsevier Inc. All rights reserved.

Calcineurin (Cn),2 a calcium- and calmodulin-dependent serine/threonine phosphatase, is a heterodimer formed by a catalytic sub-unit called calcineurin A (CnA) and a regulatory subunit called calci-neurin B (CnB) [1]. This enzyme regulates several biologicalprocesses such as heart valve morphogenesis, angiogenesis, and neu-ral and muscle development [2]. In human T cells, Cn dephosphoryl-ates the cytoplasmic nuclear factor of activated T cells (NFAT) [3],facilitating their translocation into the nucleus and consequently ini-tiating the immune response. Currently, immunosuppressive proto-

cols used in transplantation and treatment of autoimmune diseasesare based on the administration of the Cn inhibitors cyclosporine A(CsA) and FK506. Unfortunately, their mechanism of action, com-pletely blocking the Cn phosphatase activity while also requiringcontinuous administration, bears severe side effects. Thus, the iden-tification of new and more selective molecules with fewer side ef-fects than those currently in use is one of the main goals in thedesired pharmacological control of immune response activation.

New methods that focus on recent discoveries on the molecularmechanism of Cn activity need to be devised so as to uncover mod-ulators. In that sense, several endogenous Cn inhibitors, such as thefamily of regulators of calcineurin (RCAN, formerly known asDSCR1 or calcipressins), have been identified [4,5]. Recently, ourgroup demonstrated the in vivo immunosuppressive role of humanRCAN1 and RCAN3 proteins in human Jurkat T lymphocyte cellsactivated with ionomycin and phorbol 12-myristate 13-acetate(PMA) [6,7]. In addition, we reported on identification and charac-terization of the RCAN amino acid sequence that inhibits Cn–NFATsignaling in activated Jurkat T cells, which in RCAN1 spans aminoacids 198 to 218 [8]. It is worth noting that a synthetic peptidederived from such region (RCAN1198–218) interacts with CnA and

0003-2697/$ - see front matter � 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2009.10.045

* Corresponding author. Fax: +34 932607414.E-mail address: mpr@idibell.cat (M. Pérez-Riba).

1 These authors contributed equally to this work.2 Abbreviations used: Cn, calcineurin; CnA, calcineurin A; CnB, calcineurin B; NFAT,

nuclear factor of activated T cells; CsA, cyclosporine A; RCAN, regulators ofcalcineurin; PMA, phorbol 12-myristate 13-acetate; CF, carboxyfluorescein; GST,glutathione S-transferase; IPTG, isopropyl-b-D-thiogalactopyranoside; PBS, phos-phate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol;EGTA, ethyleneglycoltetraacetic acid; EDTA, ethylenediaminetetraacetic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; MALDI–TOF,matrix-assisted laser desorption/ionization time-of-flight; DMSO, dimethyl sulfoxide;mP, millipolarization units; IgG, immunoglobulin G; BSA, fraction V bovine serumalbumin; INCA, inhibitors of calcineurin.

Analytical Biochemistry 398 (2010) 99–103

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Author's personal copy

inhibits the Cn–NFAT signaling without affecting general Cn pro-tein phosphatase activity [8]. With these antecedents, an in vitrofluorescence polarization high-throughput screening assay basedon the displacement of the interaction between carboxyfluorescein(CF)–RCAN1198–218 and CnA was developed, leading to the identifi-cation of dipyridamole as a novel immunosuppressive molecule[8]. In the current study, we describe in detail such assay and theidentification of three small molecules that significantly displacethe RCAN1198–218–CnA interaction.

Materials and methods

CnA large-scale production

pGEX-6P-1-CnAa (human amino acids 2–347) plasmid con-struct was kindly provided by Patrick Hogan [9]. Escherichia coliBL21(DE3) transformed with this plasmid was grown in Luria–Ber-tani medium at 37 �C and 250 rpm. After reaching an optical den-sity of 0.6 to 0.8 at 600 nm, glutathione S-transferase (GST)–CnAproduction was induced with 0.5 mM isopropyl-b-D-thiogalactopy-ranoside (IPTG, Sigma–Aldrich, St. Louis, MO, USA) overnight at25 �C. Then cells were centrifuged at 5000g for 10 min at 4 �C,washed once in phosphate-buffered saline (PBS) supplementedwith 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Sigma–Al-drich), and centrifuged again. Pellets were frozen immediately indry ice and stored at �80 �C. These pellets were resuspended in alysis buffer containing 0.5 mM dithiothreitol (DTT, (Roche, India-napolis, IN, USA), 0.2 mM MgCl2, 1 mM ethyleneglycoltetraaceticacid (EGTA), 2 lg/ml aprotinin, 2 lg/ml leupeptin, 2 mM PMSF,10 lg/ml DNase I (Roche), and 1 mg/ml lysozyme in PBS and wereincubated in a rotary shaker for 30 min at room temperature. Thensamples were sonicated, and after adding 1% (v/v) Triton X-100,cells were incubated in a rotary shaker for 30 min at 4 �C. Finally,samples were subjected to three consecutive cycles of freezing at�80 �C and defreezing in ice-water. After centrifugation of samplesat 13.000g for 30 min at 4 �C, total bacterial protein extracts wereobtained. Then glutathione Sepharose 4B (Amersham Biosciences,Uppsala, Sweden) was used for purifying GST–CnA recombinantprotein for 4 h at 4 �C following the manufacturer’s instructions.GST–CnA–Sepharose beads were collected after centrifugationand washed in a buffer containing 150 mM NaCl, 1 mM ethylenedi-aminetetraacetic acid (EDTA), 1 mM DTT, 2 lg/ml aprotinin, 2 lg/ml leupeptin, and 2 mM PMSF in 50 mM Tris–HCl (pH 7.0) andthen packed in a column. GST–CnA–Sepharose beads were washedtwice with 10 bed volumes of this buffer and eight times morewith the same buffer without protein inhibitors. Then GST–CnA–Sepharose beads were incubated with PreScission Protease (Amer-sham Biosciences), which cleaves the specific protease cleavagesite localized between the GST and CnA protein, in a rotary shakerovernight at 4 �C. The purified CnA fractions were concentratedand quantified by spectrometry, and protein integrity was con-firmed by sodium dodecyl sulfate–polyacrylamide gel electropho-resis (SDS–PAGE).

Peptide synthesis and molecules

The RCAN1198–218 peptide, Ac-Lys-Tyr-Glu-Leu-His-Ala-Ala-Thr-Asp-Thr-Thr-Pro-Ser-Val-Val-Val-His-Val-Cys-Glu-Ser-NH2; its N-terminal CF derivative, the labeled CF–control peptide, CF–Gly-Gly-Met-Ala-Gly-Pro-His-Pro-Val-Ile-Val-Ile-Thr-Gly-Pro-His-Glu-Glu-NH2; and the unlabeled control peptide, Ac-Ser-Ala-Val-Thr-His-Lys-Leu-Glu-Ser-Val-Asp-Pro-Ala-Thr-Val-Tyr-Cys-Glu-Thr-His-Val-NH2 were synthesized as described previously [8]. Ma-trix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry in a 4700 Proteomics Analyzer (Applied

Biosystems, Foster City, CA, USA) was used to confirm peptide iden-tity. Several libraries of peptides and small molecules were screenedto identify disruptor candidates of the CF–RCAN1198–218–CnA inter-action: a hexapeptide-based library, a diversity-oriented positionalscanning library of N-alkylglycine trimers [10], and the PrestwickChemical Library.

All molecules identified after library deconvolution were di-luted in 10% (v/v) dimethyl sulfoxide (DMSO) in water and firstanalyzed in the assay at 100 lM. Only those compounds promotinga displacement of the CF–RCAN1198–218–CnA interaction higherthan 45% were further assayed to evaluate their effect in a dose-dependent manner in the same assay.

MALDI–TOF mass spectrometry

CnA (2.5 lM) was incubated in the absence or presence of25 lM RCAN1198–218 peptide in PBS in a final volume of 100 ll.Reactions were incubated for 15 min at 25 �C and 250 rpm. Afteradding 0.37% formaldehyde, samples were incubated for 10 minmore in the same assay conditions. Finally, reactions were stoppedby adding 125 mM glycine. Samples were identified by MALDI–TOFmass spectrometry, and data obtained were analyzed using DataExplorer 4.5 software (Applied Biosystems).

Fluorescence polarization binding assay

The CF–RCAN1198–218–CnA interaction was analyzed in black96-well flat-bottom plates (OptiPlate-96F(PB), PerkinElmer, Bos-ton, MA, USA) using a Wallac 1420 Victor2 multilabel counter(PerkinElmer) and Wallac 1420 Manager software. Parameters ofthe program used were as follows: 480 nm excitation wavelength;535 nm observed emission wavelength; normal polarizer aperture;0.1 s counting time; 1.2 factor G. Results obtained, indicated in mil-lipolarization units (mP), were calculated with the followingequation:

mP ¼ 103 � ðIpa � 1:2� IpeÞðIpa þ 1:2� IpeÞ

Ipa and Ipe correspond to the intensity of the parallel and perpendic-ular emitted light, respectively. The optimal binding protocol wasobtained by adding 30 nM CF–RCAN1198–218 peptide to 200 ll ofPBS (pH 7.4) containing increasing amounts of CnA for 15 min atroom temperature. Then 0.01% (w/v) fraction V bovine serum albu-min (BSA, Roche) was added to the assay. To analyze the ability todisplace the CF–RCAN1198–218–CnA interaction, molecules wereincubated for 15 min at room temperature with 1.5 lM CnA, withthe aim of facilitating their interaction, and then 30 nM CF–RCAN1198–218 peptide was added to the assay. The final DMSO con-centration in all of the experiments was less than 2% (v/v).

Results

The RCAN proteins play an immunosuppressive role in the hu-man T lymphocyte Jurkat cell line by inhibiting the Cn–NFATsignaling pathway in vivo [6,7]. As mentioned before, the identifi-cation of the RCAN1 sequence responsible for its biological role [8]tempted us to evaluate whether an RCAN1-derived peptide(RCAN1198–218) spanning such amino acidic sequence could be auseful tool for identifying novel molecules with immunosuppres-sant therapeutic potential. With this aim, we decided to set upand optimize an in vitro assay for analyzing the interaction be-tween the RCAN1198–218 peptide and CnA, taking advantage ofthe fluorescence polarization technology.

First, we evaluated the behavior of a CF-labeled RCAN1198–218

peptide (CF–RCAN1198–218) in 96-well plates in fluorescence

100 Assay for identifying disruptors of RCAN1–CnA complex / M. Carme Mulero et al. / Anal. Biochem. 398 (2010) 99–103

Author's personal copy

polarization-based experiments in the absence of CnA (Fig. 1). Wedetected a high background when compared with a nonrelatedfluorescein-labeled control peptide (Fig. 1; cf. black bar in CF–RCAN1198–218 peptide vs. CF–control peptide). This result suggeststhat the CF–RCAN1198–218 peptide has a tendency to self-aggregatein solution or, alternatively, becomes immobilized to the assayplates. To overcome this problem, we evaluated the efficiency ofdifferent blocking agents in the assay buffer. We tested two differ-ent reagents: 10% immunoglobulin G (IgG) and 1% fraction V bo-vine serum albumin (BSA) (Fig. 1; cf. white bar and gray bar,corresponding to IgG and BSA, respectively, vs. black bar, repre-senting absence of blocking agent). Our results show that addingblocking agents significantly reduces the background of the fluo-rescence polarization values obtained with the CF–RCAN1198–218

peptide alone. Further characterization of the CF–RCAN1198–218

peptide–CnA interaction was performed supplementing the assaywith BSA.

To study the interaction between the CF–RCAN1198–218 peptideand CnA, we performed titration curves at a constant CF–RCAN1198–218 peptide concentration (30 or 60 nM) and increasingCnA concentrations (in a range from 0.01 to 10 lM). In both cases,the CF–RCAN1198–218–CnA interaction is established with similarefficiency and the polarization units increase in a CnA-dependentmanner, confirming the specificity of such interaction. Both condi-tions evaluated showed comparable millipolarization values(Fig. 2A). Thus, a 30-nM concentration of CF–RCAN1198–218 peptidewas routinely used in the assay. Furthermore, the actual binding ofthe RCAN1198–218 peptide to CnA was also confirmed by peptide–protein crosslinking followed by mass spectrometry (MALDI–TOF) analysis (Fig. 2B). In the absence of the RCAN1198–218 peptideor when CnA was incubated with the crosslinker agent and anunlabeled control peptide, the mass spectrum obtained showedonly one peak corresponding to free CnA with molecular mass of41 kDa (Fig. 2B; see CnA and CnA–control peptide correspondinggraphs). However, when CnA in the presence of the RCAN1198–218

peptide was crosslinked, a second peak appeared in the mass spec-trum (43 kDa). This peak was assigned to the complex betweenRCAN1198–218 peptide and CnA (Fig. 2B; see RCAN1198–218–CnA cor-responding graph).

Next, we evaluated the steady-state optimal conditions for theCF–RCAN1198–218–CnA interaction-based assay. As depicted in

Fig. 3A, the millipolarization values obtained were constant after15, 30, and 60 min at room temperature, suggesting the formationof a stable complex after 15 min. The stabilization of a protein–protein or peptide–protein interaction has a strong hydrophobiccomponent arising from the interaction between the two largeamino acidic surfaces provided. These complexes are sensitive tothe presence of the organic solvents of chemical library aliquots(with DMSO being the preferred one). Then, the DMSO concentra-tion allowed by the CF–RCAN1198–218–CnA interaction was ana-lyzed and turned out to be very sensitive, with the maximumDMSO concentration allowed at the assay being only 2% (Fig. 3B).

Once optimized, all parameters of the CF–RCAN1198–218–CnAinteraction in the in vitro fluorescence polarization assay, bindingparameters for the CF–RCAN1198–218 peptide, were determinedperforming titration curves until reaching saturation. The resultsobtained showed that the Kd (dissociation constant) for theRCAN1198–218 peptide is 1.25 ± 0.09 lM and the IC50 is 3.72 ±0.28 lM [8]. This fact, together with the relatively large increasein millipolarization units shown (from 45 to 195 mP) and the smallstandard deviation obtained (<5%), permitted us to develop arobust in vitro assay.

Different libraries composed of peptides or small moleculeswere used to identify molecules capable of disrupting the CF–RCAN1198–218–CnA interaction, among them the Prestwick Chemi-cal Library. This library includes 880 biologically active compoundswith high chemical and pharmacological diversity. An initialscreening assay led us to identify some hit compounds that werealso analyzed in a dose-dependent manner to confirm their efficacy(Fig. 4). Table 1 shows the structure of the hit candidates analyzed.Further in vivo characterization of the most efficient molecule,dypiridamole (which is clinically used as an antiplatelet agent toprevent heart stroke), revealed a previously unreported immuno-suppressant role for this molecule [8]. Taken together, these resultsdemonstrate that the in vitro assay described here is a useful toolfor identifying novel molecules with immunosuppressive effect.

Discussion

Nowadays, immunosuppressive protocols used in transplanta-tion and treatment of autoimmune diseases always include theuse of Cn inhibitors due to the pivotal role of this enzyme in theactivation of the immune response. Unfortunately, the multipleand severe side effects that their long-life administration pro-duces—cancer, nephrotoxicity and neurotoxicity—make necessaryfinding novel effective and less toxic agents. With this aim, Roehrland coworkers developed an in vitro assay based on the interactionestablished between CnA and the VIVIT peptide [9,11] that inhibitsNFAT signaling. The VIVIT peptide amino acid sequence was se-lected from the natural SPRIEIT sequence on NFAT1 due to its highaffinity to binding to Cn [12,13]. From this assay, several immuno-suppressant small molecules called INCA (inhibitors of calcineurin)were identified. Unfortunately, most of those molecules were reac-tive quinones that promoted severe cytotoxic effects in all evalu-ated cell models [9]. Thus, we decided to direct our discoveryefforts in finding new immunosuppressants toward the novelRCAN1–CnA interaction recently described [4]. Our results demon-strate that the RCAN1198–218 peptide binds to CnA with high affin-ity and, more important, that this peptide inhibits Cn signalingtoward NFAT, thereby preventing NFAT from being activated, with-out affecting general Cn phosphatase activity [8]. The RCAN1198–218

peptide arises as an alternative and efficient tool for identifying no-vel immunosuppressive molecules. More important, its specificmechanism of inhibition toward the Cn–NFAT signaling indicatesthat the molecules identified might also be specific for the Cn–NFAT pathway in cultured T cells.

Fig. 1. Determination of the CF–RCAN1198–218 peptide fluorescence background inthe fluorescence polarization assay. The CF–RCAN1198–218 peptide or a controlnonrelated carboxyfluoresceinated peptide were incubated in the assay with noblocking agent (black bars) or in the presence of two different blocking agents: 10%IgG (white bars) or 1% BSA (gray bars). The graphs show the means ± standarddeviations of three independent experiments performed in triplicate.

Assay for identifying disruptors of RCAN1–CnA complex / M. Carme Mulero et al. / Anal. Biochem. 398 (2010) 99–103 101

Author's personal copy

With this aim, we set up and optimized the first in vitro fluores-cence polarization-based assay targeting the interaction betweenthe RCAN1198–218 peptide and CnA. The results presented here indi-cate that this assay is specific, fast, highly reproducible, and robustand that it could be easily standardized for high-throughputscreening analysis. As a consequence, it will increase the numberof hit candidates that, in turn, will offer new possibilities asidefrom those molecules that promoted a total displacement of theCn–NFAT interaction. Up to now, our group has evaluated dypirida-mole as one compound identified from a chemical library usingsuch an assay. The specific inhibitory capacity of dipyridamole ob-tained in in vivo experiments confirms the effectiveness of the as-say in identifying novel immunosuppressive drugs [8].Furthermore, three additional hit candidates were also identified:coralyne chloride hydrate, daunorubicin hydrochloride, and Chi-cago sky blue 6B (Fig. 4). Coralyne chloride hydrate and daunoru-bicin hydrochloride are drugs clinically used as antileukemicagents [14,15]. Chicago sky blue 6B is a potent inhibitor of L-gluta-

A

Fig. 2. CF–RCAN1198–218 peptide interacting with CnA. (A) Determination of the optimal CF–RCAN1198–218 peptide concentration required to achieve the higher differencebetween the minimum and maximum millipolarization values. Titration experiments were performed fixing the CF–RCAN1198–218 peptide concentration (30 or 60 nM) in thepresence of increasing CnA concentrations (from 0.01 to 10 lM). Experiments were performed three times in triplicate. (B) Evaluation of the RCAN1198–218–CnA interaction bypeptide–protein crosslinking followed by mass spectrometry (MALDI–TOF) analysis. The left panel corresponds to the assay containing CnA alone, the middle panelcorresponds to the assay containing RCAN1198–218 and CnA, and the right panel corresponds to the assay containing an unlabeled RCAN nonrelated peptide together with CnA.CnA molecular mass is approximately 41 kDa, and RCAN1198–218 and control peptide molecular mass is approximately 2.2 kDa.

A B

Fig. 3. Characterization of the optimal assay conditions for the CF–RCAN1198–218–CnA interaction. (A) A 15 min incubation for a sample containing CF–RCAN1198–218 and CnAis sufficient for the protein–protein interaction to take place. White bars correspond to free CF–RCAN1198–218 peptide, and black bars correspond to the CF–RCAN1198–218–CnAestablished interaction. (B) A DMSO concentration above 2% affects the CF–RCAN1198–218–CnA fluorescence polarization assay. White bars correspond to the free CF–RCAN1198–218 peptide, and black bars correspond to the CF–RCAN1198–218–CnA. All experiments were performed twice in triplicate.

Fig. 4. Dose-dependent analysis of disruptors of the CF–RCAN1198–218–CnA inter-action identified from screening the Prestwick Chemical Library by the fluorescencepolarization assay. Data shown correspond to three independent experimentsperformed in triplicate.

102 Assay for identifying disruptors of RCAN1–CnA complex / M. Carme Mulero et al. / Anal. Biochem. 398 (2010) 99–103

Author's personal copy

mate uptake into synaptic vesicles [16] and recently was describedas an inhibitor of the Ca2+/calmodulin-dependent protein kinasephosphatase family but not calcineurin [17]. Thus, to date thereis no evidence suggesting the existence of a novel immunosuppres-sive role for any of these three compounds. In a similar manner ashas been described for dipyridamole [8], we expect to further con-firm their in vivo inhibitory effect toward the Cn–NFAT signalingpathway using cell lines as well as animal models.

Acknowledgments

We thank Ana Giménez and the Proteomic Unit from the Centrode Investigación Príncipe Felipe for their skillful technical assis-tance. This work was supported by grants from Fundació La Maratóde TV3 (030830), the Spanish Ministry of Education and Science(SAF2006-04815, BIO2004-00998, BIO2007-60066, and CTQ2005-00995/BQU), the Fundación Mutua Madrileña 2007, and the Gener-alitat de Catalunya (2006 BE 00051).

References

[1] C.B. Klee, T.H. Crouch, M.H. Krinks, Calcineurin: a calcium- and calmodulin-binding protein of the nervous system, Proc. Natl. Acad. Sci. USA 76 (1979)6270–6273.

[2] S. Martinez-Martinez, J.M. Redondo, Inhibitors of the calcineurin/NFATpathway, Curr. Med. Chem. 11 (2004) 997–1007.

[3] M.M. Winslow, J.R. Neilson, G.R. Crabtree, Calcium signalling in lymphocytes,Curr. Opin. Immunol. 15 (2003) 299–307.

[4] J.J. Fuentes, L. Genesca, T.J. Kingsbury, K.W. Cunningham, M. Perez-Riba, X.Estivill, S. de la Luna, DSCR1, overexpressed in Down syndrome, is an inhibitorof calcineurin-mediated signaling pathways, Hum. Mol. Genet. 9 (2000) 1681–1690.

[5] B. Rothermel, R.B. Vega, J. Yang, H. Wu, R. Bassel-Duby, R.S. Williams, A proteinencoded within the Down syndrome critical region is enriched in striated

muscles and inhibits calcineurin signalling, J. Biol. Chem. 275 (2000) 8719–8725.

[6] A. Aubareda, M.C. Mulero, M. Perez-Riba, Functional characterization of thecalcipressin 1 motif that suppresses calcineurin-mediated NFAT-dependentcytokine gene expression in human T cells, Cell. Signal. 18 (2006) 1430–1438.

[7] M.C. Mulero, A. Aubareda, A. Schluter, M. Perez-Riba, RCAN3, a novelcalcineurin inhibitor that down-regulates NFAT-dependent cytokine geneexpression, Biochim. Biophys. Acta 1773 (2007) 330–341.

[8] M.C. Mulero, A. Aubareda, M. Orzaez, J. Messeguer, E. Serrano-Candelas, S.Martinez-Hoyer, A. Messeguer, E. Perez-Paya, M. Perez-Riba, Inhibiting thecalcineurin–NFAT (nuclear factor of activated T cells) signaling pathway with aregulator of calcineurin-derived peptide without affecting general calcineurinphosphatase activity, J. Biol. Chem. 284 (2009) 9394–9401.

[9] M.H. Roehrl, S. Kang, J. Aramburu, G. Wagner, A. Rao, P.G. Hogan, Selectiveinhibition of calcineurin–NFAT signalling by blocking protein–proteininteraction with small organic molecules, Proc. Natl. Acad. Sci. USA 101(2004) 7554–7559.

[10] M. Humet, T. Carbonell, I. Masip, F. Sanchez-Baeza, P. Mora, E. Canton, M.Gobernado, C. Abad, E. Perez-Paya, A. Messeguer, A positional scanningcombinatorial library of peptoids as a source of biological active molecules:Identification of antimicrobials, J. Comb. Chem. 5 (2003) 597–605.

[11] S. Kang, H. Li, A. Rao, P.G. Hogan, Inhibition of the calcineurin–NFATinteraction by small organic molecules reflects binding at an allosteric site, J.Biol. Chem. 280 (2005) 37698–37706.

[12] J. Aramburu, F. Garcia-Cozar, A. Raghavan, H. Okamura, A. Rao, P.G. Hogan,Selective inhibition of NFAT activation by a peptide spanning the calcineurintargeting site of NFAT, Mol. Cell 1 (1998) 627–637.

[13] J. Aramburu, M.B. Yaffe, C. Lopez-Rodriguez, L.C. Cantley, P.G. Hogan, A. Rao,Affinity-driven peptide selection of an NFAT inhibitor more selective thancyclosporin A, Science 285 (1999) 2129–2133.

[14] K.Y. Zee-Cheng, K.D. Paull, C.C. Cheng, Experimental antileukemic agents:coralyne, analogs, and related compounds, J. Med. Chem. 17 (1974) 347–351.

[15] C. Tan, H. Tasaka, K.P. Yu, M.L. Murphy, D.A. Karnofsky, Daunorubicin, anantitumor antibiotic, in the treatment of neoplastic disease: clinical evaluationwith special reference to childhood leukemia, Cancer 20 (1967) 333–353.

[16] S. Roseth, E.M. Fykse, F. Fonnum, Uptake of L-glutamate into rat brain synapticvesicles: effect of inhibitors that bind specifically to the glutamate transporter,J. Neurochem. 65 (1995) 96–103.

[17] N. Sueyoshi, T. Takao, T. Nimura, Y. Sugiyama, T. Numano, Y. Shigeri, T.Taniguchi, I. Kameshita, A. Ishida, Inhibitors of the Ca2+/calmodulin-dependentprotein kinase phosphatase family (CaMKP and CaMKP-N), Biochem. Biophys.Res. Commun. 363 (2007) 715–721.

Table 1Summary of the molecules with immunosuppressant potential responsible for disrupting the CF–RCAN1198–218–CnA interaction identified from the Prestwick Chemical Library.

Disruptor molecules of the RCAN198–218–CnA interaction

1. DipyridamoleC24H40N8O4

MW: 504,6376 N

N

N

N

N

N

N

N

OH

OH

OH

OH

7. Coralyne chloride hydrateC22H24CINO5

MW: 417,89328

N+

CH3

O

O

CH3

O

CH3

O

CH3

CH3

Cl

OH2H2O

2. Chicago sky blue 6BC34H24N6Na4O16S4

MW: 992,81618S

NH2

OH

O

OO

S

N

OO

O

N

OCH3

O

N

CH3

N

OH

NH2

S

S

OO

O

O

OO

Na+

Na+

Na+

Na+

8. Daunorubicin hydrochlorideC27H30CINO10

MW: 563,99385O

O

OOCH3

OH

OH

CH3

O

OH

O

OH

CH3

NH2

ClH

Assay for identifying disruptors of RCAN1–CnA complex / M. Carme Mulero et al. / Anal. Biochem. 398 (2010) 99–103 103