| Title | Novel fluorescent polarization-based assay for the identification of disruptors of the RCAN1/calcineurin A protein complex. | | Author(s) | Mulero MC, Orzáez M, Messeguer J, Messeguer A, Pérez-Payá E, Pérez-Riba M | | Institution | From Medical and Molecular Genetics Center, Institut d'Investigació Biomèdica de Bellvitge, IDIBELL, Gran Via s/n km. 2.7, 08907 L'Hospitalet de Llobregat (Barcelona), Spain. | | Source | Anal Biochem 2009 Nov 2. | | Abstract | Calcineurin is a Ca(2+)-calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programmes, including immune response activation. One of the most studied substrates of calcineurin is the transcription factors NFAT (Nuclear Factor of Activated T cell) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A or FK506, are the most commonly immunosuppressants used in transplantation therapies. Unfortunately, their mechanism of action, blocking completely the calcineurin phosphatase activity together with the requirement of continuous administration, bears severe side effects. In the last years, the family of Regulators of Calcineurin (RCAN) has been described and extensively studied as modulators of calcineurin signalling pathways. The RCAN1-1 region, from amino acids 198 to 218, responsible for inhibiting in vivo the calcineurin-NFAT signaling pathway has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. We report here the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction whose immunosuppressive effect was confirmed in cellular models. Moreover, three small molecules with a potential immunosuppressive effect were also identified. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19891949 |
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