title: A fluorescent polarization-based assay for the identification of disruptors of the RCAN1-calcineurin A protein complex.
abstract: Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly 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 been described 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 pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report 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. Moreover, three small molecules with a potential immunosuppressive effect were also identified.
authors.affiliation: Molecular Genetics Laboratory, Institut d'Investigació Biomèdica de Bellvitge, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Carme Mulero
authors.forename: Mar
authors.initials: M
authors.lastname: Orzáez
authors.forename: Joaquim
authors.initials: J
authors.lastname: Messeguer
authors.forename: Angel
authors.initials: A
authors.lastname: Messeguer
authors.forename: Enrique
authors.initials: E
authors.lastname: Pérez-Payá
authors.forename: Mercè
authors.initials: M
authors.lastname: Pérez-Riba
mesh_terms: D000595:Amino Acid Sequence; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004176:Dipyridamole / Q000032:analysis / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000032:analysis / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D010455:Peptides; C108612:RCAN1 protein, human; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D004176:Dipyridamole; D019703:Calcineurin