title: Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.
abstract: The protein complex formed by the Ca2+sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.
authors.affiliation: Departamento de Neurobiología del Desarrollo, Instituto Cajal, Spanish National Research Council, 28002 Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Mansilla
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano, Spanish National Research Council, 28006 Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Chaves-Sanjuan
authors.affiliation: Centro de Investigaciones Biológicas, Spanish National Research Council, 28040 Madrid, Spain.
authors.forename: Nuria E
authors.initials: NE
authors.lastname: Campillo
authors.affiliation: Division of Neuroscience, Biomedical Sciences Research Centre Alexander Fleming, 16672 Vari, Greece.
authors.forename: Ourania
authors.initials: O
authors.lastname: Semelidou
authors.affiliation: Centro de Investigaciones Biológicas, Spanish National Research Council, 28040 Madrid, Spain.
authors.forename: Loreto
authors.initials: L
authors.lastname: Martínez-González
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano, Spanish National Research Council, 28006 Madrid, Spain.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Infantes
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano, Spanish National Research Council, 28006 Madrid, Spain.
authors.forename: Juana María
authors.initials: JM
authors.lastname: González-Rubio
authors.affiliation: Centro de Investigaciones Biológicas, Spanish National Research Council, 28040 Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Gil
authors.affiliation: Instituto de Química Médica, Spanish National Research Council, 28006 Madrid, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Conde
authors.affiliation: Division of Neuroscience, Biomedical Sciences Research Centre Alexander Fleming, 16672 Vari, Greece.
authors.forename: Efthimios M C
authors.initials: EM
authors.lastname: Skoulakis
authors.affiliation: Departamento de Neurobiología del Desarrollo, Instituto Cajal, Spanish National Research Council, 28002 Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ferrús
authors.affiliation: Centro de Investigaciones Biológicas, Spanish National Research Council, 28040 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Martínez
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano, Spanish National Research Council, 28006 Madrid, Spain; xmjose@iqfr.csic.es.
authors.forename: María José
authors.initials: MJ
authors.lastname: Sánchez-Barrena
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D014150:Antipsychotic Agents / Q000737:chemistry / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004195:Disease Models, Animal; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010640:Phenothiazines / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000235:genetics / Q000378:metabolism*
chemical_list: D014150:Antipsychotic Agents; D029721:Drosophila Proteins; D020662:Guanine Nucleotide Exchange Factors; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D010640:Phenothiazines; C101464:frequenin calcium sensor proteins
keywords: NCS-1; X-ray crystallography; fragile X syndrome; protein–protein interaction inhibitor; synapse regulation