INVESTIGADORES
ARAMENDIA Pedro Francisco
congresos y reuniones científicas
Título:
AZA-BODIPY Fluorescent Marker for CRHR1: Computer Design and Synthesis, Signaling Effects, and Binding Constant Estimation in Cells
Autor/es:
SZALAI, ALAN M.; ARMANDO, NATALIA G.; BARABAS, FEDERICO M.; STEFANI, FERNANDO D.; GIORDANO, LUCIANA; BARI, SARA E.; CAVASOTTO, CLAUDIO N.; SILBERSTEIN, SUSANA; ARAMEND√ćA, PEDRO F.
Lugar:
Dublin
Reunión:
Congreso; 27th IUPAC Symposium on Photochemistry; 2018
Institución organizadora:
IUPAC
Resumen:
Corticotropin-releasinghormone type 1 Receptor (CRHR1) belongs to the class B family ofG-protein-coupled receptors (GPCRs). GPCRs are the largest group ofmembrane proteins, targets for about one third of pharmaceuticalagents [1]. The corticotropin-releasing hormone (CRH) is a 41-aapeptide ligand for CRHR1, crucial in the integration ofneuroendocrine, autonomic, and behavioral responses to stress [2].Dysregulation of the CRH/CRHR1 system in the central nervous systemis related to mood disorders as anxiety and depression [3]. Owingto the lack of reliable specific antibodies for CRHR1, studying itsdynamics and distribution by fluorescence microscopy requires adifferent labeling approach.Here,we describe the strategy to label CRHR1 with a small fluorescentantagonist that permits the performance of stochastic opticalreconstruction microscopy (STORM) with 25 nm resolution. Smallmolecules as fluorescent markers display many advantages, such asdirect application in living cells or the possibility to carry outexperiments under endogenous cellular protein expression. Inaddition, a recent advance in fluorescence nanoscopy brought thespatial resolution to 1 nm [4], enhancing the need for development ofsmall fluorescent labels. Based on docking studies, we designed andsynthesized an organic fluorescent antagonist (ABP-09) for CRHR1, andtested its performance in cells expressing the receptor. Thefluorophore is an asymmetrically substituted azaBODIPY that shows anexcellent performance in STORM imaging, comparable to the referencefor this technique: AlexaFluor-647. Experiments in neuronalhippocampal cells demonstrate antagonist effects in similarconcentrations as the well-established antagonist CP-376395,co-crystallized with the protein. Finally, we evaluated the bindingaffinity of CRHR1 to ABP-09 in the cellular environment by aquantitative analysis of two color STORM images, obtaining a value of12 Mfor the dissociation equilibrium constant. p { margin-bottom: 0.25cm; direction: ltr; line-height: 120%; text-align: left; }a:link { color: rgb(0, 0, 255); }
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