INVESTIGADORES
ARAMENDIA Pedro Francisco
congresos y reuniones científicas
Título:
NEW AZA-BORON-DIPYRROMETHENECOMPOUND FOR SINGLE MOLECULE FLUORESCENCE STUDIES IN CELLS
Autor/es:
ALAN SZALAI; NATALIA ARMANDO; LUCIANA GIORDANO; SARA BARI; CAROLINA INDA; CLAUDIO N. CAVASOTTO; SUSANA SILBERSTEIN; PEDRO F. ARAMENDÍA
Reunión:
Congreso; XIII ENCUENTRO LATINOAMERICANO DE FOTOQUIMICA Y FOTOBIOLOGIA; 2017
Resumen:
p { margin-bottom: 0.25cm; direction: ltr; line-height: 120%; text-align: left; }p.western { }p.cjk { }a:link { }Corticotropin-releasing hormone (CRH) is a keyprotein involved in neuroendocrine response to stress. There is awidespread interest in understanding CRH type 1 receptor (CRHR1)molecular behavior, as it plays a primary role in regulatorymechanisms1.Owing to the lack of reliable specific antibodies for CRHR1, studyingits dynamics and distribution by fluorescence microscopy requires adifferent labeling strategy. A set of novel fluorescent antagonistshave been designed for this purpose. Based on non-fluorescentreported antagonists and structure of CRHR12,a set of compounds based on an aza-boron-dipyrromethene (aza-BODIPY)structure were chosen for a docking study. Aza-BODIPYs are suitablefor single molecule fluorescence microscopy (SMFM), due to their highphotostability and emission brightness3.The software used for docking studies was InternalCoordinates Mechanics (ICM), and aflexible ligand-rigid receptor approach was performed. Compound 1 was chosen as the first compound to be synthesized. Its precursor, 2,has been successfully synthesized and its spectroscopic propertieswere measured.Compound 2is an asymmetric aza-BODIPY with only one aromatic substituent in 1,7, 3 and 5 positions. There are no aza-BODIPYs with thischaracteristics described in literature. It displays negativesolvatochromism, and its emission quantum yield is around 0.80, witha lifetime of 3.9 ns. 2also shows antagonist activity in biochemical assays. In STORM(Stochastic Optical ReconstructionMicroscopy) experiments carried out infixed hippocampal cells expressing CRHR1, 2demonstrated a blinking and brightness performance comparable toAlexa647, one of the most frequent dyes used in this technique. Livecells experiments showed a short-time dye incorporation inside cells,and an exponential decrease of emission from cells, arriving to aplateau after 13 hours. This may coincide with specific bindingbetween 2and CRHR1. These results indicate that 2may be considered as an adequate fluorescent antagonist for CRHR1,and that it is a proper dye for single molecule experiments, as forsuper-resolution microscopy.References1-        Arzt,E.; Holsboer, F. Trends inPharmacological Sciences 2006,27,531-5382-        Hollenstein,K.; Kean, J.; Bortolato, A.; Cheng, R. K. Y.; Doré, A. S.; Jazayeri,A.; Cooke, R. M.; Weir, M.; Marshall, F. H. Nature 2013,499,438-4453-        Loudet,A.; Burgess, K. Chem. Rev.2007, 107,4891?4932
rds']