New aza-boron-dipyrromethene compound for single molecule fluorescence studies in cells

LUCIANA GIORDANO; NATALIA ARMANDO; CLAUDIO N. CAVASOTTO; SARA E. BARI; PAULA DOS SANTOS CLARO; PEDRO F. ARAMENDÍA; ALAN M. SZALAI; CAROLINA INDA; SUSANA SILBERSTEIN

Villa Carlos Paz

Conferencia; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; 2017

Corticotropin-releasing hormone (CRH) is a key protein involved in neuroendocrine response to stress [1]. There is a widespread interest in understanding the molecular behavior of CRH type 1 receptor (CRHR1), as it plays a primary role in regulatory mechanisms. Owing to the lack of reliable specific antibodies for CRHR1, studying its dynamics and distribution by fluorescence microscopy requires a different labeling strategy. A set of novel fluorescent antagonists have been designed for this purpose. Based on non-fluorescent reported antagonists and structure of CRHR1 [2], a backbone based on an aza-boron-dipyrromethene (aza-BODIPY) structure was chosen for a docking study. The software used for docking studies was Internal Coordinates Mechanics (ICM), and a flexible ligand-rigid receptor approach was performed. Aza-BODIPYs are suitable for single molecule fluorescence microscopy (SMFM), due to their high photostability and emission brightness. Substitution to provide adequate photochemical stability was also taken into account.Compound 1 was chosen as the first one to be synthesized. Its precursor, 2, has been successfully synthesized and its spectroscopic properties were measured.Compound 2 is an asymmetric aza-BODIPY with only one aromatic substituent in 1, 7, 3 and 5 positions. There are no aza-BODIPYs with this characteristics described in literature. It displays negative solvatochromism, and its emission quantum yield is around 0.80, with a lifetime of 3.9 ns. 2 also shows antagonist activity in biochemical assays. In STORM (Stochastic Optical Reconstruction Microscopy) experiments carried out in fixed hippocampal cells expressing CRHR1, 2 demonstrated a blinking and brightness performance comparable to Alexa647, one of the most frequent dyes used in this technique. Live cells experiments showed a short-time dye incorporation inside cells, and an exponential decrease of emission from cells, arriving to a plateau after 13 hours. This may coincide with specific binding between 2 and CRHR1.These results indicate that 2 may be considered as an adequate fluorescent antagonist for CRHR1, and that it is a proper dye for single molecule experiments, as for super-resolution microscopy.[1] E. Arzt, F. Holsboer, Trends in Pharmacological Sciences 2006, 27, 531.[2] K. Hollenstein, J. Kean, A. Bortolato, R. K. Y. Cheng, A. S. Doré, A. Jazayeri, R. M. Cooke, M. Weir, F. H. Marshall, Nature 2013, 499, 438.