AZA-BODIPY FLUORESCENT MARKER FOR CRHR1: COMPUTER DESIGN AND SYNTHESIS, SIGNALING EFFECTS, AND BINDING CONSTANT ESTIMATION IN CELLS.

ALAN M. SZALAI; FERNANDO D. STEFANI; CLAUDIO N. CAVASOTTO; NATALIA G. ARMANDO; LUCIANA GIORDANO; SUSANA SILBERSTEIN; FEDERICO M. BARABAS; SARA E. BARI; PEDRO F. ARAMENDÍA

Dublin

Simposio; 27th IUPAC Symposium on Photochemistry; 2018

University College Dublin

Corticotropin-releasing hormone type 1 Receptor (CRHR1) belongs to the class B family of G-protein-coupled receptors (GPCRs). GPCRs are the largest group of membrane proteins, targets for about one third of pharmaceutical agents [1]. The corticotropin-releasing hormone (CRH) is a 41-aa peptide ligand for CRHR1, crucial in the integration of neuroendocrine, autonomic, and behavioral responses to stress [2]. Dysregulation of the CRH/CRHR1 system in the central nervous system is related to mood disorders as anxiety and depression [3]. Owing to the lack of reliable specific antibodies for CRHR1, studying its dynamics and distribution by fluorescence microscopy requires a different labeling approach. Here, we describe the strategy to label CRHR1 with a small fluorescent antagonist that permits the performance of stochastic optical reconstruction microscopy (STORM) with 25 nm resolution. Small molecules as fluorescent markers display many advantages, such as direct application in living cells or the possibility to carry out experiments under endogenous cellular protein expression. In addition, a recent advance in fluorescence nanoscopy brought the spatial resolution to 1 nm [4], enhancing the need for development of small fluorescent labels. Based on docking studies, we designed and synthesized an organic fluorescent antagonist (ABP-09) for CRHR1, and tested its performance in cells expressing the receptor. The fluorophore is an asymmetrically substituted azaBODIPY that shows an excellent performance in STORM imaging, comparable to the reference for this technique: AlexaFluor-647. Experiments in neuronal hippocampal cells demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395, co-crystallized with the protein. Finally, we evaluated the binding affinity of CRHR1 to ABP-09 in the cellular environment by a quantitative analysis of two color STORM images, obtaining a value of 12 M for the dissociation equilibrium constant.