Development and use of a high-throughput screen to identify novel modulators of the corticotropin releasing factor binding protein

Abstract

Background: Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF₁ and CRF₂), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF₁, the role of CRF₂ in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF₂, interacting with a C terminal fragment of CRFBP, CRFBP(10kD), may have a role in the modulation of neuronal activity. However, the mechanism by which CRF interacts with CRFBP(10kD) and CRF₂ has not been fully elucidated due to the lack of useful chemical tools to probe CRFBP.

Methods: We miniaturized a cell-based assay, where CRFBP(10kD) is fused as a chimera with CRF₂, and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF₂ complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF₂ in the absence of CRFBP(10kD), and toward CRF₁ in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF₁⁺ neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF₁ promoter, and 2) the CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in dopamine neurons in the ventral tegmental area (VTA).

Results: We found that CRFBP(10kD) potentiates CRF-intracellular Ca²⁺ release specifically via CRF₂, indicating that CRFBP may possess excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP, CRFBP(27kD). We identified novel small molecule CRFBP-CRF₂ NAMs that do not alter the CRF₁-mediated effects of exogenous CRF but blunt CRF-induced potentiation of NMDAR-mediated synaptic transmission in dopamine neurons in the VTA, an effect mediated by CRF₂ and CRFBP.

Conclusion: These results provide the first evidence of specific roles for CRF₂ and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF₂ NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.

Keywords: AUD; CRF(2); CRFBP; HTS; NAM; Stress.