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

BACKGROUND: Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF(1) and CRF(2)), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF(1,) the role of CRF(2) in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF(2,) 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 CRF(2) 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 CRF(2,) and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF(2) complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF(2) in the absence of CRFBP(10kD), and toward CRF(1) in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF(1)(+) neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF(1) 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 Ca(2+) release specifically via CRF(2,) 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-CRF(2) NAMs that do not alter the CRF(1)-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 CRF(2) and CRFBP. CONCLUSION: These results provide the first evidence of specific roles for CRF(2) and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF(2) NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.