Stress responses involve corticotropin releasing factor (CRF), the two cognate receptors (CRF1 and CRF2) and the CRF-
binding protein (CRFBP). Utilizing a novel
cell-based
assay, a C-terminal CRFBP fragment [CRFBP(10 kD)] was found to potentiates CRF-
intracellular Ca2+ release, demonstrating that CRFBP possesses excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP [CRFBP(27 kD)]. This interaction was CRF2-specific, as CRF1 responses were not potentiated by CRFBP(10 kD). As there were currently no
small molecule ligands available that selectively interact with either CRFBP or CRF2, a
cell-based
assay was miniaturized, wherein CRFBP(10 kD) was fused as a chimera with CRF2α, that allowed us to a perform a high-
throughput screen (HTS) of approximately 350,000 small molecules. This resulted in the identification of negative allosteric modulators (NAMs) of the CRFBP(10 kD)-CRF2 complex that blunt CRF-induced potentiation of N-Methyl-D-
aspartic acid receptor (NMDAR)-mediated synaptic transmission in
dopamine neurons in the ventral tegmental area (VTA). These results provide the first evidence of specific roles for CRF2 and CRFBP in the modulation of neuronal activity and suggest that NMDARs in the VTA may be a target for the treatment of stress and
substance abuse disorders such as
alcohol use disorder.