Stress responses involve corticotropin releasing factor (CRF), the two cognate receptors (CRFi and CRF 2 ) and the CRF-binding protein (CRFBP). Utilizing a novel cell-based assay, a C-terminal CRFBP fragment [CRFBP(IOkD)] was found to potentiates CRF-intracellular Ca 2+ release, demonstrating that CRFBP possesses excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP [CRFBP(27kD)]. This interaction was CRFa-specific, as CRF1 responses were not potentiated by CRFBP(IOkD). 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(IOkD) was fused as a chimera with CRF 2a, 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(10kD)-CRF2 complex that blunt CRF-induced potentiation of /V-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 GRF 2 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.
COMPOSITIONS AND METHODS FOR THE MODULATION OF CRFBP AND THE TREATMENT OF ALCOHOLISM