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Updated: Jun 26, 2026
Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Published on: January 27, 2014
Development and use of a high-throughput screen to identify novel modulators of the corticotropin releasing factor
Carolina L Haass-Koffler1, T Chase Francis2, Pauravi Gandhi3
1Department of Psychiatry and Human Behavior, Alpert Medical School; Department of Behavioral and Social Sciences, School of Public Health; Center for Alcohol and Addiction Studies; Carney Institute for Brain Science, Brown University, Providence RI, United States.
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.
Insights
New research identifies small molecules that modulate corticotropin-releasing factor receptor 2 (CRF2) and CRF-binding protein (CRFBP). These compounds may offer novel treatments for stress-related disorders.
Area of Science:
- Neuroscience
- Pharmacology
Background:
- Stress responses involve corticotropin-releasing factor (CRF), CRF receptors (CRF1 and CRF2), and CRF-binding protein (CRFBP).
- Research has primarily focused on CRF1, with the role of CRF2 in the central nervous system (CNS) being less understood.
- CRF2's interaction with a CRFBP fragment (CRFBP(10kD)) suggests a role in modulating neuronal activity, but the mechanism remains unclear due to a lack of chemical tools.
Purpose of the Study:
- To investigate the role of CRF2 and CRFBP(10kD) in neuronal activity modulation.
- To identify negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF2 complex using high-throughput screening.
- To characterize the identified NAMs for their effects on CRF1 and CRF2 signaling and neuronal transmission.
Main Methods:
- Miniaturized a cell-based assay with a CRFBP(10kD)-CRF2 chimera for high-throughput screening of 350,000 small molecules.
- Confirmed hits by assessing activity in parental cells, on CRF2 alone, and on CRF1 in vitro.
- Utilized ex vivo electrophysiology in CRF1:GFP mice and on dopamine neurons in the ventral tegmental area (VTA) to evaluate effects on neuronal activity.
Main Results:
- CRFBP(10kD) was found to potentiate CRF-induced intracellular Ca2+ release specifically via CRF2, suggesting excitatory roles.
- Novel small molecule CRFBP-CRF2 NAMs were identified.
- These NAMs selectively blunted CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in VTA dopamine neurons, an effect mediated by CRF2 and CRFBP, without affecting CRF1-mediated responses.
Conclusions:
- Provided the first evidence for specific roles of CRF2 and CRFBP(10kD) in modulating neuronal activity.
- Demonstrated that CRFBP(10kD)-CRF2 NAMs can selectively target CRF2-mediated pathways.
- These findings suggest potential therapeutic applications for CRFBP(10kD)-CRF2 NAMs in treating stress-related disorders, including alcohol and substance use disorders.
