@article{9cb043d56f0c4714867af8c51de8cbf5,
title = "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, CRFBP, HTS, NAM, Stress",
author = "Haass-Koffler, {Carolina L.} and Francis, {T. Chase} and Pauravi Gandhi and Reesha Patel and Mohammad Naemuddin and Nielsen, {Carsten K.} and Bartlett, {Selena E.} and Antonello Bonci and Stefan Vasile and Hood, {Becky L.} and Eigo Suyama and Hedrick, {Michael P.} and Smith, {Layton H.} and Limpert, {Allison S.} and Marisa Roberto and Cosford, {Nicholas D.P.} and Sheffler, {Douglas J.}",
note = "Funding Information: The authors would like to thank the following for funding: the National Institute on Alcohol Abuse and Alcoholism (NIAAA): R01 AA026589 (DJS); K01 AA023867, R01 AA027760, R21 AA027614 (CLH-K), R01 AA021491 and R01 AA029841 (MR), T32 AA007456 (RP); the National Institute of General Medical Sciences (NIGMS), Center of Biomedical Research Excellence (COBRE, P20 GM130414) (CLH-K); the National Institute of Mental Health (NIMH) K99 MH123673 and NIGMS FI2 GM128622 (TCF); the National Institute Drug Abuse (NIDA) R21 DA209966 and R33 DA029966 (NDPC; SEB); NIDA Intramural Research Program (TCF); NIH Fast Track Award (SEB); and the National Cancer Institute (NCI) Cancer Center Support Grant P30 CA030199. K i determinations and receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. The high-throughput screen was funded by the MLPCN (HG005033). LHS acknowledges support for the Conrad Prebys Center for Chemical Genomics at Lake Nona from the Florida Translational Research Program (COHK8). SEB acknowledges the State of California for Medical Research on Alcohol and Substance Abuse through the University of California, San Francisco. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: The authors would like to thank the following for funding: the National Institute on Alcohol Abuse and Alcoholism (NIAAA): R01 AA026589 (DJS); K01 AA023867, R01 AA027760, R21 AA027614 (CLH-K), R01 AA021491 and R01 AA029841 (MR), T32 AA007456 (RP); the National Institute of General Medical Sciences (NIGMS), Center of Biomedical Research Excellence (COBRE, P20 GM130414) (CLH-K); the National Institute of Mental Health (NIMH) K99 MH123673 and NIGMS FI2 GM128622 (TCF); the National Institute Drug Abuse (NIDA) R21 DA209966 and R33 DA029966 (NDPC; SEB); NIDA Intramural Research Program (TCF); NIH Fast Track Award (SEB); and the National Cancer Institute (NCI) Cancer Center Support Grant P30 CA030199. Ki determinations and receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. The high-throughput screen was funded by the MLPCN (HG005033). LHS acknowledges support for the Conrad Prebys Center for Chemical Genomics at Lake Nona from the Florida Translational Research Program (COHK8). SEB acknowledges the State of California for Medical Research on Alcohol and Substance Abuse through the University of California, San Francisco. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2022",
year = "2022",
month = dec,
doi = "10.1016/j.slasd.2022.09.005",
language = "English (US)",
volume = "27",
pages = "448--459",
journal = "SLAS Discovery",
issn = "2472-5552",
publisher = "Sage Publications",
number = "8",
}