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. 2022 Dec;27(8):448-459.
doi: 10.1016/j.slasd.2022.09.005. Epub 2022 Oct 7.

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

Carolina L Haass-Koffler  1 , T Chase Francis  2 , Pauravi Gandhi  3 , Reesha Patel  3 , Mohammad Naemuddin  4 , Carsten K Nielsen  4 , Selena E Bartlett  5 , Antonello Bonci  6 , Stefan Vasile  7 , Becky L Hood  7 , Eigo Suyama  7 , Michael P Hedrick  7 , Layton H Smith  7 , Allison S Limpert  8 , Marisa Roberto  3 , Nicholas D P Cosford  9 , Douglas J Sheffler  10
Affiliations

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

Carolina L Haass-Koffler et al. SLAS Discov. 2022 Dec.

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(2); CRFBP; HTS; NAM; Stress.

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Conflict of interest statement

Declaration of Competing Interests LHS is currently an employee of Fate Therapeutics, San Diego, CA, United States. SV and BSH are currently employees of Alchem Laboratories Corp, Alachua, FL, United States. ES is currently an employee of Chugai Pharmaceutical Co. Ltd., Tokyo, Japan. MPH is currently an employee of Boundless Bio, La Jolla, CA, United States. MM is an employee of Kite Pharmaceuticals, CA, United States. AB is currently an employee of GIA (Global Institutes on Addiction), Miami, FL. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.. Optimization of cell-based assay expressing FLAG-CRFBP(10kD)-HA-CRF2α using the [35S]GTPγS binding assay.
(A) CRF produced a dose dependent stimulation of [35S]GTPγS-binding FLAG-CRF-BP(10kD)-HA-CRF2α. (B) AS-30 inhibits CRF-stimulated (1 μM) [35S]GTPγS FLAG-CRF-BP(10kD)-HA-CRF2α binding. The values are expressed as M ± SEM percentage increase in basal [35S]GTPγS binding.
Fig. 2.
Fig. 2.. Miniaturization of the calcium assay in 384-well format.
(A) Dose response curves for CRF-induced (1 pM-10 μM) intracellular calcium release in HEK293 cells expressing the FLAG-CRFBP(10kD)-HA-CRF2α (EC50 = 451 ± 1 nM). (B) Inhibition of CRF-induced (1 μM) intracellular calcium release in HEK293 cells expressing CRFBP(10kD)-CRF2α by CRF2 antagonist, AS-30 (2.2 nM - 0.54 μM) (IC50 = 27 ± 1 nM). Results are expressed as the M ± SEM relative fluorescence units (RFU), using 384-well assay format, calculated as agonist-induced maximum calcium peak/cell number × 1000.
Fig. 3.
Fig. 3.. Quality control of cell-based assay expressing FLAG-CRFBP(10kD)-HA-CRF2.
Assay quality control analysis from: (A) 96-well format (Z’ = 0.62) and (B) 384-well format (Z’ = 0.54) in HEK293 cells stably expressing the CRFBP(10kD)-CRF2. CRF (1 μM)-induced intracellular calcium (maximum RFU, EC80 concentration) was consistently inhibited by AS-30 (1 μM) (minimum RFU), n = 35. Results are expressed as the M ± SEM relative fluorescence units (RFU), calculated as agonist-induced maximum calcium peak/cell number × 1000.
Fig. 4.
Fig. 4.. CRFBP(10kD)-CRF2 antagonist hits.
Compounds fell into two series: (A) represented by the tetrazole-thiomethyl-oxadiazole like MLS-0046818, and (B) represented by the quinazolinone like MLS-0219419.
Fig. 5.
Fig. 5.. MLS-0046818 and MLS-0219419 selectively antagonize CRFBP-CRF2 responses.
(A) MLS-0046818 and (B) MLS-0219419 dose-responses were performed in the presence of an EC80 concentration of CRF in CRF1 (Red), CRF2 (Green), and CRFBP-CRF2 (Blue) Ca2+ assays. MLS-0046818 and MLS-0219419 only inhibit the CRFBP-CRF2 response. Results are expressed as the M ± SEM of the % of the EC80 CRF response.
Fig. 6.
Fig. 6.. MLS-0046818 and MLS-0219419 noncompetitively antagonize CRF responses in CRFBP-CRF2 Ca2+ assays.
CRF responses were performed for (A) ± MLS-0046818 or (B) ± MLS-0219419 as indicated. CRF maximal responses are decreased with increasing concentrations of either MLS-0046818 or MLS-0219419. Results are expressed as the M ± SEM of the % of the vehicle treated maximal CRF response.
Fig. 7.
Fig. 7.. In vivo PK properties in mouse plasma.
Mice were dosed 10 mg/kg i.p. with either (A) MLS-0046818 or (B) MLS-0219419 and drug levels were monitored over 24h in the mouse plasma. Data represented as M ± SEM of the blood plasma levels from three independent mice with AUC shown in light blue.
Fig. 8.
Fig. 8.. CRFBP-CRF2 modulators do not affect CRF1 activity on action potential dependent GABA transmission in the CeA.
A) Electrophysiological recordings were performed from CRF1+ labeled neurons in CeA slices. B) Representative traces of mIPSCs at baseline, in the presence of R121219 (1 μM) and MLS-0046818 (30 μM), and R121219 (1 μM) + MLS-0046818 (30 μM) + CRF (200 nM). C) Application of R121219 (1 μM) and MLS-0046818 (30 μM) for 15 min does not induce significant changes: one sample t-test, p = 0.5575 (frequency), p = 0.0908 (amplitude), p = 0.2017 (rise time) and p = 0.4208 (decay) in mIPSC properties relative to baseline (one-sample t-test; n = 9 neurons/6 mice). D) Application of CRF (200 nM) following pre-treatment of brain slices with R121219 (1 μM) and MLS-0046818 (30 μM) for 15 min do not produce significant changes: one sample t-test, p = 0.8948 (frequency), p = 0.3619 (amplitude), p = 0.7754 (rise time) and p = 0.1513 (decay) in mIPSC properties relative to baseline (one-sample t-test, n = 8 neurons/4 mice).
Fig. 9.
Fig. 9.. NMDAR potentiation by CRF and blockade by CRFBP-CRF2 NAMs.
(A) CRF potentiated NMDA receptor EPSCs recorded from VTA-DA neurons (n = 8, 11). 30 μM of (B) MLS-0046818 (n = 4, 9) and (C) MLS-0219419 (n = 4, 8) block CRF potentiation of NMDAR EPSCs. (D) Summary of the M EPSC ± SEM. The asterisk indicates significant differences between treatment and vehicle samples (** p < 0.01), (n = mice, cells). Traces: Black, pre-CRF; Red, Post-CRF; Scale bar 25pA/ 25msec.
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