Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

Abstract

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

Genetically Encoded Sensor Cells for the Screening of Glucocorticoid Receptor (GR) Effectors in Herbal Extracts

“…Protein 2 containing the fluorescence signal is initially located in the nucleus of the sensor cells owing to the NLS peptide (KRPAATKKAGQAKKKKLD), whereas protein 1 is located in the cytosol. Cortisol binds to GR and prompts the nuclear-translocation of protein 1 to trigger an intein-mediated conditional protein splicing (CPS) reaction, resulting in the reconstitution of the NES signal peptide (KVYPIILRLCFNLSL) [ 19 , 27 ]. The reconstituted signal peptide is instantly activated without tedious refolding steps with the consequent translocation of the fluorescent cargo to the cytosol, reporting the presence of the target, cortisol ( Figure 1 C).…”

“…In order to overcome the limitations of BiFC and FRET-based approaches, we have previously designed a reporting system based on an intein-mediated reconstitution of the signal peptide [ 17 , 18 , 19 ]. Complemented split-inteins self-catalytically react to form and break specific peptide bonds without requiring an energy source or cofactors [ 20 , 21 ].…”

“…Split-signal peptides are instantly activated through covalent bond formation without a tedious refolding step and cannot be activated by merely binding the split-fragments; therefore, this approach can address the limitations of BiFC and FRET-based reporting systems. These reporter systems have been designed into cell-based sensors that can detect various bioactive targets, including Ca 2+ , cortisol, and rapamycin [ 17 , 18 , 19 ]. These sensor cells demonstrated excellent performance in screening targets with enhanced sensitivity with a short response time.…”

The long winding road to the safer glucocorticoid receptor (GR) targeting therapies

“…In some cases, more thorough investigations included analysis of effects on GR expression, major steps in GR activation: its phosphorylation (mostly at activating Ser211) and nuclear translocation; and GR ligand properties assessed by molecular docking or ligand-binding assays [87][88][89][90][91][92][93][94]. Recently, focused screens for natural mimetics of cortisol were performed utilizing genetically engineered sensor cells detecting GR nuclear translocation, which demonstrated that decursin from Dong quai (Angelica sinensis, commonly known as female ginseng), and L-limonene from peppermint oil induce GR translocation similar to cortisol [95,96].…”

Cell-Based Sensors for the Detection of EGF and EGF-Stimulated Ca2+ Signaling

“…To overcome these limitations, we designed a cell-based sensor for the detection of EGF-EGFR interaction using a reporting strategy based on intein-mediated reconstitution of the signal peptide, and consequent fluorescence translocation. Intein (a self-processing protein) is utilized to form or break specific amide bonds to activate the signal peptide [ 39 , 40 , 41 ]. Split-intein-mediated conditional protein splicing (CPS) or conditional protein cleavage (CPC) reaction is initiated by the presence of target molecules as a trigger [ 42 ].…”