qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate more ..
BioActive Compounds: 352
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn, NIH
NCGC Assay Overview:
Thyroid Stimulating Hormone Receptor
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate (cAMP). The TSH receptor (THSR) is mainly expressed in thyroid follicular cells and regulates their growth and function. Recombinant TSH is used to activate TSHR in patients with thyroid cancer receiving thyroid hormone suppression therapy and to screen for residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as an invaluable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages in therapeutic settings. However, no selective small molecule agonist of the TSH receptor exists, and no small molecule screen for TSHR agonists has ever been reported.
This cell based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction by potential TSHR agonists.
TSHR cell line
HEK 293 cells stably expressing the TSHR as well as a modified CNG were purchased from BD biosciences. Stimulation of cAMP production causes the CNG to open and subsequent membrane depolarization to occur. A fluorescent membrane potential dye (BD biosciences) was utilized to detect cAMP stimulation in TSHR cells.
The cells were maintained in DMEM medium (Invitrogen) containing 10 % FBS (Hyclone), 100 units/ml Penicillin, 100 ug/ml Streptomycin (Invitrogen), and 250 ug/ml Geneticin (Invitrogen) and 1 ug/ml Puromycin (Invitrogen) at 37C in 5% CO2. HEK 293 cells expressing CNG but without TSHR (parental cells) were used in concert to control for compounds signaling through endogenous receptors and targets of HEK 293 cells.
NCGC Assay Protocol Summary:
High Throughput Screening. Freshly passaged cells (TSHR or parental) were dispensed into 1536-well assay plates at 4 ul/well in DMEM containing phenol red (Invitrogen), 10% FCS (Hyclone), and 100 units/ml Penicillin, 100 ug/ml Streptomycin (Invitrogen) and incubated at 37 C with 5% CO2 for 20 to 36 hrs before the robotic screening. The screen was initiated by addition of 4 ul/well membrane potential dye containing 100 uM RO 20-1724 (Sigma Aldrich), followed by 60 min incubation at room temperature to allow for dye equilibration across the cell membrane. 23 nl/well compound or control was then added to the assay plates, which were incubated for additional 30 min before reading a Envision fluorescence plate reader (Perkin Elmer) in bottom-read mode for fluorescence intensity (emission at 590 nm).
Keywords: Thyroid-stimulating hormone TSH, TSHR, TSHR agonist, quantitative high throughput screening, qHTS, HTS, probe identification, CNG, cAMP, cyclic nucleotide gated ion channel PubChem
1. Compounds are first classified as activators, inconclusive, and inactive based on activity in the TSHR and parental cell lines. TSHR agonists (activators) are expected to increase signal in TSHR cells, but not to potentiate signal in parental cells. Signal activators potentiate signal in both cell lines (inconclusive).
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Classification". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds (activators), a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)