Sequence: thyroid stimulating hormone receptor [Homo sapiens]

Gene:TSHR     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
1402	Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Luteinizing Hormone Receptor Cell Line	confirmatory
1403	Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Follicle Stimulating Hormone Receptor Cell Line	confirmatory
1401	Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summary	summary

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn

NCGC Assay Overview:

This cell based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction. HEK 293 cells stably expressing the modified CNG were purchased from BD biosciences (http://www.atto.com/products/actone/features_benefits.shtml) . 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 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. This assay was used as a counterscreen to "Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor", to control for compounds signaling through endogenous receptors and other targets of HEK 293 cells.

NCGC Assay Protocol Summary:

High Throughput Screening. Freshly passaged cells 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: cAMP, CNG Ion Channel, quantitative high throughput screening, qHTS, HTS, probe identification, CNG, cAMP, cyclic nucleotide gated ion channel PubChem

Compound Ranking:

1. Compounds are first 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 Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds increasing signal (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.