qHTS Assay for Antagonists of the Thyroid Stimulating Hormone Receptor: SAR for Probe
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: 4
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. This cell based assay was conducted on a TSHR transfected HEK 293 cell line, measuring cAMP stimulation using antibody-based Homogeneous Time Resolved Fluorescence (HTRF).
Our goal is to identify small molecules that will inhibit (antagonize) the thyroid-stimulating hormone receptor (TSHR), a member of the 7 transmembrane-spanning receptor (7TMR) family of cell surface receptors. TSHR-mediated hyperthyroidism is important in thyroid pathology. The development of small molecule antagonists of TSHR may lead to therapeutic approaches for TSHR-mediated hyperthyroidism caused by constitutively activating mutations or stimulating auto-antibodies associated with Graves' disease. Graves' disease is the leading cause of hyperthyroidism and is caused by autoantibodies that stimulate TSHR (TsAbs) causing thyroid growth and unregulated overproduction of thyroid hormones and existing treatments have liabilities (agranulocytosis). In addition, TSHRs are known to be expressed in multiple extrathyroidal tissues including bone, brain, kidney, testis, fat and cells of the immune system but the role of the TSHR in these tissues is not clear. Therefore, TSHR antagonists/inverse agonists could be used as probes of extrathyroidal TSHR function. Finally, TSHR inverse agonists, which are a subclass of antagonists that inhibit agonist-independent (or basal or constitutive) TSHR signaling, could be used to inhibit TSH-independent signaling in patients with recurrent or metastatic thyroid cancer who are receiving thyroid hormone to suppress TSH.
The present assay presents confirmation data for analogues of series of interest in the primary screening assay.
An anti cAMP antibody contains a fluorescent cryptate tag (excitation 337 nm; emission 620 nm) and the acceptor is cAMP-d2 (excitation 620 nm; emission 665 nm). When antibody and cAMP-d2 interact and are excited by a 337 nm light, a FRET occurs resulting in emission at 665 nm. The assay is competition between cAMP-d2 and cAMP (from the cell lysate). The homogeneous protocol allows for one step dispense after stimulation that is automation friendly.
Step, Parameter, Value, Description
1, Cells, 3 uL, 2000 TSHR freshly harvested cells/well 1536 TC treated White solid bottom plate (Cells resuspended in 10 mM HEPES buffered HBSS and 70 uM RO 20-1724)
2, Compound, 23 nL, Libraries and controls
3, Compound, 1 uL, EC80 TSH (4 nM final)
4, Time, 2 hours, 37C
5, Reagent, 2 uL, cAMP antibody diluted 1:20 in lysis buffer
6, Time, 30 minutes, Room temperature
7, Detector, HTRF settings, Viewlux
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 inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)