Antagonists of the Thyroid Stimulating Hormone Receptor: HTS campaign
The thyroid-stimulating hormone receptor (TSHR) is a member of the 7 trans-membrane-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 more ..
BioActive Compounds: 670
The thyroid-stimulating hormone receptor (TSHR) is a member of the 7 trans-membrane-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.
In collaboration between scientist from the NCGC and the NIDDK, a miniaturized HTRF high-throughput screen was developed. It's a cell based assay, where 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.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH090855-01
Assay Submitter (PI): Marvin Gerhengorn
1. 3ul of 200 TSHR freshly harvested cells/well are dispensed into a 1536 TC-treated white solid bottom plate.
2. Through pintool addition, 23nl of compounds and control in 100% DMSO are added to the plate.
3. Then 1ul of EC80 TSH (4nM final) is added to the plate.
4. Assay is incubated at 37 deg C for 2 hours.
5. 2ul of cAMP antibody diluted 1:20 in lysis buffer is added.
6. Immediately following, 2ul of d2 diluted 1:20 in HBSS is added seperately.
7. The assay is incubated for 30 minutes at ambient room temperature.
8. The result is obtained by using a ViewLux read-out with HTRF settings.
This assay looks at inhibition, hence SIDs are "active" if activity at 11.50uM is below -70%; "inconclusive" if activity at 11.50uM is between -50% but below -70%; and inactive if activity at 11.50uM is above -50%.
PUBCHEM_ACTIVITY_SCORE is the absolute value of activity at 11.50uM, except if the activity at 11.50 uM was below -100% the activity score was set to 100. If a compound had an active above 0 at 11.50, the activity score was set to 0. The most active compound gets a score of 100 and an inactive compound gets a score of 0.
Data Table (Concise)