qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation in HTRF Activity in a Luteinizing Hormone Receptor Cell Line
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: 29
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. In order to confirm specificity of the compounds to the TSHR, a confirmatory assay was conducted on a similar family member, the Luteinizing Hormone (LH) receptor, transfected into a HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: antibody-based Homogeneous Time Resolved Fluorescence (HTRF). This line did not contain the CNG and was therefore incapable of being used in the primary assay format.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
Assay Provider: Marvin Gershengorn, NIDDK
Homogeneous Time Resolved Fluorescence (HTRF) cAMP Assay. HEK 293 cells stably transfected with a vector containing the LH receptor and maintained under hygromycin selection were obtained from the laboratory of Dr. Marvin Gershengorn. Compounds were assayed using a HTRF cAMP detection kit (Cisbio) on the LH receptor transfected cell line. Briefly, 750 cells were plated in 2.5 ul/well of complete media (DMEM containing 10 % FCS, and 100 uM RO 20-1724) in 1536 well solid bottom white plates and 23 nl/well compound in DMSO solution or controls was added. Following 30 minute incubation at room temperature, 2.5 ul/well of labeled d2 cAMP and 2.5 ul/well of anti-cAMP antibody (both diluted 1:20 in lysis buffer) were added to each well using a flying reagent dispenser (Aurora Discovery, San Diego) . Plates were measured using the Envision plate reader (PerkinElmer, Boston, MA) with excitation at 330 nm and emissions of 615 nm and 660 nm.
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)