Counterscreen for antagonists of the thyroid stimulating hormone receptor: ELISA activity in a Follicle Stimulating Hormone receptor cell line
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.
In order to confirm specificity of the compounds to the TSHR, a confirmatory assay was conducted on a similar family member, the Follicle Stimulating Hormone (FSH) receptor, transfected into a HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: an ELISA based cAMP competition assay using cAMP-Screen Direct System (Applied Biosystems, Foster City, CA).
Transiently transfected HEK-EM 293 cells or cells stably expressing FSHR were seeded into 96-well plates at a density of 7 x104 cells per well in DMEM containing 10% FBS. Cells were cultured for 24 h before incubation for 20 min in HBSS/10 mM HEPES (pH 7.4). To determine the effect of the compound on FSH induced FSHR activity, cells were incubated for 20 min with the compound in HBSS/HEPES without IBMX and subsequently for 40 min with bovine FSH (Sigma) and the compound in HBSS/HEPES supplemented with 1 mM IBMX at 37 C. The levels of cAMP in cells incubated in HBSS/HEPES without IBMX were subtracted in all experiments. After aspiration of the incubation medium, cells were lysed using lysis buffer of the cAMP-Screen Direct system (Applied Biosystems, Foster City, CA). The cAMP content of the cell lysate was determined using the method described in the manufacturer's protocol. The chemiluminescence signal was measured in a VICTOR3 V 1420 multilabel counter (PerkinElmer,
Norwalk, CT). The potencies (EC50 and IC50) of the ligands were obtained from dose-response curves by data analysis with GraphPad Prism 4 for Windows.
Active compounds were given a score of 100, inactive compounds were given a score of 0.
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* Activity Concentration.
Data Table (Concise)