Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: ELISA Activity Detection
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: 12
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn, NIH
NCGC Assay Overview:
Confirmation of Thyroid Stimulating Hormone Receptor Agonists
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. Recombinant TSH is used to activate TSHR in patients with thyroid cancer receiving thyroid hormone suppression therapy and to screen for residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as an invaluable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages in therapeutic settings. However, no selective small molecule agonist of the TSH receptor exists, and no small molecule screen for TSHR agonists has ever been reported.
A confirmatory assay of putative TSHR agonists was conducted on a HEK 293 cell line stably expressing the TSHR, 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).
NCGC Assay Protocol Summary:
HEK 293 cells expressing TSHR were cultured for 24 h before incubation for 1 h in serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) (SIGMA) and small molecule ligands in a humidified 5% CO2 incubator at 37C. Following aspiration of the medium, cells were lysed using lysis buffer of the cAMP-Screen DirectTM System (Applied Biosystems). The cAMP content of the cell lysate was determined using the method described in the manufacturer#s protocol. An enzyme substrate with a luminescent enhancer generates glow light emission kinetics. Light signal intensity, which is inversely proportional to the cAMP level in the sample, is measured in a luminometer (Perkin Elmer, 1420 Multilabel, Victor 3) for 3 seconds/well 30 min after substrate addition. Potencies (i.e., EC50) of the ligands were obtained from dose-response curves by data analysis with GraphPad Prism 4 software for Windows.
Compounds tested with an AC50 below one micomolar were given a score of 100. Less active compounds were given a score of 10 and inactive compounds were given a score of 0.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)