Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summary
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 ..
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:
Thyroid Stimulating Hormone Receptor
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.
The primary assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction by potential TSHR agonists. A confirmatory assay of putative TSHR agonists was conducted on a different TSHR transfected HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: antibody-based Homogeneous Time Resolved Fluorescence (HTRF). In order to confirm specificity of the compounds to the TSHR, similar family members were also assayed including the Follicle Stimulating Hormone (FSH) receptor and Luteinizing Hormone (LH) receptor using transfected HEK 293 cell lines and measured using the HTRF format.
Please refer to other AIDs (926, 938, 939, 953, 933, 1402, 1403, 2104) for detailed assay protocols.
For the purposes of summarizing the probe activities from the project, MLSCN probes were given a score of 100. Molecules in the prior art were given a score of 80. Other, less active molecules in the same chemical series as the probe molecules were given a score of 50. Inactive analogues from these series were given a score of 0.
Categorized Comment - additional comments and annotations
Assay Cell Type: HEK293
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML067
PubChem Substance ID (SID) for probe 1: 3714076
PubChem Compound ID (CID) for probe 1: 2998659
Probe type for probe 1: Agonist
IC50/EC50 (nM) for probe 1: 11500
Anti-target for probe 1: FSHR, LHCGR
Fold selectivity for probe 1: Inactive @100 uM
Grant number for probe 1: MH080680-01
MLP Probe ML# for probe 2: ML109
PubChem Substance ID (SID) for probe 2: 26755506
PubChem Compound ID (CID) for probe 2: 25246343
Probe type for probe 2: Agonist
IC50/EC50 (nM) for probe 2: 7943
Target for probe 2: TSH receptor (gi: 38016895)
Disease relevance for probe 2: Inflammation and cancer
Anti-target for probe 2: FSHR, LHR
Fold selectivity for probe 2: >10
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK56234/ (ID: 2509788)
Grant number for probe 2: MH080680-01
PubMed Publication ID (PMID) for probe 1: 18216391
PubMed Publication ID (PMID) for probe 2: 19592511
NCBI Book chapter title for probe 2: Identification of Potent and Selective Thyroid Stimulating Hormone Receptor Agonists
Data Table (Concise)