Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptor.
The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.
Titus
Steve
S
National Institutes of Health Chemical Genomics Center, National Human Genome Research Institute, NIH, Bethesda, MD 20892-3370, USA.
Neumann
Susanne
S
Zheng
Wei
W
Southall
Noel
N
Michael
Sam
S
Klumpp
Carleen
C
Yasgar
Adam
A
Shinn
Paul
P
Thomas
Craig J
CJ
Inglese
James
J
Gershengorn
Marvin C
MC
Austin
Christopher P
CP
D000465:Algorithms; D002138:Calibration; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005189:False Positive Reactions; D006801:Humans; D008904:Miniaturization; D008954:Models, Biological; D021281:Quantitative Structure-Activity Relationship; D011989:Receptors, Thyrotropin / Q000819:agonists* / Q000235:genetics; D054852:Small Molecule Libraries / Q000032:analysis*; D014162:Transfection
D011989:Receptors, Thyrotropin; D054852:Small Molecule Libraries; D000242:Cyclic AMP