Quantitative High-Throughput Screening Using a Live-Cell cAMP Assay Identifies Small-Molecule Agonists of the TSH Receptor

Abstract

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. (Journal of Biomolecular Screening 2008:120-127)

Keywords

thyroid-stimulating hormone TSH TSHR TSHR agonist quantitative high-throughput screening qHTS HTS probe identification CNG PubChem

References

Vassart G. , Pardo L. , Costagliola S. : A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 2004;29(3):119-126.

Latif R. , Ando T. , Daniel S. , Davies TF : Localization and regulation of thyrotropin receptors within lipid rafts. Endocrinology 2003;144(11): 4725-4728.

Davies TF , Ando T. , Lin RY , Tomer Y. , Latif R: Thyrotropin receptor-associated diseases: from adenomata to Graves disease. J Clin Invest 2005;115(8):1972-1983.

Fernandes JK , Day TA , Richardson MS , Sharma AK Overview of the management of differentiated thyroid cancer. Curr Treat Options Oncol 2005 ;6(1):47-57.

Chazenbalk GD , Nagayama Y. , Kaufman KD , Rapoport B: The functional expression of recombinant human thyrotropin receptors in nonthyroidal eukaryotic cells provides evidence that homologous desensitization to thyrotropin stimulation requires a cell-specific factor. Endocrinology 1990 ;127(3):1240-1244.

Tate RL , Schwartz HI , Holmes JM , Kohn LD : Thyrotropin receptors in thyroid plasma membranes: characteristics of thyrotropin binding and solubilization of thyrotropin receptor activity by tryptic digestion . J Biol Chem 1975;250(16):6509-6515.

Ludgate M. , Gire V. , Crisp M. , Aijan R. , Weetman A. , Ivan M. , et al: Contrasting effects of activating mutations of GalphaS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells. Oncogene 1999; 18(34):4798-4807.

Demeure MJ , Doffek KM , Wilson SD : Defective thyrotropin receptor G-protein cyclic adenosine monophosphate signaling mechanism in the FTC human follicular thyroid cancer cell line. Surgery 1997;122(6):1195-1201.

Lonze BE , Ginty DD : Function and regulation of CREB family transcription factors in the nervous system. Neuron 2002;35(4):605-623.

10.

Prystay L. , Gagne A. , Kasila P. , Yeh LA , Banks P: Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP. J Biomol Screen 2001;6(2):75-82.

11.

Gabriel D. , Vernier M. , Pfeifer MJ , Dasen B. , Tenaillon L. , Bouhelal R: High throughput screening technologies for direct cyclic AMP measurement . Assay Drug Dev Technol 2003 ;1(2):291-303.

12.

Weber M. , Ferrer M. , Zheng W. , Inglese J. , Strulovici B. , Kunapuli P: A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation. Assay Drug Dev Technol 2004;2(1):39-49.

13.

Reinscheid RK , Kim J. , Zeng J. , Civelli O. : High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels . Eur J Pharmacol 2003;478(1):27-34.

14.

Inglese J. , Auld DS , Jadhav A. , Johnson RL , Simeonov A. , Yasgar A. , et al: Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries . Proc Natl Acad Sci U S A 2006;103(31):11473-11478.

15.

Yao Y. , Lu J. , Llorente I. , DeBernardi M. , Cao L. : ACT:One technology: a live cAMP assay. Poster presented at the 10th Anniversary Conference of Society of Biomolecular Sciences, Orlando, FL, September 2004 .

16.

Zheng W. , Spencer RH , Kiss L: High throughput assay technologies for ion channel drug discovery . Assay Drug Dev Technol 2004;2(5):543-552.

17.

Thomas P. , Smart TG : HEK293 cell line: a vehicle for the expression of recombinant proteins . J Pharmacol Toxicol Methods 2005;51(3):187-200.

18.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4(2):67-73.