Background: Novel small molecular ligands (SMLs) to the TSH receptor (TSHR) have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction and thyroid cancer.

Methods: To identify novel SMLs to the TSHR, we developed a transcription based luciferase-cAMP high-throughput screening (HTS) system.

Results: We screened 48,224 compounds of a 100K library in duplicate, which resulted in 62 hits using the cut-off criteria of meaną3SDs above the baseline. 20 molecules with the greatest activity were re-screened against the parent CHO-luciferase cell for non7specific activation and we selected 2 molecules (MS437 & MS438) with highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against LH/hCG receptor and FSH receptor expressing cells. Molecule MS437 had a TSHR stimulating potency with an EC₅₀ of 13 x 10^-8M and molecule MS438 had an EC₅₀ of 5.3 x 10^-8M.

The ability of these small molecule agonists to bind to the transmembrane domain of the receptor and initiate signal transduction was suggested by their activation of a chimeric receptor consisting of an LH receptor ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 & E506 for MS438 and T501 for MS437 in the intra-helical region of transmembrane helix 3 (TMH3). We also examined the G protein activating ability of these molecules using CHO cells co-expressing TSHRs transfected with luciferase reporter vectors in order to quantitatively measure G_sa, G_bg, G_aq and G_a12 activation. The MS437 and MS438 molecules showed potent activation of G_sa, G_aq and G_a12 similar to TSH but neither the small molecule agonists nor TSH showed activation of the G_bg pathway. The small molecules MS437 and MS438 also showed up-regulation of thyroglobulin (Tg), sodium iodine symporter (NIS) and TSH receptor (TSHR) gene expression.

Conclusions: Pharmacokinetic (PK) analysis of MS437 and MS438 indicated their pharmacotherapeutic potential and their intra-peritoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules, therefore, can serve as lead molecules for further development of powerful TSH agonists.