New Small Molecule Agonists to the Thyrotropin Receptor

Abstract

Background: Novel small molecular ligands (SMLs) to the thyrotropin 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 highthroughput screening system and we screened 48,224 compounds from a 100K library in duplicate. Results: We obtained 62 hits using the cut-off criteria of the mean -three standard deviations above the baseline. Twenty molecules with the greatest activity were rescreened against the parent CHO-luciferase cell for nonspecific activation, and we selected two molecules (MS437 and MS438) with the highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against luteinizing hormone (LH)/human chorionic gonadotropin receptor and follicle stimulating hormone receptor-expressing cells. Molecule MS437 had a TSHR-stimulating potency with an EC 50 of 13 · 10 -8 M, and molecule MS438 had an EC 50 of 5.3 · 10 -8 M. 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 LHR ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 and E506 for MS438 and T501 for MS437 in the intrahelical region of transmembrane helix 3. 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 measure G sa , G bc , G aq , and G a12 activation quantitatively. 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 bc pathway. The small molecules MS437 and MS438 also showed upregulation of thyroglobulin (Tg), sodium iodine symporter (NIS), and TSHR gene expression. Conclusions: Pharmacokinetic analysis of MS437 and MS438 indicated their pharmacotherapeutic potential, and their intraperitoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules can therefore serve as lead molecules for further development of powerful TSH agonists.

Reflections on Thyroid Autoimmunity: A Personal Overview from the Past into the Future

“…Synthetic 'small molecule' TSHR antagonists have been isolated by several groups [95][96][97]. More antigen specific than systemic IGF-1R blockade, these molecules enter a pocket in the transmembrane domain and have relative in vitro specificity for the TSHR compared to the closely related gonadotropin hormone receptors.…”

Targeting the thyroid-stimulating hormone receptor with small molecule ligands and antibodies

“…Our own extensive studies on the TSHR for several years lead us also to discover agonists to the TSHR by performing high-throughput screening (HTS) of large chemical libraries. Our efforts using a transcriptionally based luciferase assay based on TSHR transfected CHO cells [60] and screening 50,000 compounds revealed two novel and specific agonists against the TSHR which are also allosteric ligands with ‘drug-like’ characteristics (pharmacokinetics and pharmacodynamics -PKs/PDs) and showing in vivo potency in animal studies. These lead TSHR agonists (MS437 and MS438) (Table 2) have different binding sites than the agonist described earlier but are also allosteric ligands binding to the TMD (Figure 3) [60].…”

“…Our efforts using a transcriptionally based luciferase assay based on TSHR transfected CHO cells [60] and screening 50,000 compounds revealed two novel and specific agonists against the TSHR which are also allosteric ligands with ‘drug-like’ characteristics (pharmacokinetics and pharmacodynamics -PKs/PDs) and showing in vivo potency in animal studies. These lead TSHR agonists (MS437 and MS438) (Table 2) have different binding sites than the agonist described earlier but are also allosteric ligands binding to the TMD (Figure 3) [60]. These data suggest that chemical modifications or ligands with novel scaffolds targeting signal-sensitive amino acids surrounding the allosteric binding sites within the TSHR may lead to agonists of even greater activity [61].…”

Mutations of TSHR and TP53 Genes in an Aggressive Clear Cell Follicular Carcinoma of the Thyroid

“…TSHR L272V mutation has not been reported before and its functional consequences are unknown. However, a concomitant overexpression of the TG and NIS genes in this tumor, usually decreased in thyroid carcinomas [17,18], suggests that the TSHR L272V may be a gain-offunction mutation. Gain-of-function mutants of TSHR can overstimulate thyroid follicular cells by the cAMP signaling cascade as the elevated level of TSH does.…”