A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice.

Because the TSH receptor (TSHR) plays an important role in the pathogenesis of thyroid disease, a TSHR antagonist could be a novel treatment. We attempted to develop a small molecule, drug-like antagonist of TSHR signaling that is selective and active in vivo. We synthesized NCGC00242364 (ANTAG3) by chemical modification of a previously reported TSHR antagonist. We tested its potency, efficacy, and selectivity in a model cell system in vitro by measuring its activity to inhibit stimulation of cAMP production stimulated by TSH, LH, or FSH. We tested the in vivo activity of ANTAG3 by measuring its effects to lower serum free T4 and thyroid gene expression in female BALB/c mice continuously treated with ANTAG3 for 3 days and given low doses of TRH continuously or stimulated by a single administration of a monoclonal thyroid-stimulating antibody M22. ANTAG3 was selective for TSHR inhibition; half-maximal inhibitory doses were 2.1 µM for TSHR and greater than 30 µM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease.

Constitutively active thyrotropin and thyrotropin-releasing hormone receptors and their inverse agonists.

Receptors for thyrotropin-releasing hormone (TRH) and thyrotropin (thyroid-stimulating hormone-TSH) are important regulators of the function of the TSH-producing cells of the anterior pituitary gland and the thyroid gland, respectively, and thereby play a central role in thyroid hormone homeostasis. Although the roles of TRH- and TSH-stimulated signaling in these endocrine glands are well understood, these receptors are expressed in other sites and their roles in these extraglandular tissues are less well known. Moreover, one of the two subtypes of TRH receptors (TRH-R2) and the single TSH receptor (TSHR) exhibit constitutive signaling activity and the roles of constitutive signaling by these receptors are poorly understood. One approach to studying constitutive signaling is to use inverse agonists. In this chapter, we will describe the experimental procedures used to measure constitutive signaling by TRH-R2 and TSHR and the effects of their specific inverse agonists.

A virtual screen for diverse ligands: discovery of selective G protein-coupled receptor antagonists.

Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.

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.

Conformationally restricted TRH analogues: constraining the pyroglutamate region.

A modified synthetic route has been developed so that the steric size of constraints added to the pyroglutamate region of TRH (pGluHisProNH(2)) can be varied. Both an analogue with a smaller ethylene bridge and a larger, more flexible propane bridge in this region have been synthesized. These analogues were synthesized in order to probe why the initial incorporation of an ethane bridge into this region of the molecule had led to an analogue with a binding constant and potency three times lower than that of an directly analogous unconstrained analogue. The data for both analogues indicated that the fall off in activity caused by the ethane bridge in the initial analogue was not caused by the size of the bridge.