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.
Thyrotropin-releasing hormone receptor type 2 (TRH-R2), not TRH-R1, has been proposed to mediate the CNS effects of TRH and its more effective analog taltirelin (TAL). Consistent with this idea, TAL exhibited higher binding affinity and signaling potency at mouse TRH-R2 than TRH-R1 in a model cell system. We used TRH-R1 knockout (R1ko), R2ko and R1/R2ko mice to determine which receptor mediates the CNS effects of TAL. There was no TRH-R1 mRNA in R1ko and R1/R2ko mice and no TRH-R2 mRNA in R2ko and R1/R2ko mice. Specific [ 3 H]MeTRH binding to whole brain membranes was 5% of wild type (WT) for R1ko mice, 100% for R2ko mice and 0% for R1/R2ko mice, indicating TRH-R1 is the predominant receptor expressed in the brain. In arousal assays, TAL shortened sleep time with pentobarbital sedation in WT and R2ko mice by 44 and 49% and with ketamine/xylazine sedation by 66 and 55%, but had no effect in R1ko and R1/R2ko mice. In a tail flick assay of nociception, TAL increased response latency by 65 and 70% in WT and R2ko mice, but had no effect in R1ko and R1/R2ko mice. In a tail suspension test of depression-like behavior, TAL increased mobility time by 49 and 37% in WT and R2ko mice, but had no effect in R1ko and R1/R2ko mice. Thus, in contrast to the generally accepted view that the CNS effects of TAL are mediated by TRH-R2, these effects are mediated primarily if not exclusively by TRH-R1 in mice. Neuropsychopharmacology (2013) 38, 950-956;
We are developing an orally available small-molecule, allosteric TSH receptor (TSHR) agonist for follow-up diagnostics of patients with thyroid cancer. The agonist C2 (NCGC00161870) that we have studied so far is a racemic mixture containing equal amounts of two enantiomers, E1 and E2. As enantiomers of many drugs exhibit different pharmacologic properties, we assessed the properties of E1 and E2. We separated the two enantiomers by chiral chromatography and determined E2 as the (S)-(+) isomer via crystal structure analysis. E1 and E2 were shown to bind differently to a homology model of the transmembrane domain of TSHR in which E2 was calculated to exhibit lower binding energy than E1 and was, therefore, predicted to be more potent than E1. In HEK293 cells expressing human TSHRs, C2, E1, and E2 were equally efficacious in stimulating cAMP production, but their potencies were different. E2 was more potent (EC50 = 18 nM) than C2 (EC50 = 46 nM), which was more potent than E1 (EC50 = 217 nM). In primary cultures of human thyrocytes, C2, E1, and E2 stimulated increases in thyroperoxidase mRNA of 92-, 55-, and 137-fold and in sodium–iodide symporter mRNA of 20-, 4-, and 121-fold above basal levels, respectively. In mice, C2 stimulated an increase in radioactive iodine uptake of 1.5-fold and E2 of 2.8-fold above basal level, whereas E1 did not have an effect. C2 stimulated an increase in serum T4 of 2.4-fold, E1 of 1.9-fold, and E2 of 5.6-fold above basal levels, and a 5-day oral dosing regimen of E2 increased serum T4 levels comparable to recombinant human TSH (rhTSH, Thyrogen®). Thus, E2 is more effective than either C2 or E1 in stimulating thyroid function and as efficacious as rhTSH in vivo. E2 represents the next step toward developing an oral drug for patients with thyroid cancer.
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