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

Abstract

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.

Figure 1.

ANTAG3 structure and selectivity. A, Structure of ANTAG3. B, Antagonist activity of ANTAG3 in cells in culture. Human embryonic kidney 293 cells stably expressing TSHRs, LHRs, or FSHRs were preincubated with various doses of ANTAG3 for 20 minutes in HBSS without IBMX. Subsequently the cells were stimulated with the EC₅₀ of TSH (1.8 nM), LH (1 nM), or FSH (1 nM) in the presence of varying doses of ANTAG3 in the presence of IBMX. cAMP production was measured after 60 minutes. The results are the mean ± SE in three experiments.

Figure 2.

ANTAG3 lowers serum FT4 levels and thyroidal mRNAs for TPO and NIS in mice continuously stimulated by TRH. A, TRH (2.4 μg/d for 3 d) was administered ip via osmotic pump with or without ANTAG3 (2 mg/d). The animals were euthanized on day 4. B, Serum FT4 levels. C, mRNA levels of TPO and NIS in thyroid gland lysates. The bars represent the mean ± SE of TRH-treated (n = 20) and ANTAG3+TRH-treated mice (n = 16) in three experiments. ***, P < .001, **, P < .01 ANTAG3 vs vehicle.

Figure 3.

ANTAG3 lowers serum FT4 levels and thyroidal mRNAs for TPO and NIS in mice stimulated by a single injection of M22. A, T₃ (5 μg/d for 4 d) was administered by daily ip injection, and ANTAG3 was given for 3 days (2 mg/d) via an osmotic pump. On day 4, the animals were given an ip injection of vehicle or ANTAG3 (2 mg) and 4 hours later an ip injection of M22 (0.5 μg) or vehicle. The animals were euthanized 24 hours after the M22 injection on day 5. B, Serum FT4 levels. C, mRNA levels of TPO and NIS in thyroid gland lysates. The bars represent the mean ± SE of vehicle only (n = 10), vehicle + M22-treated (n = 15), and ANTAG3 + M22-treated mice (n = 14) in two experiments. ***, P < .001, **, P < .01 T₃ vs T₃/M22 and T₃/M22 vs T₃/ANTAG3/M22.