A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor

Abstract

Context: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD.

Objective: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon.

Design: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA.

Results: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%.

Conclusion: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Fig. 1.

Synthetic scheme and structure of 1. See Subjects and Methods for details of the synthesis.

Fig. 2.

1 is an inverse agonist at TSHR and a competitive antagonist of TSH-stimulated signaling. A, HEKTSHR cells were exposed to the noted concentrations of 1 for 60 min in HBSS with 1 mm IBMX (Basal Signaling). Total cAMP levels were measured by ELISA. The data are from three experiments with duplicate samples and are presented as mean ± se. B, HEKTSHR cells were exposed to the indicated concentrations of 1 for 20 min in HBSS and then were incubated in HBSS with 1 mm IBMX, 1, and 1 mU/ml hTSH. After 40 min, the incubation was stopped, and total cAMP levels were measured by ELISA. The data are from two experiments with duplicate samples and are presented as mean ± se. C, HEKTSHR cells were incubated in HBSS (Control or) exposed to 30 μm 1 for 30 min in HBSS and then 1 mm IBMX, and various concentrations of hTSH were added. After an additional 45 min, the incubation was stopped, and total cAMP levels were measured by ELISA. The data are from two experiments with duplicate samples.

Fig. 3.

1 does not inhibit TSH or TSAb binding to TSHR. Specific binding of [¹²⁵I]TSH to HEKTSHR cells was performed in the absence or presence of 30 μm 1 as described in Subjects and Methods. The data are from two experiments with triplicate samples. There was no effect of 1 on TSH binding (P > 0.1). Binding of 2C11 or 4C1 antibodies to HEKTSHR cells was performed in the absence or presence of 30 μm 1 and analyzed by FACS as described in Subjects and Methods. The data are from two experiments with triplicate samples. There was no effect of 1 on antibody binding (P > 0.1).

Fig. 4.

1 is an antagonist of GD sera stimulation of cAMP production. A, HEKTSHR cells were incubated without (control) or with 30 μm 1 for 20 min, and then 1 mm IBMX and 1:30 or 1:100 dilutions of each of three GD sera were added. After an additional 40 min, the incubation was stopped, and total cAMP levels were measured by ELISA. The data are from one experiment with triplicate samples and are representative of three experiments. The effects of 1 were significant by paired t test (P < 0.01). B, HEKTSHR cells were incubated without (control) or with 30 μm 1 for 20 min, and then 1 mm IBMX and 1:30 dilutions of 30 GD sera were added. After an additional 40 min, the incubation was stopped, and total cAMP levels were measured by ELISA. The data are presented as percent inhibition = 100 − (100 × samples exposed to 1/control). The data are representative of two experiments performed in duplicate.

Fig. 5.

Inhibition of basal and TSAb-induced up-regulation of the expression of TPO mRNA by 1 in primary cultures of human thyrocytes. Thyrocytes were incubated in DMEM containing 2% FBS without (control) or with 30 μm 1 without (basal) or with 1:10 dilutions of GD sera at 37 C. After 48 h, the buffers were aspirated, the cells were lysed, and the levels of TPO mRNA were measured and normalized to GAPDH mRNA. The mRNA levels are presented as fold of basal levels (control) (A) and as percent inhibition = 100 − (100 × samples exposed to 1/control) (B). The data from two independent experiments with duplicate samples are shown.