A small molecule inverse agonist for the human thyroid-stimulating hormone receptor

Abstract

Small molecule inverse agonists for the TSH receptor (TSHR) may be used as probes of the role of basal (or agonist-independent or constitutive) signaling and may have therapeutic potential as orally active drugs to inhibit basal signaling in patients with thyroid cancer and in some patients with hyperthyroidism. We describe the first small-molecule ligand [1;2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one] that exhibits inverse agonist properties at TSHR. 1 inhibits basal and TSH-stimulated signaling, measured as cAMP production, by TSHRs in HEK-EM 293 cells stably expressing wild-type TSHRs; the antagonism of TSH-mediated signaling is competitive. 1 also inhibits basal signaling by wild-type TSHRs, and four constitutively active mutants of TSHR expressed transiently in HEK-EM 293 cells. 1 was active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs where it inhibited basal levels of mRNA transcripts for thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR. These data serve as proof of principle that small, drug-like molecules can inhibit basal signaling by TSHR. We suggest that this small molecule is a lead compound for the development of higher-potency inverse agonists that can be used as probes of TSHR biology with therapeutic potential.

Figure 1

Synthesis of 1 [2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one (NCGC00161856)]. ACN, Acetonitrile; EtOH, ethanol; Me, methyl; MW, microwave.

Figure 2

Structure and effect of 1 on agonist-independent signaling by TSHR. A, Chemical structure of 1 [2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one (NCGC00161856)]. B, HEK-EM 293 cells stably expressing TSHRs were exposed to the noted concentrations of 1 for 60 min in HBSS with 1 mm IBMX (basal signaling) as described in Materials and Methods. Nontreated cells, incubated with IBMX only, were used as control. After 60 min, the cells were lysed, and cAMP levels were measured by ELISA. The data from two independent experiments with duplicate samples are shown as percentage of control.

Figure 3

1 is a competitive antagonist of TSH-stimulated signaling. A, HEK-EM 293 cells stably expressing TSHRs were exposed to the noted concentrations of 1 20 min before the addition of a half-maximally effective concentration of TSH (1.8 nm, control) and 1 mm IBMX. After 40 min incubation with 1 and TSH, the cells were lysed and cAMP levels were measured by ELISA. B, HEK-EM 293 cells stably expressing TSHRs were incubated in the absence of 1 or in the presence of 3, 10, or 30 μm 1 that was added 20 min before the addition of increasing concentrations of TSH (0.18 nm to 1.8 μm) and 1 mm IBMX. After 40 min, the cells were lysed and cAMP levels were measured by ELISA. The data from two independent experiments with duplicate samples are shown as percentage of maximum. A Schild plot of these data was linear with a slope not different from 1.0 (not shown). bTSH, Bovine TSH.

Figure 4

1 inhibits the basal activities of constitutively active mutant TSHRs (CAMs). HEK-EM 293 cells transiently expressing wild-type TSHRs and mutant TSHRs S281N, M453T, I568T, or F631T, were incubated without 1 (control activity) or with the noted concentrations of 1 in HBSS with 1 mm IBMX for 60 min. Subsequently, cells were lysed and cAMP levels were measured by ELISA. The control activities of the CAMs were as follows: S281N, 15 ± 3.3-fold; M453T, 27 ± 7.3-fold; I568T, 24 ± 0.71-fold; and F631I, 20 ± 4.5-fold above wild-type TSHR basal activity. The data from two independent experiments with duplicate samples are shown as percentage of control activities.

Figure 5

Inhibition of basal cAMP production and of the basal expression of mRNAs for TPO, TSHR, TG, NIS, and DIO2 by 1 in primary cultures of human thyrocytes. A, Thyrocytes were incubated in HBSS without or with 1 mm IBMX and without (control) or with increasing concentrations of 1 for 2 h at 37 C. Thereafter, the buffers were aspirated, the cells were lysed, and intracellular cAMP was measured by ELISA. The basal activity without IBMX was subtracted from all samples. B, Thyrocytes were incubated in DMEM containing 2% FBS and 1 mm IBMX without or with 30 μm 1 as described in Materials and Methods. After 48 h, the cells were lysed and the levels of the mRNAs were measured and normalized to GAPDH mRNA. The mRNA levels are presented as percentage of basal activity (control). The data from two independent experiments with duplicate samples are shown.