A low-molecular-weight antagonist for the human thyrotropin receptor with therapeutic potential for hyperthyroidism

Abstract

Low-molecular-weight (LMW) antagonists for TSH receptor (TSHR) may have therapeutic potential as orally active drugs to block stimulating antibodies (TsAbs) in Graves' hyperthyroidism. We describe an approach to identify LMW ligands for TSHR based on Org41841, a LMW partial agonist for the LH/choriogonadotropin receptor and TSHR. We used molecular modeling and functional experiments to guide the chemical modification of Org41841. We identified an antagonist (NIDDK/CEB-52) that selectively inhibits activation of TSHR by both TSH and TsAbs. Whereas initially characterized in cultured cells overexpressing TSHRs, the antagonist was also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs in which it inhibited TSH- and TsAb-induced up-regulation of mRNA transcripts for thyroperoxidase. Our results establish this LMW compound as a lead for the development of higher potency antagonists and serve as proof of principle that LMW ligands that target TSHR could serve as drugs in patients with Graves' disease.

Figure 1

TSHR antagonist NIDDK/CEB-52 (compound 52; c52): rational design and pharmacology. A, Structures of Org41841 and compound 52. B, Comparison of docking modes of compound 52 (C atoms orange) and Org41841 (light purple) in a model of TSHR. The binding pocket of compound 52 is within the extracellular half of the transmembrane helical bundle between TMH3, -5, -6, and -7 (white) and close to ECL2 (white). In contrast to Org41841, the t-butyl group of compound 52 sits higher in the binding cleft and points toward P6.50. Therefore, movement of TMH6 during TSHR activation is not enforced by compound 52. This structural constraint may cause an antagonistic effect on TSHR signaling (C). Noteworthy, experimental data reveal that in the TSHR single mutant Y7.42A, compound 52 acts as an agonist (D). In Y7.42A, the alanine is less bulky compared with tyrosine; therefore, compound 52 moves downward to TMH6 and TMH7, similar to Org41841, and presses the kinked TMH6 apart below P6.50, which likely leads to movement of the intracellular part of TMH6 and TSHR activation. C, Antagonistic activity of compound 52 at TSHR and LHCGR. Intracellular cAMP accumulation was determined in response to increasing concentrations of compound 52. EC₅₀ concentrations of native ligands were as follows: TSH, 1.8 nm; LH, 0.34 nm. D, Compound 52 activates TSHR mutants Y7.42A and M9 in contrast to TSHR. Intracellular cAMP accumulation was determined without ligands (basal) or in response to 30 μm of compound 52. In M9 (7); TSHR residues within and covering the Org41841 binding cleft were replaced by the corresponding residues of LHCGR: I560V (LHCGR: V505), L570F (LHCGR: F515), P5.34T, A5.36S, L5.37Q, A5.38V, F5.42T, Y6.54F, and I5.59A. The data are presented as mean ± sem of three independent experiments, each performed in duplicate.

Figure 2

Compound 52 (c52) inhibits TSHR activation by TSH and TsAbs of Graves’ disease sera. A, Intracellular cAMP accumulation in HEK-EM 293 cells stably expressing TSHR was determined in response to a 1:50 dilution of sera from patients with Graves’ disease (GD) or the EC₅₀ concentration of bovine TSH (1.8 nm) in the presence or absence of c52. Serum from a patient with multinodular goiter was used as a control. The data are presented as mean ± sem of four independent experiments. B, c52 inhibits TPO mRNA expression in primary cultures of human thyrocytes from donor 2 stimulated by bovine TSH or GD sera. Thyrocytes were incubated with bovine TSH (1.8 nm) or a 1:50 dilution of GD sera and 10 μm of c52 for 24 h. Cells receiving 10 μm c52 were preincubated for 1 h with the same concentration of c52 before the 24-h incubation with bovine TSH. Data are presented as mean ± sem of two independent experiments.