Abstract
Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves’ disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3–0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin – a post receptor activator of adenylyl cyclase – confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in GD.
Highlights
The TSH receptor (TSHR) is primarily expressed in the basolateral surface of thyroid follicular cells and induces thyroid cell growth, hormone synthesis, and hormone secretion and happens to be a primary autoantigen in autoimmune thyroid disease; especially Graves’ disease (GD) [1,2,3,4]
The pathogenic effects of GD are driven, in part, by the interaction of stimulating antibodies to the TSHR, which bind to its large extracellular domain (ECD) [6]
Evaluation of the high-throughput screening (HTS) Luciferase-Based Inhibition Assay for Screening and Identification of TSHR-Specific Inhibitors
Summary
The TSH receptor (TSHR) is primarily expressed in the basolateral surface of thyroid follicular cells and induces thyroid cell growth, hormone synthesis, and hormone secretion and happens to be a primary autoantigen in autoimmune thyroid disease; especially Graves’ disease (GD) [1,2,3,4]. GD is one of the most common organ-specific autoimmune diseases with a prevalence of ~2% in the general population [5] It is an antibody and T cell-mediated disease where hyperstimulation of the thyroid gland leads to excess thyroid hormone production. The pathogenic effects of GD are driven, in part, by the interaction of stimulating antibodies to the TSHR, which bind to its large extracellular domain (ECD) [6]. Such autoantibodies come in different varieties that can stimulate, block, or lead to apoptosis via induction of cellular stress [2, 7]. The presence of the TSHR in these and other extra thyroidal depots [10] makes it an important candidate receptor for several undefined roles secondary to the cascade of effects that may result from its chronic stimulation in GD
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