title: TSH Receptor Signaling Abrogation by a Novel Small Molecule.
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 andin vitrocharacterization 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 aZscore 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 IC50of 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 silicodocking 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.
authors.affiliation: Thyroid Research Unit, James J. Peters VA Medical Center, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors.forename: Rauf
authors.initials: R
authors.lastname: Latif
authors.affiliation: Sulzberger Columbia Genome Center, Columbia University , New York, NY , USA.
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Realubit
authors.affiliation: Sulzberger Columbia Genome Center, Columbia University , New York, NY , USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Karan
authors.affiliation: Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors.forename: Mihaly
authors.initials: M
authors.lastname: Mezei
authors.affiliation: Thyroid Research Unit, James J. Peters VA Medical Center, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
authors.forename: Terry F
authors.initials: TF
authors.lastname: Davies
keywords: TSH receptor; antagonist; small molecule