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TSH Receptor Signaling Abrogation by a Novel Small Molecule

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Hit validation. This bar graph illustrates the testing of the fourteen chosen compounds for inhibition of TSH and forskolin (FSK) signaling (mean ± SEM). The percent inhibition of the maximum TSH or forskolin signal is represented on the y-axis. Indicated by the arrows were three potentially specific candidates that showed minimal inhibition of forskolin (black bars) but significant inhibition of TSH (red bars). These three candidate molecules were then subjected to dose–response analyses.
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Figure 3: Hit validation. This bar graph illustrates the testing of the fourteen chosen compounds for inhibition of TSH and forskolin (FSK) signaling (mean ± SEM). The percent inhibition of the maximum TSH or forskolin signal is represented on the y-axis. Indicated by the arrows were three potentially specific candidates that showed minimal inhibition of forskolin (black bars) but significant inhibition of TSH (red bars). These three candidate molecules were then subjected to dose–response analyses.

Mentions: Using our selection criteria, we identified 14 molecules as potential inhibitory molecules. These 14 molecules were further tested in triplicate at 16.7 μM against stimulation with 20 μU of TSH and 10 μM of FSK to confirm their specific inhibitory potency as indicated (Figure 3). Three molecules (marked by the arrows) appeared to have potential and were subjected to dose–response studies against TSH and FSK.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Hit validation. This bar graph illustrates the testing of the fourteen chosen compounds for inhibition of TSH and forskolin (FSK) signaling (mean ± SEM). The percent inhibition of the maximum TSH or forskolin signal is represented on the y-axis. Indicated by the arrows were three potentially specific candidates that showed minimal inhibition of forskolin (black bars) but significant inhibition of TSH (red bars). These three candidate molecules were then subjected to dose–response analyses.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037132&req=5

Figure 3: Hit validation. This bar graph illustrates the testing of the fourteen chosen compounds for inhibition of TSH and forskolin (FSK) signaling (mean ± SEM). The percent inhibition of the maximum TSH or forskolin signal is represented on the y-axis. Indicated by the arrows were three potentially specific candidates that showed minimal inhibition of forskolin (black bars) but significant inhibition of TSH (red bars). These three candidate molecules were then subjected to dose–response analyses.
Mentions: Using our selection criteria, we identified 14 molecules as potential inhibitory molecules. These 14 molecules were further tested in triplicate at 16.7 μM against stimulation with 20 μU of TSH and 10 μM of FSK to confirm their specific inhibitory potency as indicated (Figure 3). Three molecules (marked by the arrows) appeared to have potential and were subjected to dose–response studies against TSH and FSK.

View Article: PubMed Central - PubMed

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.

No MeSH data available.