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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.


Related in: MedlinePlus

Inhibition assay for detection of TSHR antagonists and overview of positive hits. (A) The HTS luciferase assay that was developed for detection of antagonists against the TSHR was first tested for its TSH inhibitory activity using bovine TSH (10 and 100 μU) against a control antagonist. We screened a diverse chemical library of ~80,000 compounds for novel antagonists using this assay. (B) All positive hits were tested in parallel against TSH (y-axis) and forskolin (x-axis) as shown in this x–y plot. Molecules that showed >50% inhibition of TSH signal but 0 or <5% inhibition of forskolin signal were considered as potential inhibitors of the TSHR. Fourteen such randomly selected representative molecules are shown as green dots on this plot.
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Figure 1: Inhibition assay for detection of TSHR antagonists and overview of positive hits. (A) The HTS luciferase assay that was developed for detection of antagonists against the TSHR was first tested for its TSH inhibitory activity using bovine TSH (10 and 100 μU) against a control antagonist. We screened a diverse chemical library of ~80,000 compounds for novel antagonists using this assay. (B) All positive hits were tested in parallel against TSH (y-axis) and forskolin (x-axis) as shown in this x–y plot. Molecules that showed >50% inhibition of TSH signal but 0 or <5% inhibition of forskolin signal were considered as potential inhibitors of the TSHR. Fourteen such randomly selected representative molecules are shown as green dots on this plot.

Mentions: Throughout the screen, the signal to background ratio was linear and the mean CV was 5.4% and the Z′ factor was in the range of 0.3–0.6 based on the positive control Antag3 (19) (also kindly provided by Dr. M. Gershengorn, NIH, Bethesda, MD, USA) used in the plate. This exceeded the commonly accepted threshold for validation of high-throughput assays (24). When we challenged the cells with two different concentrations of bovine TSH (10 and 100 μU) (Figure 1A), we found that stimulation with 10 μU of TSH gave an inhibition of ~30–40% compared with less than 10% inhibition observed by stimulation with 100 μU of TSH. However, on optimization of the HTS, we found 20 μU TSH as the best stimulation because it gave similar inhibition in the HTS. We used an arbitrary fixed criteria for selecting molecules as positive hits if they showed ≥50% inhibition of TSH activity. Medium with <1% of DMSO was the negative control, whereas the control molecule with TSH and just TSH alone acted as positives in the assay for normalization of the signal and identification of positives hits.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Inhibition assay for detection of TSHR antagonists and overview of positive hits. (A) The HTS luciferase assay that was developed for detection of antagonists against the TSHR was first tested for its TSH inhibitory activity using bovine TSH (10 and 100 μU) against a control antagonist. We screened a diverse chemical library of ~80,000 compounds for novel antagonists using this assay. (B) All positive hits were tested in parallel against TSH (y-axis) and forskolin (x-axis) as shown in this x–y plot. Molecules that showed >50% inhibition of TSH signal but 0 or <5% inhibition of forskolin signal were considered as potential inhibitors of the TSHR. Fourteen such randomly selected representative molecules are shown as green dots on this plot.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037132&req=5

Figure 1: Inhibition assay for detection of TSHR antagonists and overview of positive hits. (A) The HTS luciferase assay that was developed for detection of antagonists against the TSHR was first tested for its TSH inhibitory activity using bovine TSH (10 and 100 μU) against a control antagonist. We screened a diverse chemical library of ~80,000 compounds for novel antagonists using this assay. (B) All positive hits were tested in parallel against TSH (y-axis) and forskolin (x-axis) as shown in this x–y plot. Molecules that showed >50% inhibition of TSH signal but 0 or <5% inhibition of forskolin signal were considered as potential inhibitors of the TSHR. Fourteen such randomly selected representative molecules are shown as green dots on this plot.
Mentions: Throughout the screen, the signal to background ratio was linear and the mean CV was 5.4% and the Z′ factor was in the range of 0.3–0.6 based on the positive control Antag3 (19) (also kindly provided by Dr. M. Gershengorn, NIH, Bethesda, MD, USA) used in the plate. This exceeded the commonly accepted threshold for validation of high-throughput assays (24). When we challenged the cells with two different concentrations of bovine TSH (10 and 100 μU) (Figure 1A), we found that stimulation with 10 μU of TSH gave an inhibition of ~30–40% compared with less than 10% inhibition observed by stimulation with 100 μU of TSH. However, on optimization of the HTS, we found 20 μU TSH as the best stimulation because it gave similar inhibition in the HTS. We used an arbitrary fixed criteria for selecting molecules as positive hits if they showed ≥50% inhibition of TSH activity. Medium with <1% of DMSO was the negative control, whereas the control molecule with TSH and just TSH alone acted as positives in the assay for normalization of the signal and identification of positives hits.

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&rsquo; 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&ndash;0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7&thinsp;&mu;M. The selection criteria for a positive hit were based on a mean signal threshold of &ge;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 &ndash; a post receptor activator of adenylyl cyclase &ndash; confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3&thinsp;&mu;M 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.


Related in: MedlinePlus