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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

Testing of selected lead molecules. These four panels indicate the dose–responses of three likely (from Figures 3 and 1) and one control unlikely lead molecule against TSH (red) and FSK (gray). VA-K14 was the only molecule that effectively inhibited the TSH signal and had no inhibition for FSK compared with L13, F9, and D22. The toxicity of these molecules was tested by measuring viability (black) using the Cell titer Fluor assay within the same assay.
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Figure 4: Testing of selected lead molecules. These four panels indicate the dose–responses of three likely (from Figures 3 and 1) and one control unlikely lead molecule against TSH (red) and FSK (gray). VA-K14 was the only molecule that effectively inhibited the TSH signal and had no inhibition for FSK compared with L13, F9, and D22. The toxicity of these molecules was tested by measuring viability (black) using the Cell titer Fluor assay within the same assay.

Mentions: Dose–responses of each of the selected molecules (K14, L13 and F9) are represented in the different panels in Figure 4 along with one non-specific molecule (D22). The dose–response curves of the molecules strongly indicated that K14 had a 30–40% inhibition of TSH with negligible inhibition of FSK and low cytotoxicity compared with molecules L13, F9, or D22. Since K14 showed specific inhibition in the range examined, this was regarded as our most specific lead antagonist against the TSHR (now referred to as VA-K-14). VA-K-14 had an IC50 = 12.32 μM, and, although it was specific to the TSHR, it appeared to have a narrow inhibitory range.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Testing of selected lead molecules. These four panels indicate the dose–responses of three likely (from Figures 3 and 1) and one control unlikely lead molecule against TSH (red) and FSK (gray). VA-K14 was the only molecule that effectively inhibited the TSH signal and had no inhibition for FSK compared with L13, F9, and D22. The toxicity of these molecules was tested by measuring viability (black) using the Cell titer Fluor assay within the same assay.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Testing of selected lead molecules. These four panels indicate the dose–responses of three likely (from Figures 3 and 1) and one control unlikely lead molecule against TSH (red) and FSK (gray). VA-K14 was the only molecule that effectively inhibited the TSH signal and had no inhibition for FSK compared with L13, F9, and D22. The toxicity of these molecules was tested by measuring viability (black) using the Cell titer Fluor assay within the same assay.
Mentions: Dose–responses of each of the selected molecules (K14, L13 and F9) are represented in the different panels in Figure 4 along with one non-specific molecule (D22). The dose–response curves of the molecules strongly indicated that K14 had a 30–40% inhibition of TSH with negligible inhibition of FSK and low cytotoxicity compared with molecules L13, F9, or D22. Since K14 showed specific inhibition in the range examined, this was regarded as our most specific lead antagonist against the TSHR (now referred to as VA-K-14). VA-K-14 had an IC50 = 12.32 μM, and, although it was specific to the TSHR, it appeared to have a narrow inhibitory range.

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