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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.


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Inhibition of GD sera by VA-K-14. (A) The bar graph shows the inhibition of TSH signal (50 μU/ml) by a human blocking monoclonal antibody (K1-70). Increasing doses of antibody caused inhibition of TSH signal. Significant inhibition (P = 0.0116) was observed at 10 μg/ml of K1-70 monoclonal antibody. This suggested that the inhibition assay was capable of measuring TSHR-Ab inhibition of cAMP generation. (B) We tested a series of GD serum samples at 1:10 dilution for inhibition by VA-K-14. The cells were first preincubated with 10 μM of VA-K-14 or just medium and then challenged with diluted serum in triplicate wells. As indicated here, there was a varied degree of inhibition observed in the presence of 10 μM of VA-K-14 (black filled bars) compared with untreated serum (gray bars) with the luciferase assay. As seen here, P3 and P5 showed the most significant suppression of their stimulating responses in the presence of antagonist, but inhibition of P12 was poor.
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Figure 6: Inhibition of GD sera by VA-K-14. (A) The bar graph shows the inhibition of TSH signal (50 μU/ml) by a human blocking monoclonal antibody (K1-70). Increasing doses of antibody caused inhibition of TSH signal. Significant inhibition (P = 0.0116) was observed at 10 μg/ml of K1-70 monoclonal antibody. This suggested that the inhibition assay was capable of measuring TSHR-Ab inhibition of cAMP generation. (B) We tested a series of GD serum samples at 1:10 dilution for inhibition by VA-K-14. The cells were first preincubated with 10 μM of VA-K-14 or just medium and then challenged with diluted serum in triplicate wells. As indicated here, there was a varied degree of inhibition observed in the presence of 10 μM of VA-K-14 (black filled bars) compared with untreated serum (gray bars) with the luciferase assay. As seen here, P3 and P5 showed the most significant suppression of their stimulating responses in the presence of antagonist, but inhibition of P12 was poor.

Mentions: We first tested our luciferase assay for inhibition of signal response in the presence of potent blocking TSHR antibody that binds to the ectodomain of the receptor. Figure 6A shows the inhibition of luciferase signal observed on stimulation of cells with 50 μU of TSH in the absence of antibody (gray bar) and in the presence of increasing doses of a monoclonal human TSHR blocking antibody (K1-70) (31) (kindly provided by Dr Bernard Rees Smith, RSR Ltd., Cardiff, Wales). Nearly 40% inhibition of stimulation was observed at 10 μg of the K1-70 blocking antibody.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Inhibition of GD sera by VA-K-14. (A) The bar graph shows the inhibition of TSH signal (50 μU/ml) by a human blocking monoclonal antibody (K1-70). Increasing doses of antibody caused inhibition of TSH signal. Significant inhibition (P = 0.0116) was observed at 10 μg/ml of K1-70 monoclonal antibody. This suggested that the inhibition assay was capable of measuring TSHR-Ab inhibition of cAMP generation. (B) We tested a series of GD serum samples at 1:10 dilution for inhibition by VA-K-14. The cells were first preincubated with 10 μM of VA-K-14 or just medium and then challenged with diluted serum in triplicate wells. As indicated here, there was a varied degree of inhibition observed in the presence of 10 μM of VA-K-14 (black filled bars) compared with untreated serum (gray bars) with the luciferase assay. As seen here, P3 and P5 showed the most significant suppression of their stimulating responses in the presence of antagonist, but inhibition of P12 was poor.
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Related In: Results  -  Collection

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Figure 6: Inhibition of GD sera by VA-K-14. (A) The bar graph shows the inhibition of TSH signal (50 μU/ml) by a human blocking monoclonal antibody (K1-70). Increasing doses of antibody caused inhibition of TSH signal. Significant inhibition (P = 0.0116) was observed at 10 μg/ml of K1-70 monoclonal antibody. This suggested that the inhibition assay was capable of measuring TSHR-Ab inhibition of cAMP generation. (B) We tested a series of GD serum samples at 1:10 dilution for inhibition by VA-K-14. The cells were first preincubated with 10 μM of VA-K-14 or just medium and then challenged with diluted serum in triplicate wells. As indicated here, there was a varied degree of inhibition observed in the presence of 10 μM of VA-K-14 (black filled bars) compared with untreated serum (gray bars) with the luciferase assay. As seen here, P3 and P5 showed the most significant suppression of their stimulating responses in the presence of antagonist, but inhibition of P12 was poor.
Mentions: We first tested our luciferase assay for inhibition of signal response in the presence of potent blocking TSHR antibody that binds to the ectodomain of the receptor. Figure 6A shows the inhibition of luciferase signal observed on stimulation of cells with 50 μU of TSH in the absence of antibody (gray bar) and in the presence of increasing doses of a monoclonal human TSHR blocking antibody (K1-70) (31) (kindly provided by Dr Bernard Rees Smith, RSR Ltd., Cardiff, Wales). Nearly 40% inhibition of stimulation was observed at 10 μg of the K1-70 blocking antibody.

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