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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.


(A) Specificity and TSHR docking of the lead molecule (A) CHO-TSHr stable cells, HEK-LH/CGr stable cells, and FSHr- Sertoli cells (TM4) were stimulated with the maximum effective concentrations of their respective ligands as described in Section “Materials and Methods” for 1 h at 37°C in the presence of 1 mM IBMX and increasing concentrations of our lead antagonist VA-K-14. As indicated by the red line, VA-K-14 inhibited TSH by 75% at 10 μM in contrast to an inhibition of 10–15% with the hCG/LHr cells and FSHr cells. These data were average plots of two independent experiments performed in triplicate. (B) The structure of VA-K-14 is N-methyl-4-(2-phenyl-1H-indol-3-yl)-thiazole-2-amine with a molecular weight of 305.406 Da. (C)In silico docking performed on the homology model of the TSHR transmembrane domain (26) using Autodock 4 strongly suggested that VA-K-14 docks into the hydrophobic pocket of the TSHR–TMD, thus making contact with two residues in extracellular loop 1 (ECL1) (residues Asparagine 483 and tryptophan 488) and further contacts with Leucine 468 on TMH 2, Threonine 500 on TMH 3, and Valine 664 on TMH 7.
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Figure 5: (A) Specificity and TSHR docking of the lead molecule (A) CHO-TSHr stable cells, HEK-LH/CGr stable cells, and FSHr- Sertoli cells (TM4) were stimulated with the maximum effective concentrations of their respective ligands as described in Section “Materials and Methods” for 1 h at 37°C in the presence of 1 mM IBMX and increasing concentrations of our lead antagonist VA-K-14. As indicated by the red line, VA-K-14 inhibited TSH by 75% at 10 μM in contrast to an inhibition of 10–15% with the hCG/LHr cells and FSHr cells. These data were average plots of two independent experiments performed in triplicate. (B) The structure of VA-K-14 is N-methyl-4-(2-phenyl-1H-indol-3-yl)-thiazole-2-amine with a molecular weight of 305.406 Da. (C)In silico docking performed on the homology model of the TSHR transmembrane domain (26) using Autodock 4 strongly suggested that VA-K-14 docks into the hydrophobic pocket of the TSHR–TMD, thus making contact with two residues in extracellular loop 1 (ECL1) (residues Asparagine 483 and tryptophan 488) and further contacts with Leucine 468 on TMH 2, Threonine 500 on TMH 3, and Valine 664 on TMH 7.

Mentions: We, next, analyzed the specificity of VA-K-14 against other closely homologous glycoprotein receptors – the FSH receptor and LH/hCG receptor – using a cAMP femto HTRF bioassay (Cat # 62AM5PEB, Cisbio Inc.). For the LH receptor cells, we used HEK 293 cells transfected with the rat LH/hCG receptor, and, for the FSH receptor, we used a murine Sertoli cell line (TM4), which expresses the FSHR and responds to human FSH in a dose-dependent manner. Inhibition of intracellular cAMP generation was measured after stimulation of these cells with maximal responsive doses of their respective ligands (TSH, FSH, and hCG), after preincubation with VA-K-14 (0.01–100 μM). The TSHR-CHO cells were stimulated with 20 μU of bovine TSH, LH/hCC receptor cells with 1000 μU/ml of hCG, and Sertoli cells were stimulated with 700 μU/ml of human FSH, which had previously been titrated for optimum stimulation of cAMP under our experimental conditions (18). VA-K-14 showed more than 40% inhibition on the TSHR-expressing cells (Figure 5A). VA-K-14 showed a minor degree of inhibition (~10–15%) against the hCG/LH and FSH receptor-expressing cells, suggesting small molecules that are strong antagonists against the TSHR might have inhibitory effects against their homologous glycoprotein hormone receptors as seen previously (19).


TSH Receptor Signaling Abrogation by a Novel Small Molecule
(A) Specificity and TSHR docking of the lead molecule (A) CHO-TSHr stable cells, HEK-LH/CGr stable cells, and FSHr- Sertoli cells (TM4) were stimulated with the maximum effective concentrations of their respective ligands as described in Section “Materials and Methods” for 1 h at 37°C in the presence of 1 mM IBMX and increasing concentrations of our lead antagonist VA-K-14. As indicated by the red line, VA-K-14 inhibited TSH by 75% at 10 μM in contrast to an inhibition of 10–15% with the hCG/LHr cells and FSHr cells. These data were average plots of two independent experiments performed in triplicate. (B) The structure of VA-K-14 is N-methyl-4-(2-phenyl-1H-indol-3-yl)-thiazole-2-amine with a molecular weight of 305.406 Da. (C)In silico docking performed on the homology model of the TSHR transmembrane domain (26) using Autodock 4 strongly suggested that VA-K-14 docks into the hydrophobic pocket of the TSHR–TMD, thus making contact with two residues in extracellular loop 1 (ECL1) (residues Asparagine 483 and tryptophan 488) and further contacts with Leucine 468 on TMH 2, Threonine 500 on TMH 3, and Valine 664 on TMH 7.
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Figure 5: (A) Specificity and TSHR docking of the lead molecule (A) CHO-TSHr stable cells, HEK-LH/CGr stable cells, and FSHr- Sertoli cells (TM4) were stimulated with the maximum effective concentrations of their respective ligands as described in Section “Materials and Methods” for 1 h at 37°C in the presence of 1 mM IBMX and increasing concentrations of our lead antagonist VA-K-14. As indicated by the red line, VA-K-14 inhibited TSH by 75% at 10 μM in contrast to an inhibition of 10–15% with the hCG/LHr cells and FSHr cells. These data were average plots of two independent experiments performed in triplicate. (B) The structure of VA-K-14 is N-methyl-4-(2-phenyl-1H-indol-3-yl)-thiazole-2-amine with a molecular weight of 305.406 Da. (C)In silico docking performed on the homology model of the TSHR transmembrane domain (26) using Autodock 4 strongly suggested that VA-K-14 docks into the hydrophobic pocket of the TSHR–TMD, thus making contact with two residues in extracellular loop 1 (ECL1) (residues Asparagine 483 and tryptophan 488) and further contacts with Leucine 468 on TMH 2, Threonine 500 on TMH 3, and Valine 664 on TMH 7.
Mentions: We, next, analyzed the specificity of VA-K-14 against other closely homologous glycoprotein receptors – the FSH receptor and LH/hCG receptor – using a cAMP femto HTRF bioassay (Cat # 62AM5PEB, Cisbio Inc.). For the LH receptor cells, we used HEK 293 cells transfected with the rat LH/hCG receptor, and, for the FSH receptor, we used a murine Sertoli cell line (TM4), which expresses the FSHR and responds to human FSH in a dose-dependent manner. Inhibition of intracellular cAMP generation was measured after stimulation of these cells with maximal responsive doses of their respective ligands (TSH, FSH, and hCG), after preincubation with VA-K-14 (0.01–100 μM). The TSHR-CHO cells were stimulated with 20 μU of bovine TSH, LH/hCC receptor cells with 1000 μU/ml of hCG, and Sertoli cells were stimulated with 700 μU/ml of human FSH, which had previously been titrated for optimum stimulation of cAMP under our experimental conditions (18). VA-K-14 showed more than 40% inhibition on the TSHR-expressing cells (Figure 5A). VA-K-14 showed a minor degree of inhibition (~10–15%) against the hCG/LH and FSH receptor-expressing cells, suggesting small molecules that are strong antagonists against the TSHR might have inhibitory effects against their homologous glycoprotein hormone receptors as seen previously (19).

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.