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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.


Mechanistic action of VA-K-14. (A) Indicated in this dose–response graph is a comparison of our lead molecule VA-K-14 and the reported Antag3 in order to compare their inhibition of TSH signaling. Although the two molecules are structurally different, they clearly have the potential to inhibit TSH action in a similar manner. However, the area under the curve calculation of VA-K-14 versus Antag3 indicated VA-K-14 to be significantly different in its inhibitory characteristics of the TSH response in this assay. (B) This diagram indicates the possible mechanism for the inhibitory effect of VA-K-14 on binding to the hydrophobic pocket in the TMD of the TSHR. The molecule contacts tryptophan 488 (W488) in ECL1 (pink) and valine 664 (V664) on helix 7 (blue). W488 is a naturally occurring inactivating mutation where V664 is a critical partner with Isoleucine 568 in ELC2 in stabilization of the receptor conformation. Thus, it would seem that this charge interaction network formed by VA-K-14 in the hydrophobic pocket must play a role in stabilizing an inactive conformation of the receptor transmembrane and, thus, leading to dampening of the signal.
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Figure 7: Mechanistic action of VA-K-14. (A) Indicated in this dose–response graph is a comparison of our lead molecule VA-K-14 and the reported Antag3 in order to compare their inhibition of TSH signaling. Although the two molecules are structurally different, they clearly have the potential to inhibit TSH action in a similar manner. However, the area under the curve calculation of VA-K-14 versus Antag3 indicated VA-K-14 to be significantly different in its inhibitory characteristics of the TSH response in this assay. (B) This diagram indicates the possible mechanism for the inhibitory effect of VA-K-14 on binding to the hydrophobic pocket in the TMD of the TSHR. The molecule contacts tryptophan 488 (W488) in ECL1 (pink) and valine 664 (V664) on helix 7 (blue). W488 is a naturally occurring inactivating mutation where V664 is a critical partner with Isoleucine 568 in ELC2 in stabilization of the receptor conformation. Thus, it would seem that this charge interaction network formed by VA-K-14 in the hydrophobic pocket must play a role in stabilizing an inactive conformation of the receptor transmembrane and, thus, leading to dampening of the signal.

Mentions: When we compared VA-K-14 with the control antagonist (Antag 3), which is structurally dissimilar and makes contact with disparate residues in the TMD region, we found similar degrees of inhibition at concentrations between 1 and 100 μM, but comparison of the area under the curves (AUC) showed that the two molecules were significantly (P = 0.003) different in their degree of inhibition (Figure 7A). This significant difference in their dose–response relationships also most likely indicated their binding to different residues in the TSHR-TM domain. Since VA-K-14 and Antag 3 have different binding sites within the hydrophobic pocket of the receptor, we examined the effect on inhibition of stimulation by combining the two. This analysis clearly indicated that combining two antagonists did not enhance the degree of inhibition (data not shown), suggesting that the complex dynamic molecular interactions that the small molecules make with the receptor allosteric site has limits to its distortion potential.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Mechanistic action of VA-K-14. (A) Indicated in this dose–response graph is a comparison of our lead molecule VA-K-14 and the reported Antag3 in order to compare their inhibition of TSH signaling. Although the two molecules are structurally different, they clearly have the potential to inhibit TSH action in a similar manner. However, the area under the curve calculation of VA-K-14 versus Antag3 indicated VA-K-14 to be significantly different in its inhibitory characteristics of the TSH response in this assay. (B) This diagram indicates the possible mechanism for the inhibitory effect of VA-K-14 on binding to the hydrophobic pocket in the TMD of the TSHR. The molecule contacts tryptophan 488 (W488) in ECL1 (pink) and valine 664 (V664) on helix 7 (blue). W488 is a naturally occurring inactivating mutation where V664 is a critical partner with Isoleucine 568 in ELC2 in stabilization of the receptor conformation. Thus, it would seem that this charge interaction network formed by VA-K-14 in the hydrophobic pocket must play a role in stabilizing an inactive conformation of the receptor transmembrane and, thus, leading to dampening of the signal.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Mechanistic action of VA-K-14. (A) Indicated in this dose–response graph is a comparison of our lead molecule VA-K-14 and the reported Antag3 in order to compare their inhibition of TSH signaling. Although the two molecules are structurally different, they clearly have the potential to inhibit TSH action in a similar manner. However, the area under the curve calculation of VA-K-14 versus Antag3 indicated VA-K-14 to be significantly different in its inhibitory characteristics of the TSH response in this assay. (B) This diagram indicates the possible mechanism for the inhibitory effect of VA-K-14 on binding to the hydrophobic pocket in the TMD of the TSHR. The molecule contacts tryptophan 488 (W488) in ECL1 (pink) and valine 664 (V664) on helix 7 (blue). W488 is a naturally occurring inactivating mutation where V664 is a critical partner with Isoleucine 568 in ELC2 in stabilization of the receptor conformation. Thus, it would seem that this charge interaction network formed by VA-K-14 in the hydrophobic pocket must play a role in stabilizing an inactive conformation of the receptor transmembrane and, thus, leading to dampening of the signal.
Mentions: When we compared VA-K-14 with the control antagonist (Antag 3), which is structurally dissimilar and makes contact with disparate residues in the TMD region, we found similar degrees of inhibition at concentrations between 1 and 100 μM, but comparison of the area under the curves (AUC) showed that the two molecules were significantly (P = 0.003) different in their degree of inhibition (Figure 7A). This significant difference in their dose–response relationships also most likely indicated their binding to different residues in the TSHR-TM domain. Since VA-K-14 and Antag 3 have different binding sites within the hydrophobic pocket of the receptor, we examined the effect on inhibition of stimulation by combining the two. This analysis clearly indicated that combining two antagonists did not enhance the degree of inhibition (data not shown), suggesting that the complex dynamic molecular interactions that the small molecules make with the receptor allosteric site has limits to its distortion potential.

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.