Limits...
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.


Evaluation of the HTS assay. (A) The Z factor was calculated using the positive control and basal control responses in each plate as per the formula described by Zhang et al (24) (B) The signal to background ratio was obtained using the total signal from the positive control well against those wells receiving medium plus DMSO. (C) The coefficient of variation (% CV) was calculated as the SD from the wells with the basal medium divided by the wells of the positive control. The data indicated that the HTS assay performed within the limits of a reliable screening.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037132&req=5

Figure 2: Evaluation of the HTS assay. (A) The Z factor was calculated using the positive control and basal control responses in each plate as per the formula described by Zhang et al (24) (B) The signal to background ratio was obtained using the total signal from the positive control well against those wells receiving medium plus DMSO. (C) The coefficient of variation (% CV) was calculated as the SD from the wells with the basal medium divided by the wells of the positive control. The data indicated that the HTS assay performed within the limits of a reliable screening.

Mentions: Using this HTS inhibition assay, we screened a total of 80,328 molecules consisting of all three libraries as described earlier at a single concentration of 16.7 μM. We obtained 450 positives hits from this initial screen with a hit ratio of 0.56%. The performance of the assay throughout the screen is indicated by the Z score, signal to background ratio, and % CV plots (Figure 2). Further, to eliminate false positives and to obtain a secondary confirmation of the positives, we performed a secondary testing at 16.7 μM against 20 μU of TSH and 10 μM of FSK. Figure 1B is an x–y plot showing the results of such a screen where percent of TSH luciferase signal is indicated in the y-axis with percent of FSK signal in the x-axis. Molecules that showed 50% or greater inhibition of TSH and none or very little inhibition against FSK were generally regarded as potential inhibitory molecules specific to the TSHR and marked out for dose–response analysis. Fourteen such potential hits (marked by green circles) in Figure 1B are represented in the plot.


TSH Receptor Signaling Abrogation by a Novel Small Molecule
Evaluation of the HTS assay. (A) The Z factor was calculated using the positive control and basal control responses in each plate as per the formula described by Zhang et al (24) (B) The signal to background ratio was obtained using the total signal from the positive control well against those wells receiving medium plus DMSO. (C) The coefficient of variation (% CV) was calculated as the SD from the wells with the basal medium divided by the wells of the positive control. The data indicated that the HTS assay performed within the limits of a reliable screening.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Evaluation of the HTS assay. (A) The Z factor was calculated using the positive control and basal control responses in each plate as per the formula described by Zhang et al (24) (B) The signal to background ratio was obtained using the total signal from the positive control well against those wells receiving medium plus DMSO. (C) The coefficient of variation (% CV) was calculated as the SD from the wells with the basal medium divided by the wells of the positive control. The data indicated that the HTS assay performed within the limits of a reliable screening.
Mentions: Using this HTS inhibition assay, we screened a total of 80,328 molecules consisting of all three libraries as described earlier at a single concentration of 16.7 μM. We obtained 450 positives hits from this initial screen with a hit ratio of 0.56%. The performance of the assay throughout the screen is indicated by the Z score, signal to background ratio, and % CV plots (Figure 2). Further, to eliminate false positives and to obtain a secondary confirmation of the positives, we performed a secondary testing at 16.7 μM against 20 μU of TSH and 10 μM of FSK. Figure 1B is an x–y plot showing the results of such a screen where percent of TSH luciferase signal is indicated in the y-axis with percent of FSK signal in the x-axis. Molecules that showed 50% or greater inhibition of TSH and none or very little inhibition against FSK were generally regarded as potential inhibitory molecules specific to the TSHR and marked out for dose–response analysis. Fourteen such potential hits (marked by green circles) in Figure 1B are represented in the plot.

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.