TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate more ..
Target
Tested Compounds:
Depositor Specified Assays
| AID | Name | Type | Probe | Comment |
|---|---|---|---|---|
| 926 | qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor | confirmatory | ||
| 953 | Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293 | confirmatory | ||
| 938 | qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293 | confirmatory | ||
| 933 | Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: HTRF Activity Detection | confirmatory | ||
| 939 | Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor | confirmatory | ||
| 1401 | Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summary | summary | 3 | |
| 2104 | Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: ELISA Activity Detection | confirmatory |
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn, NIH
NCGC Assay Overview:
Confirmation of Thyroid Stimulating Hormone Receptor Agonists
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate (cAMP). The TSH receptor (THSR) is mainly expressed in thyroid follicular cells and regulates their growth and function. Recombinant TSH is used to activate TSHR in patients with thyroid cancer receiving thyroid hormone suppression therapy and to screen for residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as an invaluable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages in therapeutic settings. However, no selective small molecule agonist of the TSH receptor exists, and no small molecule screen for TSHR agonists has ever been reported.
In order to confirm specificity of the compounds to the TSHR, a confirmatory assay was conducted on a similar family member, the Luteinizing Hormone (LH) receptor, transfected into a HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: antibody-based Homogeneous Time Resolved Fluorescence (HTRF). This line did not contain the CNG and was therefore incapable of being used in the primary assay format.
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn, NIH
NCGC Assay Overview:
Confirmation of Thyroid Stimulating Hormone Receptor Agonists
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate (cAMP). The TSH receptor (THSR) is mainly expressed in thyroid follicular cells and regulates their growth and function. Recombinant TSH is used to activate TSHR in patients with thyroid cancer receiving thyroid hormone suppression therapy and to screen for residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as an invaluable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages in therapeutic settings. However, no selective small molecule agonist of the TSH receptor exists, and no small molecule screen for TSHR agonists has ever been reported.
In order to confirm specificity of the compounds to the TSHR, a confirmatory assay was conducted on a similar family member, the Luteinizing Hormone (LH) receptor, transfected into a HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: antibody-based Homogeneous Time Resolved Fluorescence (HTRF). This line did not contain the CNG and was therefore incapable of being used in the primary assay format.
Protocol
NCGC Assay Protocol Summary:
Homogeneous Time Resolved Fluorescence (HTRF) cAMP Assay. HEK 293 cells stably transfected with a vector containing the LH receptor and maintained under hygromycin selection were obtained from the laboratory of Dr. Marvin Gershengorn. Compounds were assayed using a HTRF cAMP detection kit (Cisbio) on the LH receptor transfected cell line. Briefly, 750 cells were plated in 2.5 ul/well of complete media (DMEM containing 10 % FCS, and 100 uM RO 20-1724) in 1536 well solid bottom white plates and 23 nl/well compound in DMSO solution or controls was added. Following 30 minute incubation at room temperature, 2.5 ul/well of labeled d2 cAMP and 2.5 ul/well of anti-cAMP antibody (both diluted 1:20 in lysis buffer) were added to each well using a flying reagent dispenser (Aurora Discovery, San Diego) . Plates were measured using the Envision plate reader (PerkinElmer, Boston, MA) with excitation at 330 nm and emissions of 615 nm and 660 nm.
Keywords: Luteinizing hormone LH, Thyroid-stimulating hormone TSH, TSHR, TSHR agonist, quantitative high throughput screening, qHTS, HTS, probe identification, CNG, cAMP, cyclic nucleotide gated ion channel PubChem
Homogeneous Time Resolved Fluorescence (HTRF) cAMP Assay. HEK 293 cells stably transfected with a vector containing the LH receptor and maintained under hygromycin selection were obtained from the laboratory of Dr. Marvin Gershengorn. Compounds were assayed using a HTRF cAMP detection kit (Cisbio) on the LH receptor transfected cell line. Briefly, 750 cells were plated in 2.5 ul/well of complete media (DMEM containing 10 % FCS, and 100 uM RO 20-1724) in 1536 well solid bottom white plates and 23 nl/well compound in DMSO solution or controls was added. Following 30 minute incubation at room temperature, 2.5 ul/well of labeled d2 cAMP and 2.5 ul/well of anti-cAMP antibody (both diluted 1:20 in lysis buffer) were added to each well using a flying reagent dispenser (Aurora Discovery, San Diego) . Plates were measured using the Envision plate reader (PerkinElmer, Boston, MA) with excitation at 330 nm and emissions of 615 nm and 660 nm.
Keywords: Luteinizing hormone LH, Thyroid-stimulating hormone TSH, TSHR, TSHR agonist, quantitative high throughput screening, qHTS, HTS, probe identification, CNG, cAMP, cyclic nucleotide gated ion channel PubChem
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds increasing signal (activators), a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Ratio-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds increasing signal (activators), a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Ratio-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more |
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed. | | Float | μM | |
| 3 | Efficacy | Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves. | | Float | % | |
| 4 | Analysis Comment | Annotation/notes on a particular compound's data or its analysis. | String | |||
| 5 | Acceptor-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control. | String | |||
| 6 | Acceptor-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 7 | Acceptor-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 8 | Acceptor-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 9 | Acceptor-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 10 | Acceptor-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 11 | Acceptor-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 12 | Acceptor-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 13 | Acceptor-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 14 | Acceptor-Activity at 0.0000109366 uM (1.09366e-05μM**) | % Activity at given concentration. | | Float | % | |
| 15 | Acceptor-Activity at 0.0000218733 uM (2.18733e-05μM**) | % Activity at given concentration. | | Float | % | |
| 16 | Acceptor-Activity at 0.0000437465 uM (4.37465e-05μM**) | % Activity at given concentration. | | Float | % | |
| 17 | Acceptor-Activity at 0.0000874931 uM (8.74931e-05μM**) | % Activity at given concentration. | | Float | % | |
| 18 | Acceptor-Activity at 0.0001749861 uM (0.000174986μM**) | % Activity at given concentration. | | Float | % | |
| 19 | Acceptor-Activity at 0.0003499722 uM (0.000349972μM**) | % Activity at given concentration. | | Float | % | |
| 20 | Acceptor-Activity at 0.0006999444 uM (0.000699944μM**) | % Activity at given concentration. | | Float | % | |
| 21 | Acceptor-Activity at 0.00140 uM (0.00139989μM**) | % Activity at given concentration. | | Float | % | |
| 22 | Acceptor-Activity at 0.00280 uM (0.00279978μM**) | % Activity at given concentration. | | Float | % | |
| 23 | Acceptor-Activity at 0.00560 uM (0.00559956μM**) | % Activity at given concentration. | | Float | % | |
| 24 | Acceptor-Activity at 0.011 uM (0.0111991μM**) | % Activity at given concentration. | | Float | % | |
| 25 | Acceptor-Activity at 0.022 uM (0.0223982μM**) | % Activity at given concentration. | | Float | % | |
| 26 | Acceptor-Activity at 0.045 uM (0.0447964μM**) | % Activity at given concentration. | | Float | % | |
| 27 | Acceptor-Activity at 0.090 uM (0.0895929μM**) | % Activity at given concentration. | | Float | % | |
| 28 | Acceptor-Activity at 0.179 uM (0.179186μM**) | % Activity at given concentration. | | Float | % | |
| 29 | Acceptor-Activity at 0.358 uM (0.358372μM**) | % Activity at given concentration. | | Float | % | |
| 30 | Acceptor-Activity at 0.717 uM (0.716743μM**) | % Activity at given concentration. | | Float | % | |
| 31 | Acceptor-Activity at 1.433 uM (1.43349μM**) | % Activity at given concentration. | | Float | % | |
| 32 | Acceptor-Activity at 2.867 uM (2.86697μM**) | % Activity at given concentration. | | Float | % | |
| 33 | Acceptor-Activity at 5.734 uM (5.73394μM**) | % Activity at given concentration. | | Float | % | |
| 34 | Acceptor-Activity at 11.47 uM (11.4679μM**) | % Activity at given concentration. | | Float | % | |
| 35 | Acceptor-Activity at 22.94 uM (22.9358μM**) | % Activity at given concentration. | | Float | % | |
| 36 | Acceptor-Activity at 45.87 uM (45.8716μM**) | % Activity at given concentration. | | Float | % | |
| 37 | Acceptor-Activity at 91.74 uM (91.7431μM**) | % Activity at given concentration. | | Float | % | |
| 38 | Donor-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control. | String | |||
| 39 | Donor-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 40 | Donor-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 41 | Donor-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 42 | Donor-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 43 | Donor-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 44 | Donor-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 45 | Donor-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 46 | Donor-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 47 | Donor-Activity at 0.0000109366 uM (1.09366e-05μM**) | % Activity at given concentration. | | Float | % | |
| 48 | Donor-Activity at 0.0000218733 uM (2.18733e-05μM**) | % Activity at given concentration. | | Float | % | |
| 49 | Donor-Activity at 0.0000437465 uM (4.37465e-05μM**) | % Activity at given concentration. | | Float | % | |
| 50 | Donor-Activity at 0.0000874931 uM (8.74931e-05μM**) | % Activity at given concentration. | | Float | % | |
| 51 | Donor-Activity at 0.0001749861 uM (0.000174986μM**) | % Activity at given concentration. | | Float | % | |
| 52 | Donor-Activity at 0.0003499722 uM (0.000349972μM**) | % Activity at given concentration. | | Float | % | |
| 53 | Donor-Activity at 0.0006999444 uM (0.000699944μM**) | % Activity at given concentration. | | Float | % | |
| 54 | Donor-Activity at 0.00140 uM (0.00139989μM**) | % Activity at given concentration. | | Float | % | |
| 55 | Donor-Activity at 0.00280 uM (0.00279978μM**) | % Activity at given concentration. | | Float | % | |
| 56 | Donor-Activity at 0.00560 uM (0.00559956μM**) | % Activity at given concentration. | | Float | % | |
| 57 | Donor-Activity at 0.011 uM (0.0111991μM**) | % Activity at given concentration. | | Float | % | |
| 58 | Donor-Activity at 0.022 uM (0.0223982μM**) | % Activity at given concentration. | | Float | % | |
| 59 | Donor-Activity at 0.045 uM (0.0447964μM**) | % Activity at given concentration. | | Float | % | |
| 60 | Donor-Activity at 0.090 uM (0.0895929μM**) | % Activity at given concentration. | | Float | % | |
| 61 | Donor-Activity at 0.179 uM (0.179186μM**) | % Activity at given concentration. | | Float | % | |
| 62 | Donor-Activity at 0.358 uM (0.358372μM**) | % Activity at given concentration. | | Float | % | |
| 63 | Donor-Activity at 0.717 uM (0.716743μM**) | % Activity at given concentration. | | Float | % | |
| 64 | Donor-Activity at 1.433 uM (1.43349μM**) | % Activity at given concentration. | | Float | % | |
| 65 | Donor-Activity at 2.867 uM (2.86697μM**) | % Activity at given concentration. | | Float | % | |
| 66 | Donor-Activity at 5.734 uM (5.73394μM**) | % Activity at given concentration. | | Float | % | |
| 67 | Donor-Activity at 11.47 uM (11.4679μM**) | % Activity at given concentration. | | Float | % | |
| 68 | Donor-Activity at 22.94 uM (22.9358μM**) | % Activity at given concentration. | | Float | % | |
| 69 | Donor-Activity at 45.87 uM (45.8716μM**) | % Activity at given concentration. | | Float | % | |
| 70 | Donor-Activity at 91.74 uM (91.7431μM**) | % Activity at given concentration. | | Float | % | |
| 71 | Ratio-Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control. | String | |||
| 72 | Ratio-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 73 | Ratio-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 74 | Ratio-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 75 | Ratio-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 76 | Ratio-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 77 | Ratio-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 78 | Ratio-Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 79 | Ratio-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 80 | Ratio-Activity at 0.0000109366 uM (1.09366e-05μM**) | % Activity at given concentration. | | Float | % | |
| 81 | Ratio-Activity at 0.0000218733 uM (2.18733e-05μM**) | % Activity at given concentration. | | Float | % | |
| 82 | Ratio-Activity at 0.0000437465 uM (4.37465e-05μM**) | % Activity at given concentration. | | Float | % | |
| 83 | Ratio-Activity at 0.0000874931 uM (8.74931e-05μM**) | % Activity at given concentration. | | Float | % | |
| 84 | Ratio-Activity at 0.0001749861 uM (0.000174986μM**) | % Activity at given concentration. | | Float | % | |
| 85 | Ratio-Activity at 0.0003499722 uM (0.000349972μM**) | % Activity at given concentration. | | Float | % | |
| 86 | Ratio-Activity at 0.0006999444 uM (0.000699944μM**) | % Activity at given concentration. | | Float | % | |
| 87 | Ratio-Activity at 0.00140 uM (0.00139989μM**) | % Activity at given concentration. | | Float | % | |
| 88 | Ratio-Activity at 0.00280 uM (0.00279978μM**) | % Activity at given concentration. | | Float | % | |
| 89 | Ratio-Activity at 0.00560 uM (0.00559956μM**) | % Activity at given concentration. | | Float | % | |
| 90 | Ratio-Activity at 0.011 uM (0.0111991μM**) | % Activity at given concentration. | | Float | % | |
| 91 | Ratio-Activity at 0.022 uM (0.0223982μM**) | % Activity at given concentration. | | Float | % | |
| 92 | Ratio-Activity at 0.045 uM (0.0447964μM**) | % Activity at given concentration. | | Float | % | |
| 93 | Ratio-Activity at 0.090 uM (0.0895929μM**) | % Activity at given concentration. | | Float | % | |
| 94 | Ratio-Activity at 0.179 uM (0.179186μM**) | % Activity at given concentration. | | Float | % | |
| 95 | Ratio-Activity at 0.358 uM (0.358372μM**) | % Activity at given concentration. | | Float | % | |
| 96 | Ratio-Activity at 0.717 uM (0.716743μM**) | % Activity at given concentration. | | Float | % | |
| 97 | Ratio-Activity at 1.433 uM (1.43349μM**) | % Activity at given concentration. | | Float | % | |
| 98 | Ratio-Activity at 2.867 uM (2.86697μM**) | % Activity at given concentration. | | Float | % | |
| 99 | Ratio-Activity at 5.734 uM (5.73394μM**) | % Activity at given concentration. | | Float | % | |
| 100 | Ratio-Activity at 11.47 uM (11.4679μM**) | % Activity at given concentration. | | Float | % | |
| 101 | Ratio-Activity at 22.94 uM (22.9358μM**) | % Activity at given concentration. | | Float | % | |
| 102 | Ratio-Activity at 45.87 uM (45.8716μM**) | % Activity at given concentration. | | Float | % | |
| 103 | Ratio-Activity at 91.74 uM (91.7431μM**) | % Activity at given concentration. | | Float | % | |
| 104 | Compound QC | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01 MH080680-01
Data Table (Concise)
Classification
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