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BioAssay: AID 1844

Summary of probe development efforts to identify inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1).

Name: Summary of probe development efforts to identify inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1). ..more
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 Tested Compounds
 Tested Compounds
All(23)
 
 
Probe(4)
 
 
Active(4)
 
 
Inactive(19)
 
 
 Tested Substances
 Tested Substances
All(23)
 
 
Probe(4)
 
 
Active(4)
 
 
Inactive(19)
 
 
AID: 1844
Data Source: The Scripps Research Institute Molecular Screening Center (SF1_INH_PROBES_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-07-01
Modify Date: 2009-09-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 4    Active: 4
Related Experiments
AIDNameTypeComment
525Primary Cell-based High Throughput Screening assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)Screeningdepositor-specified cross reference: Primary HTS in singlicate to identify SF-1 inhibitors.
599Counterscreen for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1): A cell-based dose-response assay for inhibition of the RAR-related orphan receptor A (RORA)Confirmatorydepositor-specified cross reference: titration assays in triplicate to determine selectivity against RORA.
600Dose-response cell-based assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)Confirmatorydepositor-specified cross reference: Titration assays in triplicate to determine potency.
Description:
Data Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Xiaolin Li, Orphagen Pharmaceuticals, San Diego, CA
Network: Molecular Libraries Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 X01-MH077624-01
Grant Proposal PI: Xiaolin Li, Orphagen Pharmaceuticals
External Assay ID: SF1_INH_PROBES_SUMMARY

Name: Summary of probe development efforts to identify inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1).

Description:

Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding and ligand-binding domains. Compounds that interact with the ligand-binding domain could alter receptor conformation and subsequently modify transcription of target genes. Such ligand agonists and/or antagonists have already been successfully designed for 23 nuclear receptors among the 48 previously identified in the human genome (1-3). The nuclear receptor SF-1 (steroidogenic factor-1; SF-1; NR5A1) belongs to the class of "unexplored" orphan nuclear receptors that have not been fully characterized pharmacologically. SF-1 is expressed in the pituitary, testes, ovaries, and adrenal gland and regulates steroid hormone production at many levels, including direct regulation of expression of major P450 enzymes involved in steroid hormone synthesis (4). As a result, inhibitors of SF-1 are predicted to have therapeutic utility in the treatment of metastatic prostate cancer and may be useful to probe therapeutically relevant biologies, including energy metabolism and homeostasis (5), obesity, and adrenal steroid synthesis.

Summary of Probe Development Effort:

This AID summarizes the SRIMSC's probe development efforts to identify SF-1 inhibitor probes. Following primary HTS in singlicate to identify SF-1 inhibitors (AID 525), as well as titration assays in triplicate to determine potency (AID 600) and selectivity against RORA (AID 599), certain compounds were identified as possible candidates for probe development. These efforts resulted in the identification of two isoquinolinone-based SF-1 inhibitor probes (6). Structure-activity relationship (SAR) studies and analog syntheses based on the isoquinolinone scaffold resulted in two additional probes with improved potency, selectivity, and cytotoxicity profiles (7).
Details of protocols, compound structures, and assay results can be found in PubChem at the respective AIDs listed above. The results of our probe development efforts can be found at http://molscreen.florida.scripps.edu/probes.shtml. Three papers have been published detailing these compounds (6-8).

The above efforts resulted in the identification of novel, potent, and selective small molecule inhibitors of human SF-1. Two probe reports have been submitted describing compound screening and analog syntheses. This project is now closed.

References:

1. Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol 19:1429-1438, 2005.
2. Kliewer SA, Lehmann JM, and Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science 284: 757-760, 1999.
3. Li Y, Lambert MH, and Xu HE. Activation of nuclear receptors: a perspective from structural genomics. Structure (Camb) 11: 741-746., 2003.
4. Hammer GD and Ingraham HA. Steroidogenic Factor-1: Its role in endocrine organ development and differentiation. Frontiers in Neuroendocrinology 20: 199-223, 1999.
5. Majdic G, Young M, Gomez-Sanchez E, Anderson P, Szczepaniak LS, Dobbins RL, McGarry JD, and Parker KL. Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypothalamic obesity. Endocrinology. 2002. 143: 607-614.
6. Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M., Conkright, J., Griffin, P., Thacher, S., and Hodder, P., Potent, selective and cell penetrant inhibitors of SF-1 by functional ultra-high-throughput screening. Mol. Pharmacol. 2008. 73(6): p. 1776-8.
7. Roth, J., Madoux, F., Hodder, P., and Roush, W.R., Synthesis of small molecule inhibitors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on isoquinolinone scaffolds. Bioorg Med Chem Lett, 2008. 18(8): p. 2628-32.
8. Doghman M, Cazareth J, Douguet D, Madoux F, Hodder P, Lalli E. Inhibition of adrenocortical carcinoma cell proliferation by steroidogenic factor-1 inverse agonists. J Clin Endocrinol Metab. 2009 Jun;94(6):2178-83.

Keywords:

Summary AID, Probes, steroidogenic factor, SF-1, SF1, nuclear receptor, NR5A1, FTZ1, FTZF1, ELP, AD4BP, cancer, obesity, inhibitor, inhibition, inverse agonists, transcriptional assay, transient transfection, CHO-K1, luciferase, luminescence, 1536, HTS, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Summaries of all assays are presented below. Please see AIDs 525, 599, and 600 and below for detailed protocols performed in this probe development effort.

SF-1 Inhibition and IC50 Assays (Assays 1, 2 and 4): The purpose of these assays is to determine the ability of compounds to inhibit SF-1. These assays employ CHO-K1 cells transiently transfected with a Gal4DBD::SF-1LBD plasmid. The construct expresses a chimeric SF-1 nuclear receptor with its DNA binding domain (DBD) replaced with the DBD of the yeast transcription factor Gal4. These cells also transiently express a reporter plasmid in which the Gal4 upstream activating sequence (UAS) drives luciferase expression (UAS::luciferase) (4). As designed, compounds that inhibit SF-1 activity will inhibit binding of the Gal4DBD to the Gal4 UAS, thus reducing luciferase expression and well luminescence. Compounds are tested in singlicate at a nominal concentration of 10 micromolar and in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar. Details of these assays can be found in (6).

RORA IC50 Assays (Assays 3 and 5): The purpose of these counterscreen assays is to determine compound activity against the related nuclear receptor, RORA. These assays employ CHO-K1 cells transiently transfected with a Gal4DBD::RORALBD plasmid. The construct expresses a chimeric RORA nuclear receptor with its DNA binding domain (DBD) replaced with the DBD of the yeast transcription factor Gal4. These cells also transiently express a reporter plasmid in which the Gal4 upstream activating sequence (UAS) UAS drives luciferase expression (UAS::luciferase) (4). As designed, compounds that inhibit RORA activity will inhibit binding of the Gal4DBD to the Gal4 UAS, thus reducing luciferase expression and well luminescence. Compounds are tested in singlicate at a nominal concentration of 10 micromolar and in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar. Details of these assays can be found in (6).

VP16 IC50 Assay (Assay 6). The purpose of this counterscreen assay is to determine the promiscuity of compounds to inhibit transactivation reporter systems. This assay employs CHO-K1 cells transiently transfected with a Gal4DBD::VP16LBD plasmid. The construct expresses a chimeric herpes simplex virus protein VP16 with its DNA binding domain (DBD) replaced with the DBD of the yeast transcription factor Gal4. These cells also transiently express a reporter plasmid in which the Gal4 upstream activating sequence (UAS) UAS drives luciferase expression (UAS::luciferase) (4). As designed, compounds that inhibit VP16 activity will inhibit binding of the Gal4DBD to the Gal4 UAS, thus reducing luciferase expression and well luminescence. Compounds active in this assay are considered nonselective inhibitors. Compounds are tested in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar. Details of this assay can be found in (6).

CHO CC50 Assay (Assay 7). The purpose of this counterscreen assay is to determine the cytotoxicity of compounds identified as SF-1 inhibitors. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP found in viable cells. The cell line used in this assay is the parental CHO cell line that has not been transfected with any plasmids. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. Thus, well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that induce cell death will reduce ATP levels, and therefore reduce well luminescence. Compounds are assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 99 micromolar. Compounds identified as inactive in this cytotoxicity counterscreen and active in the above SF-1 assays are considered nontoxic inhibitors of SF-1. Details of this assay can be found in (6) and (7).

SFRE IC50 Assay (Assay 8). The purpose of this assay is to determine the ability of test compounds to inhibit SF-1 activity in more relevant physiological conditions. This assay employs HEK 293T cells which transiently co-express a plasmid encoding the full length SF-1 cDNA and a second plasmid containing the luciferase reporter gene under control of five tandem repeats of the endogenous SF-1 response element (p5xSFRE). As designed, compounds that inhibit SF-1 activity will inhibit SF-1 binding to p5xSFRE, leading to reduced luciferase expression and therefore reduced well luminescence. Compounds identified as active in this assay are considered probe candidates. Compounds are tested using an 11-point 1:3 serial dilution, starting at a concentration of 33 μM. Details of this assay can be found in (6).
Solubility Assay (pH 3.5) (Assay 9). The purpose of this assay is to determine the solubility of compounds identified as SF-1 inhibitors. In this assay, 1-2 mg of compounds are incubated in a glass test tube at room temperature for 24 hours in 1 mL of pH 3.5 potassium phosphate buffer, and the reaction supernatant is analyzed by high-performance liquid chromatography against a known reference compound. As designed, compounds that demonstrate solubility are considered more desirable probe candidates. Details of this assay can be found in (6).

Solubility Assay (pH 7.4) (Assay 10). The purpose of this assay is to determine the solubility of compounds identified as SF-1 inhibitors. In this assay, 1-2 mg of compounds are incubated in a glass test tube at room temperature for 24 hours in 1 mL of pH 7.4 potassium phosphate buffer, and the reaction supernatant is analyzed by high-performance liquid chromatography against a known reference compound. As designed, compounds that demonstrate solubility are considered more desirable probe candidates. Details of this assay can be found in (6).

Permeability Assay (Assay 11). The purpose of this assay is to determine compound permeability using a commercial parallel artificial membrane permeability assay kit. In this assay compounds diluted in PBS are evaluated over a range of concentrations for their ability to pass from a donor plate to a recipient plate by penetrating an artificial membrane. Membrane permeability is determined by comparison of test compounds to reference compounds of varying permeability. As designed, compounds that demonstrate higher permeability are considered more desirable probe candidates. Compounds are tested at 10 μM. Details of this assay can be found in (6).

Hepatic Microsomal Stability Assays (Assays 12-16). The purpose of these assays is to determine the hepatic microsomal stability of compounds identified as SF-1 inhibitors. In this assay, compounds are incubated with
hepatic microsomes from either human, monkey, rat, dog, or mouse in potassium phosphate buffer at 37 degrees C with shaking, followed by analysis of reaction aliquots by liquid chromatography and tandem mass spectrometry to determine half-life. As designed, compounds that exhibit greater stability (higher t1/2) are considered more desirable probe candidates. Compounds are tested at 10 μM. Details of this assay can be found in (6).
Comment
Probe molecules are defined as the positives of the assay and assigned a score of 100.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO-K1
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML017
PubChem Substance ID (SID) for probe 1: 46499821
PubChem Compound ID (CID) for probe 1: 4131581
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 200
Anti-target for probe 1: RAR-related Orphan Receptor A (RORA)
Fold selectivity for probe 1: Inactive
Grant number for probe 1: MH077624-01
MLP Probe ML# for probe 2: ML019
PubChem Substance ID (SID) for probe 2: 46499798
PubChem Compound ID (CID) for probe 2: 4076092
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 603
Anti-target for probe 2: RAR-related Orphan Receptor A (RORA)
Fold selectivity for probe 2: Inactive
Grant number for probe 2: MH077624-01
PubMed Publication ID (PMID) for probe 1: 18334597,18374567
PubMed Publication ID (PMID) for probe 2: 19318454
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1COMPOUND_SOURCESource of the compound- MLSMR or purchased.String
2MLS_NUMBERMLS numberString
3Qualifier_1Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
4Assay 1 [SF-1 INH]Indicates normalized percent inhibition of the primary screen at a nominal compound concentration of 10 micromolar.FloatμM
5Qualifier_2Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
6Assay 2 [SF-1 IC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
7Qualifier_3Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
8Assay 3 [RORA IC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
9Qualifier_4Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
10Assay 4 [SF-1 IC50]The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
11Qualifier_5Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
12Assay 5 [RORA IC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
13Qualifier_6Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
14Assay 6 [VP16 IC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
15Qualifier_7Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
16Assay 7 [CHO CC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
17Assay 8 [SFRE IC50] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in nanomolar.FloatnM
18Assay 9 [Solubility : pH 3.5]The solubility of the test compound at pH 3.5; shown in micromolar.FloatμM
19Assay 10 [Solubility Assay: pH 7.4] The solubility of the test compound at pH 7.4; shown in micromolar.FloatμM
20Assay 11 [Permeability (logP)] The membrane permeability of the test compound at 10 micromolar; shown in logP.Float
21Assay 12 [Microsome Stability Assay (human)] The microsome stability of the test compound at 10 micromolar; half life shown in minutes.Floatmin
22 Assay 13 [Microsome Stability Assay (monkey)] The microsome stability of the test compound at 10 micromolar; half life shown in minutes.Floatmin
23Assay 14 [Microsome Stability Assay (rat)] The microsome stability of the test compound at 10 micromolar; half life shown in minutes.Floatmin
24Assay 15 [Microsome Stability Assay (dog)] The microsome stability of the test compound at 10 micromolar; half life shown in minutes.Floatmin
25Assay 16 [Microsome Stability Assay (mouse)]The microsome stability of the test compound at 10 micromolar; half life shown in minutes.Floatmin
Additional Information
Grant Number: 1 X01-MH077624-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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