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BioAssay: AID 1951

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

Name: Summary of probe development efforts to identify activators of the nuclear receptor Steroidogenic Factor 1 (SF-1). ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Inactive(6)
 
 
AID: 1951
Data Source: The Scripps Research Institute Molecular Screening Center (SF-1_ACT_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-09-23

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
522Primary Cell-based High Throughput Screening assay for activators of the nuclear receptor Steroidogenic Factor 1 (SF-1)screeningPrimary screen.
560Primary Cell-based High Throughput Screening assay for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA)screeningPrimary screen.
675Counterscreen for activators 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)confirmatoryCounterscreen.
692Dose-response cell-based assay for activators of the nuclear receptor Steroidogenic Factor 1 (SF-1)confirmatoryDose response screen.
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Xiaolin Li, Orphagen Pharmaceuticals, San Diego, CA
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 X01-MH077624-01
Grant Proposal PI: Xiaolin Li
External Assay ID: SF-1_ACT_LEADS_SUMMARY

Name: Summary of probe development efforts to identify activators 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. Small pharmacological compounds able to bind to the cleft of the ligand-binding domain could alter its 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) belongs to the class of "unexplored" orphan nuclear receptors that have been poorly investigated at a pharmacological level. 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). Ligands for SF-1 may have clinical applications as modulators of adrenal steroid synthesis, and also as modulators of energy metabolism and obesity through the regulation of the ventromedial hypothalamus (VMH) (5).

Summary of Probe Development Effort:

Following primary HTS in singlicate (AID 522), counterscreening against RORA in triplicate (AID 675), as well as titration assays in triplicate to determine potency (AID 692), three compounds (SIDs 3716696, 7973564, 7977493) were identified as active against SF-1 and inactive against RORA, suggesting they were possible candidates for probe development. A fourth compound (SID 4256740) was selected based upon its apparent SF-1 selectivity in these assays (RORAEC50 /SF-1EC50 >99 μM / >11 μM). Two additional compounds [5a (SID 56459362) and 5b from reference 6] previously reported in the literature as potent SF-1 agonists in biochemical FRET-based assays (6) were synthesized at the SRIMSC. The four uHTS compounds were ordered for testing with the two synthesized compounds in cell-based assays against SF-1, RORA, and VP16 to determine SF-1 selectivity. The cytotoxicity of all six compounds was determined using the CellTiter-Glo assay, as previously reported (7).

Two compounds (SIDs 7973564 and 7977493) confirmed SF-1 selective activity. However, because the activity of these two compounds was restricted to a narrow concentration range (biphasic dose-response curve showing activity only at a single concentration), these leads were not considered further. As a result, this SF-1 activator probe discovery project is now closed.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs, as detailed below.

References:

1. Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol. 2005 Jun;19(6):1429-38.
2. Li Y, Lambert MH, Xu HE. Activation of nuclear receptors: a perspective from structural genomics. Structure. 2003 Jul;11(7):741-6.
3. Majdic G, Young M, Gomez-Sanchez E, Anderson P, Szczepaniak LS, Dobbins RL, McGarry JD, Parker KL. Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypothalamic obesity. Endocrinology. 2002 Feb;143(2):607-14.
4. Hammer GD, Ingraham HA. Steroidogenic factor-1: its role in endocrine organ development and differentiation. Front Neuroendocrinol. 1999 Jul;20(3):199-223.
5. Kliewer SA, Lehmann JM, Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science. 1999 Apr 30;284(5415):757-60.
6. Whitby RJ, Dixon S, Maloney PR, Delerive P, Goodwin BJ, Parks DJ, Willson TM. Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1. J Med Chem. 2006 Nov 16;49(23):6652-5.
7. Madoux F, Li X, Chase P, Zastrow G, Cameron MD, Conkright JJ, Griffin PR, Thacher S, Hodder P. Potent, selective and cell penetrant Activators of SF-1 by functional ultra-high-throughput screening. Mol Pharmacol. 2008 Jun;73(6):1776-84.

Keywords:

Summary AID, leads, steroidogenic factor, SF-1, nuclear receptor, NR5A1, FTZ1, FTZF1, ELP, AD4BP, transcriptional assay, CHO-K1, luciferase, luminescence, dose response, counterscreen, agonist, activator, agonism, activation, 1536, HTS, assay, Scripps, Scripps Florida, Molecular Library Probe Production Center Network, MLPCN.
Protocol
Please see AIDs 522, 560, 675, 692, and below for protocols performed in this probe development effort.

Assay 5. SF-1 Dose Response Assay.

The purpose of this assay is to determine dose response curves for compounds against SF-1. This assay employs the same protocol and cell line as AID 692. The assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor Gal4 with the ligand-binding domain of target receptor SF-1 (encoded by the pFA-hSF-1 plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-hSF-1 and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated SF-1 nuclear receptors. Compounds that increase the SF-1 activity will increase luciferase transcription, leading to increased well luminescence. Compounds were assayed in triplicate in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar.

Assay 6. RORA Dose Response Counterscreen.

The purpose of this assay is to determine dose response curves for the 6 compounds listed above against RORA. This assay employs the same protocol and cell line as AID 675 and AID 560. The assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor Gal4 with the ligand-binding domain of target receptor RORA (encoded by the pFA-hRORA plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-hRORA and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated RORA nuclear receptors. Compounds that increase RORA activity will increase luciferase transcription, leading to increased well luminescence. Compounds were assayed in triplicate in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar.

Assay 7. VP16 Dose Response Counterscreen.

The purpose of this assay is to determine dose response curves against the herpes virus transcriptional activator, viral protein 16 (VP16). The assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor Gal4 with the ligand-binding domain of the herpesvirus transcriptional activator VP16 (encoded by the pFA-VP16 plasmid) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-VP16 and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. Compounds that increase VP16 activity will increase luciferase transcription, leading to increased well luminescence. Compounds were assayed in triplicate in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar.

Assay 8. Cytotoxicity Counterscreen.

The purpose of this assay is to determine cytotoxicity dose response curves. The assay employs the CellTiter-Glo luminescent reagent, which contains luciferase to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. As designed, cytotoxic compounds will reduce viable cell numbers and ATP levels, resulting in decreased well luminescence. Compounds were assayed in triplicate at a nominal concentration of 11 micromolar.
Comment
No probes were identified.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Assay 1: SF-1 ACT Primary Assay (%ACT) (AID 522) (10μM**)Normalized percent activation of the primary screen at a compound concentration of 10 micromolar.Float%
2Assay 1: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
3Assay 2: RORA ACT Primary Assay (%ACT) (AID 560) (10μM**)Normalized percent activation of the primary screen at a compound concentration of 10 micromolar.Float%
4Assay 2: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
5Assay 3: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
6Assay 3: SF-1 ACT Dose Response Assay (EC50) (AID 692)Qualified EC50 in micromolar: The concentration at which 50% of the activation is observed (relative to -100% activation of DMSO-treated -SF1 cells).FloatμM
7Assay 3: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
8Assay 4: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
9Assay 4: RORA ACT Dose Response Counterscreen (EC50) (AID 675)Qualified EC50 in micromolar: The concentration at which 50% of the activation is observed (relative to -100% activation of DMSO-treated -RORA cells).FloatμM
10Assay 4: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
11Assay 5: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
12Assay 5: SF-1 Dose Response Assay (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.FloatμM
13Assay 5: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
14Assay 5: (Comment)String
15Assay 6: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
16Assay 6: RORA Dose Response Counterscreen (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.Float
17Assay 6: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
18Assay 7: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
19Assay 7: VP16 Dose Response Counterscreen (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.FloatμM
20Assay 7: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
21Assay 8: Cytotoxicity Counterscreen (11μM**)The percent of non-viable cells observed at a compound concentration of 11 micromolar.Float%
22Assay 8: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String

** Test Concentration.
Additional Information
Grant Number: 1 X01-MH077624-01

Data Table (Concise)
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