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BioAssay: AID 651760

Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization

Name: Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization. ..more
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 Tested Compounds
 Tested Compounds
All(46)
 
 
Active(8)
 
 
Inactive(38)
 
 
 Tested Substances
 Tested Substances
All(48)
 
 
Active(8)
 
 
Inactive(40)
 
 
AID: 651760
Data Source: The Scripps Research Institute Molecular Screening Center (HNF4_INH_LUMI_1536_3XIC50 MDCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-11-07
Hold-until Date: 2013-10-31
Modify Date: 2013-10-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 8
Related Experiments
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AIDNameTypeComment
493035Luminescence-based biochemical high throughput validation assay to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5)Screeningdepositor-specified cross reference: Primary screen (ABHD5-MLDP inhibitors in singlicate, 2K set)
493241Summary of the probe development efforts to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with the muscle lipid droplet protein, perilipin-5 (PLIN5; MLDP)Summarydepositor-specified cross reference: Summary (ABHD5-MLDP inhibitors)
504319Luminescence-based biochemical high throughput dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) (2K validation set)Confirmatorydepositor-specified cross reference: Dose response (ABHD5-MLDP inhibitors in triplicate, 2K set)
602281Luminescence-based biochemical primary high throughput screening assay to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5)Screeningdepositor-specified cross reference: Primary screen (ABHD5-MLDP inhibitors in singlicate)
652123Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization (ROUND 2)Confirmatorydepositor-specified cross reference
652124Late stage luminescence-based biochemical dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) (ROUND 2)Confirmatorydepositor-specified cross reference
652125Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-1 (PLIN1)(ROUND 2)Confirmatorydepositor-specified cross reference
652130Late stage luminescence-based biochemical dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) produced in HEK293T cells (ROUND 2)Confirmatorydepositor-specified cross reference
652137Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization (ROUND 3)Confirmatorydepositor-specified cross reference
652138Late stage luminescence-based biochemical dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) (ROUND 3)Confirmatorydepositor-specified cross reference
651612Luminescence-based biochemical high throughput confirmation assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5)Screeningsame project related to Summary assay
651672Counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical high throughput dose response assay to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-1 (PLIN1)Confirmatorysame project related to Summary assay
651674Counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical high throughput assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerizationScreeningsame project related to Summary assay
651677Luminescence-based biochemical high throughput dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5)Confirmatorysame project related to Summary assay
651720Counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical high throughput dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerizationConfirmatorysame project related to Summary assay
651733Luminescence-based biochemical high throughput dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) produced in HEK293T cellsConfirmatorysame project related to Summary assay
651759Late stage luminescence-based biochemical dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5) produced in HEK293T cellsConfirmatorysame project related to Summary assay
651761Late stage luminescence-based biochemical dose response assay for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5)Confirmatorysame project related to Summary assay
651766Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-1 (PLIN1)Confirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: James Granneman, Wayne State University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS061634-01
Grant Proposal PI: James Granneman, Wayne State University
External Assay ID: HNF4_INH_LUMI_1536_3XIC50 MDCSRUN

Name: Late stage counterscreen for inhibitors of the interaction of the lipase co-activator protein, abhydrolase domain containing 5 (ABHD5) with perilipin-5 (MLDP; PLIN5): Luminescence-based biochemical dose response assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization.

Description:

Adipocytes are important regulators of vertebrate energy stores, in part through the storage of dietary fat (triglyceride) that is mobilized via lipolysis during fasting states for use by tissues such as heart and skeletal muscle (1, 2). However, in chronic conditions of overnutrition, such as obesity and lipid storage disorders, excess intracellular lipid accumulation and reduced lipolysis leads to cellular lipotoxicity, which contributes to diabetes, atherosclerosis, and cardiomyopathy (2, 3). The metabolism of cellular lipid is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. In adipocytes lipolysis is inhibited by the interaction of a protein called abhydrolase domain-containing 5 (ABHD5) with the lipid droplet scaffold protein perilipin A (PLIN). In cells that do not express PLIN, such as myocytes, lipolysis is blocked by similar interactions of ABHD5 with myocyte lipid droplet protein (MLDP) (4). Studies showing reduced lipotoxicity following Plin overexpression (5, 6), combined with population studies identifying ABHD5 mutations as a cause of the lipid storage disease Chanarin-Dorfman syndrome (7), suggest that activating lipolysis by blocking interactions of ABHD5 with PLIN or MLDP will increase lipid clearance from adipocytes and other cells, thereby reducing lipotoxicity. As a result, compounds that inhibit these protein interactions may have therapeutic potential for lipid disorders such as obesity, diabetes, and cardiovascular disease (8).

References:

1. Scherer, PE, Adipose tissue: from lipid storage compartment to endocrine organ. Diabetes, 2006. 55(6): p. 1537-45.
2. Vazquez-Vela, ME, Torres, N and Tovar, AR, White adipose tissue as endocrine organ and its role in obesity. Arch Med Res, 2008. 39(8): p. 715-28.
3. Lewis, GF, Carpentier, A, Adeli, K and Giacca, A, Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev, 2002. 23(2): p. 201-29.
4. Granneman, JG, Moore, HP, Mottillo, EP and Zhu, Z, Functional interactions between Mldp (LSDP5) and Abhd5 in the control of intracellular lipid accumulation. J Biol Chem, 2009. 284(5): p. 3049-57.
5. Borg, J, Klint, C, Wierup, N, Strom, K, Larsson, S, Sundler, F, Lupi, R, Marchetti, P, Xu, G, Kimmel, A, Londos, C and Holm, C, Perilipin is present in islets of Langerhans and protects against lipotoxicity when overexpressed in the beta-cell line INS-1. Endocrinology, 2009. 150(7): p. 3049-57.
6. Brasaemle, DL, Rubin, B, Harten, IA, Gruia-Gray, J, Kimmel, AR and Londos, C, Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem, 2000. 275(49): p. 38486-93.
7. Lefevre, C, Jobard, F, Caux, F, Bouadjar, B, Karaduman, A, Heilig, R, Lakhdar, H, Wollenberg, A, Verret, JL, Weissenbach, J, Ozguc, M, Lathrop, M, Prud'homme, JF and Fischer, J, Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome. Am J Hum Genet, 2001. 69(5): p. 1002-12.
8. Wang, M and Fotsch, C, Small-molecule compounds that modulate lipolysis in adipose tissue: targeting strategies and molecular classes. Chem Biol, 2006. 13(10): p. 1019-27.

Keywords:

Late stage, powder, purchased, counterscreen, secondary, dose response, DCSRUN, HNF4, hepatic nuclear factor 4, HNF, triplicate, lipolysis, lipotoxicity, ABHD5, 1-acylglycerol-3-phosphate O-acyltransferase, abhydrolase domain-containing 5, CGI58, comparative gene identification 58, NCIE2 gene, perilipin-5, PLIN, PLIN5, lipid droplet-associated protein, Mldp, MLDP, LSDA5, LSDP5, OXPAT, muscle lipid droplet protein, protein-protein, interaction, adipocyte, myocyte, G. princeps, luciferase, luminescence, complementation, complementation assay, inhibitor, inhibition, 1536, MLSMR, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this biochemical assay is to determine whether available powder samples of compounds identified as possible ABHD5-MLDP interaction inhibitor probe candidates are nonselective, as determined by inhibition of HNF4 dimerization. HNF4 is a nuclear receptor/ transcription factor that binds DNA as an homodimer. This assay monitors HNF4 dimerization using luciferase protein complementation. This assay determines HNF4 dose response curves. Any compound active in this assay will not be pursued.
In this biochemical protein complementation assay, HNF4 fused to either the C-terminus of luciferase (HNF4-LucC) or to the N-terminus of luciferase (HNF4-LucN) are incubated in the presence of test compounds. HNF4 dimerization reconstitutes full length luciferase, leading to an increase in well luminescence. As designed, compounds that inhibit HNF4 dimerization will prevent luciferase reconstitution, thereby preventing an increase in well luminescence. Compounds are tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 40 uM.
Protocol Summary:
Prior to the start of the assay 2.5 uL of protein suspension containing recombinant HNF4-LucC were dispensed into each well of 1536-well microtiter plates. Test compounds or DMSO alone were then added to the appropriate wells. The assay was started by adding 2.5 uL of lysate containing recombinant HNF4-LucN protein. The plates were incubated for 4 hours at 25 C. Next, the assay was stopped by dispensing 5 uL of Coelenterazine reagent to each well, followed by incubation at room temperature for 30 minutes. Well luminescence was measured on the ViewLux plate reader.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) ) * 100
Where:
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing 25 uM Trans-chalcone.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 40 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 40 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-80, and for inactive compounds 72-0.
List of Reagents:
Trans-chalcone control (Sigma, 136123-5G)
HNF4 protein (supplied by Assay Provider)
10X Assay Buffer (provided by Assay Provider)
1536 well plates (Corning, 7254)
Coelenterazine substrate (Prolume, 303B NF-CTZ-FB)
Comment
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity 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
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
11Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.002 uM [1] (0.002μM**)Value of % inhibition at 0.002 uM compound concentration; replicate [1]Float%
14Inhibition at 0.002 uM [2] (0.002μM**)Value of % inhibition at 0.002 uM compound concentration; replicate [2]Float%
15Inhibition at 0.002 uM [3] (0.002μM**)Value of % inhibition at 0.002 uM compound concentration; replicate [3]Float%
16Inhibition at 0.006 uM [1] (0.006μM**)Value of % inhibition at 0.006 uM compound concentration; replicate [1]Float%
17Inhibition at 0.006 uM [2] (0.006μM**)Value of % inhibition at 0.006 uM compound concentration; replicate [2]Float%
18Inhibition at 0.006 uM [3] (0.006μM**)Value of % inhibition at 0.006 uM compound concentration; replicate [3]Float%
19Inhibition at 0.018 uM [1] (0.018μM**)Value of % inhibition at 0.018 uM compound concentration; replicate [1]Float%
20Inhibition at 0.018 uM [2] (0.018μM**)Value of % inhibition at 0.018 uM compound concentration; replicate [2]Float%
21Inhibition at 0.018 uM [3] (0.018μM**)Value of % inhibition at 0.018 uM compound concentration; replicate [3]Float%
22Inhibition at 0.055 uM [1] (0.055μM**)Value of % inhibition at 0.055 uM compound concentration; replicate [1]Float%
23Inhibition at 0.055 uM [2] (0.055μM**)Value of % inhibition at 0.055 uM compound concentration; replicate [2]Float%
24Inhibition at 0.055 uM [3] (0.055μM**)Value of % inhibition at 0.055 uM compound concentration; replicate [3]Float%
25Inhibition at 0.164 uM [1] (0.164μM**)Value of % inhibition at 0.164 uM compound concentration; replicate [1]Float%
26Inhibition at 0.164 uM [2] (0.164μM**)Value of % inhibition at 0.164 uM compound concentration; replicate [2]Float%
27Inhibition at 0.164 uM [3] (0.164μM**)Value of % inhibition at 0.164 uM compound concentration; replicate [3]Float%
28Inhibition at 0.492 uM [1] (0.492μM**)Value of % inhibition at 0.492 uM compound concentration; replicate [1]Float%
29Inhibition at 0.492 uM [2] (0.492μM**)Value of % inhibition at 0.492 uM compound concentration; replicate [2]Float%
30Inhibition at 0.492 uM [3] (0.492μM**)Value of % inhibition at 0.492 uM compound concentration; replicate [3]Float%
31Inhibition at 1.5 uM [1] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [1]Float%
32Inhibition at 1.5 uM [2] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [2]Float%
33Inhibition at 1.5 uM [3] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [3]Float%
34Inhibition at 4.4 uM [1] (4.4μM**)Value of % inhibition at 4.4 uM compound concentration; replicate [1]Float%
35Inhibition at 4.4 uM [2] (4.4μM**)Value of % inhibition at 4.4 uM compound concentration; replicate [2]Float%
36Inhibition at 4.4 uM [3] (4.4μM**)Value of % inhibition at 4.4 uM compound concentration; replicate [3]Float%
37Inhibition at 13.3 uM [1] (13.3μM**)Value of % inhibition at 13.3 uM compound concentration; replicate [1]Float%
38Inhibition at 13.3 uM [2] (13.3μM**)Value of % inhibition at 13.3 uM compound concentration; replicate [2]Float%
39Inhibition at 13.3 uM [3] (13.3μM**)Value of % inhibition at 13.3 uM compound concentration; replicate [3]Float%
40Inhibition at 39.8 uM [1] (39.8μM**)Value of % inhibition at 39.8 uM compound concentration; replicate [1]Float%
41Inhibition at 39.8 uM [2] (39.8μM**)Value of % inhibition at 39.8 uM compound concentration; replicate [2]Float%
42Inhibition at 39.8 uM [3] (39.8μM**)Value of % inhibition at 39.8 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R21 NS061634-01

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