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BioAssay: AID 493035

Luminescence-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)

Name: Luminescence-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). ..more
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 Tested Compounds
 Tested Compounds
All(1981)
 
 
Active(46)
 
 
Inactive(1935)
 
 
 Tested Substances
 Tested Substances
All(1982)
 
 
Active(46)
 
 
Inactive(1936)
 
 
AID: 493035
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD5-MLDP_INH_LUMI_1536_3X%INH 2K)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-01-18
Modify Date: 2011-02-16

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 46
Depositor Specified Assays
Show more
AIDNameTypeComment
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)summary
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)confirmatory
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)screening
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)screening
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)confirmatory
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) dimerizationscreening
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)confirmatory
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) dimerizationconfirmatory
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 cellsconfirmatory
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 cellsconfirmatory
651760Late 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) dimerizationconfirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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)confirmatory
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: ABHD5-MLDP_INH_LUMI_1536_3X%INH 2K

Name: Luminescence-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).

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:

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, validation, 2K, 2K validation, HTS, high throughput screen, 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 identify compounds from the NIH 2K validation collection that inhibit the interaction of the lipase co-activator protein ABHD5 with the muscle lipid droplet protein (MLDP). In this G. princeps luciferase complementation assay, ABHD5 fused to the C-terminus of luciferase (ABHD5-LucC) and MLDP fused to the N-terminus of luciferase (MLDP-LucN) are incubated in the presence of test compounds. Binding of the two proteins reconstitutes full length luciferase, leading to an increase in well luminescence. As designed, compounds that inhibit the interaction of ABHD5 and MLDP will prevent luciferase reconstitution, thereby preventing an increase in well luminescence. Compounds are tested in singlicate at a final nominal concentration of 7.9 uM.

Protocol Summary:

Prior to the start of the assay 2.5 uL of lysate containing recombinant ABHD5 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 MLDP 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 300 uM Bufexamac.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 50-0.

List of Reagents:

Bufexamac Control (Sigma, B0760)
ABHD5-LucC and MLDP-LucN proteins (supplied by Assay Provider)
Full length G. princeps Luciferase (supplied by Assay Provider)
10X Assay Buffer (provided by Assay Provider)
1536 well plates (Corning, 7254)
Coelenterazine substrate (Prolume, 303A 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 by the MLSMR.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 7.9 uM (7.9μM**)Normalized percent inhibition of the primary screen at a compound concentration of 7.9 micromolar.Float%

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

Data Table (Concise)
Classification
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