Luminescence-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)
Name: Luminescence-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). ..more
BioActive Compounds: 32
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_3XIC50 2K
Name: Luminescence-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).
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).
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.
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, muscle lipid droplet protein, protein-protein, interaction, adipocyte, myocyte, G. princeps, luciferase, luminescence, complementation, complementation assay, inhibitor, inhibition, validation, 2K, 2K validation, dose response, titration, dilution, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to determine dose response curves for compounds identified as active in a set of previous experiments entitled, "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)" (AID 493035). 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 using a 10-point, 1:3 dilution series starting at a nominal concentration of 79.4 uM.
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
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.
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 (Symyx Technologies 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. 79.4 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 79.4 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 100-88, and for inactive compounds 88-1.
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)
Coelenterazine substrate (Prolume, 303A NF-CTZ-FB)
1536 well plates (Corning, 7254)
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. The MLSMR was not able to provide all compounds selected for testing.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: ( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
* Activity Concentration. ** Test Concentration.
Data Table (Concise)