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 DCSRUN

Name: 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 high throughput 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:

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, HTS, high throughput screen, 1536, MLSMR, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this biochemical assay is to determine whether available compounds that were identified as active in a set of previous experiments entitled, "Luminescence-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)" (AID 602281) 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 100-50, and for inactive compounds 50-0.

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)

Due to the increasing size of the MLPCN compound library, 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 in this assay.

Assay: Dictionary: Version: 0.1

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

Grant Number: 1 R21 NS061634-01