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 assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization
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 assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization. ..more
BioActive Compounds: 1736
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_3X%INH CSRUN
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 assay to identify inhibitors of Hepatocyte nuclear factor 4 (HNF4) dimerization.
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.
counterscreen, secondary, CSRUN, 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.
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. 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 at a nominal final concentration of 4 uM.
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
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.
PubChem Activity Outcome and Score:
The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated 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 100-10, and for inactive compounds 10-0.
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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)