Secondary Assay for lipid storage modulators. Cytotoxcity
NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, more ..
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: TBA
Assay Submitter (PI): Beller, Mathias; Max-Planck-Institut fur Biophysikalische Chemie, Germany.
NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, which is shielded by a droplet-specific phospholipid monolayer to which proteins are attached. The current model of LD biogenesis involves an incorporation of the lipid core into the membrane leaflets of the endoplasmic reticulum (ER) followed by a subsequent budding-like maturation of a LD, which ultimately pinches off. Once released, LD volume can increase by localized lipogenesis or fusion of existing droplet. Storage lipids are re-mobilized enzymatically by lipase activity. Lipase regulation in the adipocyte is heavily studied and involves multiple components including catecholamine signaling, the LD-associated proteins Perilipin and comparative gene identification (CGI-58) and at least two lipases named hormone sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL). LD regulation outside of the adipocyte is poorly understood and only few components are known. However, there is an urgent need to learn more about ectopic fat depots as mislocalized storage of lipids, for example in the liver or muscle, is an eminent health problem associated with insulin resistance or the metabolic syndrome. We developed a laser-scanning cytometer assay to enable 1,536-well screening and combined the results of the small molecules screen with an RNAi database based on lipid storage . The compound was tested here for cytotoxcity using CellTiter-Glo.
Assay Protocol Summary: Cytotoxcity assay was performed with embryonic Drosophila S3 cells (Bloomington Drosophila Genomics Resource Center [DGRC]). We dispensed 4 uL of cells at 1.25 x 106 cells/mL into Greiner solid white 1,536-well plates with a bottle-valve solenoid-based dispenser (Aurora) to obtain 5,000 cells/well. A total of 23 nL of compound solution of different concentrations were transferred to the assay plates using a Kalypsys pin tool equipped with a 1,536-pin array containing 10-nL slotted pins (FP1S10, 0.457-mm diameter, 50.8 mm long; V&P Scientific). One microliter of oleic acid (400 uM) was added, and the plate was lidded with stainless steel rubber gasket lined lids containing pinholes. After 24 hrs of incubation at 24 degrees Celsius and 95% humidity, 4 uL of CellTiter-Glo (Promega) was added to the wells. Luminescence was detected on the Perkin Elmer ViewLux.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve, single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all intoxic compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all cytotoxic compounds, a score range was given for each curve class type given above. Cytotoxic compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)