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BioAssay: AID 602155

Liposome Uptake Assay Measured in Biochemical System Using Scintillation - 2085-16_Inhibitor_Dose_DryPowder_Activity

Assay Overview: In the primary assay, DiI labeled HDL particles are used to measure lipid uptake by cells overexpressing the Scavenger receptor, SR-BI. This assay uses purified SR-BI protein in liposomes to measure uptake in the absence of all other cellular proteins. It will prove that the compounds are interacting directly with SR-BI and not any other accessory proteins or non-SR-BI mechanisms. more ..
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(3)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(3)
 
 
Inactive(1)
 
 
AID: 602155
Data Source: Broad Institute (2085-16_Inhibitor_Dose_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-12
Hold-until Date: 2012-12-08
Modify Date: 2012-12-08

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe Projectsummary
Description:
Keywords: liposome, radiolabel, SR-BI, HDL uptake


Assay Overview: In the primary assay, DiI labeled HDL particles are used to measure lipid uptake by cells overexpressing the Scavenger receptor, SR-BI. This assay uses purified SR-BI protein in liposomes to measure uptake in the absence of all other cellular proteins. It will prove that the compounds are interacting directly with SR-BI and not any other accessory proteins or non-SR-BI mechanisms. In addition, the HDL is radiolabeled and so no fluorescence measurement is utilized. Instead, [3H]-cholesteryl ester is used to label HDL particles and incorporated into liposomes that contain immunoaffinity purified SR-BI (with an epitope tag). Radioactivity is measured by liquid scintillation.


Expected Outcome: Compounds that inhibit SR-BI mediated lipid uptake and interact direcly with SR-BI will show decreased levels of radiolabeled cholesterol within the liposomes. The positive control, BLT-1, inhibits uptake and shows a reduction in signal. Compounds with a reduced signal are labelled as active.
Protocol
For the purification of C-terminally epitope-tagged murine SR-BI (mSR-BI-t1) with uniform, truncated N-linked oligosaccharide chains, we overexpressed mSR-BI-t1 in HEK293S cells. The mSR-BI-t1 was expressed in an N-acetylglucosaminyltransferase I (GnTI)-defective HEK293S derivative, HEK293S GnTI(2), which generates a glycoprotein with uniform, truncated N-linked oligosaccharide chains under the control of a tetracycline-inducible promoter. It was immunoaffinity purified to virtual homogeneity and the detergent-solubilized receptor was reconstituted into liposomes. Subsequently, the in vitro SR-BI-mediated selective uptake of [3H]CE from [3H]CE-HDL in the reconstituted liposomes was measured. The mSR-BI-t1 with truncated N-linked chains was purified and reconstituted into liposomes. Briefly, 20 mg of SR-BI (or an equivalent volume of protein-free buffer to generate control liposomes that are devoid of SR-BI) was reconstituted into liposomes by acetone precipitation. SR-BI liposomes were washed once by resuspension of the acetone precipitate in protein-free assay medium followed by a centrifugation step for 25 min and 48,000 g at 4 degrees Celsius. The pellet was first reconstituted in assay medium without protein, and then an equal volume of assay medium with 1% Fatty Acid Free Bovine Serum Albumin was added to yield liposomes at a nominal final concentration of 18 ng SR-BI/ml. In each reaction, 30 ml were preincubated together with 30 ml of assay medium containing 0.5% Fatty Acid Free Bovine Serum Albumin, 1% DMSO, and the indicated compounds for 60 min at 37 degrees Celsius. Subsequently, 20 ml of [3H]CE-HDL (five replicates per sample) were added to a final concentration of 10 mg protein/ml. Incubation was continued for 4 hours at 37 degrees Celsius, and then selective uptake of [3H]CE into liposomes was determined using the previously described filter binding assays. SR-BI- selective uptake values were determined. The 100% of control value represents receptor-specific activity in SR-BI-t1-containing liposomes in the presence of 0.5% DMSO without compounds, and the 0% of control value represents background selective uptake in control liposomes devoid of SR-BI-t1.
Comment
Pubchem Activity Scores are calculated as (1-(%uptake/50))*100, giving the neutral control a score of 50, and higher scores showing decreased uptake.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PERCENT_UPTAKE_1uM (1μM**)Percent of lipid uptake relative to the neutral control, averaged over three runs.Float%

** Test Concentration.
Additional Information
Grant Number: 2 R01 HL052212-11

Data Table (Concise)
Classification
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