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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 ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectSummarydepositor-specified cross reference
488896HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
493194HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540246HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
540326DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540354HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588392HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588403DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588548HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588553HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588754HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
588777HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588810HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588818HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588825HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588826HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588828HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
588829HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588830HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588831HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-14_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588833HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
588836HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588837HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588842HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588843HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
602126HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
602134HDL Counter Assay: Tf Endo Assay Measured in Cell-Based System Using Plate Reader - 2085-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602138HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602152HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602154HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602285HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
623880HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
623895HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
624007HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624010HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624015HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624017HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624022HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624024HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624026HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
624029HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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