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BioAssay: AID 624015

HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_Activity_Set2

Assay Overview: SR-BI also mediates bidirectional flux (e.g., efflux) of unesterified, or 'free' cholesterol (FC) between cells and HDL or other acceptors. In vivo, the greatest SR-BI-mediated selective uptake occurs in the liver and steroidogenic organs. BLT-1 is known to interact at serine 384. If the cysteine is mutated, BLT-1 can no longer bind. This assay is used to determine if the compounds alter the efflux of free cholesterol to HDL with the mutated SR-BI receptor. Compounds that do and do not inhibit efflux in a dose dependent manner will be of value. ..more
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 Tested Compounds
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Active(1)
 
 
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Active(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 624015
Data Source: Broad Institute (2085-10_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-04-05
Hold-until Date: 2013-03-19
Modify Date: 2013-03-19

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectSummarydepositor-specified cross reference: summary
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
602155Liposome Uptake Assay Measured in Biochemical System Using Scintillation - 2085-16_Inhibitor_Dose_DryPowder_ActivityOthersame 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
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: cholesterol efflux, Cys384 mutant


Assay Overview: SR-BI also mediates bidirectional flux (e.g., efflux) of unesterified, or 'free' cholesterol (FC) between cells and HDL or other acceptors. In vivo, the greatest SR-BI-mediated selective uptake occurs in the liver and steroidogenic organs. BLT-1 is known to interact at serine 384. If the cysteine is mutated, BLT-1 can no longer bind. This assay is used to determine if the compounds alter the efflux of free cholesterol to HDL with the mutated SR-BI receptor. Compounds that do and do not inhibit efflux in a dose dependent manner will be of value.


Expected Outcome: Active compounds in this assay reduce efflux of cholesterol. Because of the absence of the cysteine-384 residue in the mutated SR-BI receptor, active compounds could reduce cholesterol efflux through a different mechanism than that used by BLT-1. A compound which is inactive in this assay but reduces efflux mediated by the wild-type SR-BI receptor most likely uses a similar mechanism as BLT-1.
Protocol
For efflux assays, [ldlA] Cys384S CHO cells were seeded on day 0 on 24-well plates at a density of 50,000 cells per well in Ham's F12 +5% fetal bovine serum + Penicillin-Streptomycin-L-Glutamine.

On day 1, the medium was replaced with Ham's F12 medium supplemented with 10% bovine lipoprotein deficient serum, 1 uCi/ml [1,2-3H]cholesterol (40-60 Ci/mmol; NEN Life Science), 2mML-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and, for ldlA[mSR-BI] cells only, 0.25 mg/ml G418.
(Hams+P/S+Gln+10% Serum + cholesterol +/-G418)

Cholesterol, [1,2-3H(N)]-, 1mCi (37MBq)
Product number: NET139001MC Perkin Elmer - NEN

On day 3, the cells were washed twice in 1 ml Ham's F12 medium without any supplements and then cultured for another 24 h in 1 ml tissue culture medium in which fetal bovine serum was replaced with 1% fatty acid-free (FAF) BSA (Sigma catalog No. A6003).
(Hams+P/S+Gln+1% FAF-BSA +/-G418)

Day 4: the efflux assay was performed on day 4. Cells were washed twice with Ham's F12 without supplements and then preincubated for 1 h at 37C with compounds at the indicated concentrations in 1 ml assay medium (Ham's F12 + P/S + Gln + 0.5% DMSO + 25 mM HEPES, pH 7.4+ 0.5% FAF BSA). Subsequently, the cells were incubated for an additional 2 h with the same concentrations of small molecules and with the indicated concentrations unlabeled HDL a 55 ul of assay medium containing 2 mg/ml HDL added to each well (final HDL concentration of 100 ug/ml)
(Ham's F12 + P/S + Gln + 0.5% DMSO + 25 mM HEPES, pH 7.4 + 0.5% FAF BSA)

After a 2-h incubation at 37 degrees C, the [3H]cholesterol contents of the cells and the media were determined as follows. 120 ul efflux media were collected and clarified by centrifugation for 1 min with a desktop microcentrifuge, and the radioactivity in 100 ul of each supernatant was determined by liquid scintillation counting (LSC). Cells were solubilized with 400 ul of lysis buffer (1% Triton X-100 in PBS) for 30 min at room temperature, and the amount of [3H]cholesterol in 100 ul of each lysate was determined by LSC. Total cellular [3H]cholesterol was calculated as the sum of the radioactivity in the efflux medium plus the radioactivity in the cells and was used to calculate the [3H]cholesterol efflux (percent of total [3H]cholesterol released into the medium).
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC_50_uM*The concentration at which activity reaches 50% of the maximumFloatμM

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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