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BioAssay: AID 588843

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

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
 Tested Compounds
All(31)
 
 
Active(12)
 
 
Inactive(17)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(31)
 
 
Active(12)
 
 
Inactive(17)
 
 
Inconclusive(2)
 
 
AID: 588843
Data Source: Broad Institute (2085-10_Inhibitor_SinglePoint_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-02
Hold-until Date: 2012-11-22
Modify Date: 2012-11-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 12
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectsummarySummary 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: 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
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PUBCHEM_ACTIVITY_SCORE_TEST1Arithmetic mean of replicate scores from test 1Float%
2REPLICATE_A_ACTIVITY_1uM_TEST1Percent of total cellular [3H]cholesterol released via efflux in the first replicate during the first run of the test at 1uM concentrationFloat%
3REPLICATE_B_ACTIVITY_1uM_TEST1Percent of total cellular [3H]cholesterol released via efflux in the second replicate during the first run of the test at 1uM concentrationFloat%
4REPLICATE_C_ACTIVITY_1uM_TEST1 Percent of total cellular [3H]cholesterol released via efflux in the third replicate during the first run of the test at 1uM concentrationFloat%
5REPLICATE_D_ACTIVITY_1uM_TEST1Percent of total cellular [3H]cholesterol released via efflux in the fourth replicate during the first run of the test at 1uM concentrationFloat%
6PUBCHEM_ACTIVITY_SCORE_TEST2Arithmetic mean of replicate scores from test 2Float%
7REPLICATE_A_ACTIVITY_1uM_TEST2Percent of total cellular [3H]cholesterol released via efflux in the first replicate during the second run of the test at 1uM concentrationFloat%
8REPLICATE_B_ACTIVITY_1uM_TEST2Percent of total cellular [3H]cholesterol released via efflux in the second replicate during the second run of the test at 1uM concentrationFloat%
Additional Information
Grant Number: 2 R01 HL052212-11

Data Table (Concise)
Classification
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