AID	Name	Type	Comment
488952	Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe Project	summary

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.

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).

Grant Number: 2 R01 HL052212-11