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

Keywords: cholesterol uptake, HDL, SR-BI, C384 mutation

Assay Overview:
One SR-BI inhibitor, BLT-1 is known to bind SR-BI at Cysteine 384. If the Cysteine is mutated to a serine, BLT-1 is no longer active. The goal of this assay is to verify compounds that disrupt the binding of HDL particles to mutant SR-BI scavenger receptor using an alternative means of labeling HDL-cholesterol particles and avoiding any type of fluorescence measurement. To measure this binding event, HDL particles are labeled with the[H3}-cholesterol and added to C384S SR-BI cells. Cells take up HDL via the SR-BI scavenger receptor in 2 to 3 hours. After significant uptake of the HDL, the radiolabel can be detected by liquid scintillation counting. The level of radioactivity correlates with the amount of HDL uptake. The uptake of lipid particles can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The mSR-BI cells utilized in the assay are a CHO cell line lacking expression of the LDL receptors and overexpresses the scavenger receptor, SR-BI.

Expected Outcome: Inhibitors of SR-BI and HDL uptake that have activity in wild type cells and do not interact with SR-BI at Cys384 will have a reduction in liquid scinitillation counts. These inhibitors will work by a mechanism distinct from BLT-1.

Specific cellular uptake of [3H]CE from [3H]CE-HDL were measured for 2 h in the absence (total activity) or presence (non-specific activity) of a 40-fold excess of unlabeled HDL. For experiments, C384S mutant cells were seeded in the wells of 24-well plates (50,000 cells per well) on day 0, and uptake assays were performed on day 2 in Ham's F12K medium plus 0.5% (wtvol) fatty acid free BSA. For uptake assays at 37 degrees C, on the day of the assay, the cells were washed twice with prewarmed (37 degrees C) medium prior to adding radioactive lipo-protein. Specific binding or uptake is the difference between total and nonspecific activities. Data were analyzed using Prism 5 (GraphPad Software). All calculated errors represent standard errors of the mean. In assays BLT-1 (all incubations at 37 degrees C), immediately prior to incubation with radiolabeled lipoproteins, the cells were washed twice with medium, preincubated with BLT-1 for 1 h, and then incubated with radiolabeled lipoproteins as described above, all in medium plus 0.5% (volvol) DMSO in the presence of the indicated concentration of BLT-1. Results were normalized to 100% of control receptor activity in the absence of BLT-1.

Grant Number: 2 R01 HL052212-11