Indolinyl-Thiazole Based Inhibitors of Scavenger Receptor-BI (SR-BI)-Mediated Lipid Transport

A potent class of indolinyl-thiazole based inhibitors of cellular lipid uptake mediated by scavenger receptor, class B, type I (SR-BI) was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) in an assay measuring the uptake of the fluorescent lipid DiI from HDL particles. This class of compounds is represented by ML278 (17–11), a potent (average IC50 = 6 nM) and reversible inhibitor of lipid uptake via SR-BI. ML278 is a plasma-stable, noncytotoxic probe that exhibits moderate metabolic stability, thus displaying improved properties for in vitro and in vivo studies. Strikingly, ML278 and previously described inhibitors of lipid transport share the property of increasing the binding of HDL to SR-BI, rather than blocking it, suggesting there may be similarities in their mechanisms of action.


2-Chloro-1-(indolin-5-yl)ethanone (14):
A microwave tube was charged with a magnetic stirbar and 2-chloro-1-(1-(phenylsulfonyl)indolin-5-yl)ethanone (3.26 g, 9.54 mmol). Concentrated sulfuric acid (9.0 ml) was added, and the resulting suspension was microwaved for 10 minutes at 100 °C. The reaction was carefully poured into ice water (500 ml). The dark mixture was stirred while warming to room temperature then treated with 10% (w/v) aqueous sodium hydroxide (approximately 200 ml) until the pH >10. This mixture was then extracted with dichloromethane (3 x 250 ml). The combined extracts were washed with brine (200 ml), then shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give a brown solid (1.52 g). This material was used immediately without further purification. tert-Butyl 5-(2-aminothiazol-4-yl)indoline-1-carboxylate (15): 2-Chloro-1-(indolin-5yl)ethanone (1.52 g, 7.75 mmol) was placed in a microwave vial and dissolved in anhydrous ethanol (30.0 mL) to give an opaque, black solution. Thiourea (0.66 g, 8.67 mmol) was added and the resulting mixture was microwaved for 30 minutes at 120 °C. 4-(N,N-Dimethylamino)pyridine (95.0 mg, 0.78 mmol) and N, N-diisopropylethylamine (1.5 ml, 9.3 mmol) were added to the reaction mixture. Neat di-tert-butyl dicarbonate (2.0 mL, 8.52 mmol) was added last, and the reaction was stirred at room temperature for 1 hour. The opaque, redbrown mixture was concentrated under reduced pressure to give a red-brown solid. This material was partitioned between water (50 ml) and ethyl acetate (75 ml) and stirred at room temperature until everything dissolved. The layers were separated and the aqueous phase was extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give an orange-brown solid. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 40/60) to give the title compound as an orange solid (1.72 g, 56 % over three steps). 1 H NMR

Cell Lines
The following cell lines were used in this study: • ldlA[mSR-BI] is a Chinese hamster ovary (CHO) cell line that overexpresses murine SR-BI, isoform 1 (NP_058021) and lacks the LDL receptor was obtained from the Assay Provider (Krieger Laboratory). This cell line was used for the primary assay and several secondary assays. A variant of this cell line expressing mutant SR-BI, where a cysteine required for interaction with BLT-1 is mutated to serine (C384S SR-BI), was used in several secondary assays. • [ldlA7] is the parental cell line to ldlA[mSR-BI] cells, and does not overexpresses SR-BI, and can be used to rule out compound activity independent of SR-BI.

a) DiI-HDL Uptake Assay
ldlA[mSR-BI] cells were plated into 384-well plates at 30 µl per well and incubated overnight. As a measurable surrogate for cholesterol uptake, human HDL particles were treated with the lipophilic fluorescent dye DiI and exposed to ldlA[mSR-BI] cells in lipoprotein-free media (Ham's F12/0.5% fatty acid-free Bovine Serum Albumin [BSA]/25 mM HEPES pH 7.4 plus 10 µg protein/ml DiI-HDL). Cells took up the Dil via SR-BI over 3 hours in the presence of compound. After significant uptake of the DiI, the cells became fluorescent. The level of fluorescence correlates with the amount of Dil uptake and can be measured with a standard plate reader. The uptake of lipid (represented by DiI) was inhibited by the compound BLT-1 or with an excess of HDL untreated with DiI.
The ldlA[mSR-BI] cells used in the assay were a CHO cell line lacking expression of the LDL receptors and overexpressing the Scavenger receptor (SR-BI). Inhibitors of SR-BI and HDL-mediated uptake lead to a reduction in fluorescence in this assay. Fluorescence was measured using a PerkinElmer EnVision plate reader. Primary HTS data were analyzed in Genedata Screener Assay Analyzer, and were normalized against DMSO and 1 µM BLT-1 (positive control). For the HTS, the average of two replicates was used to rank order activity and to choose compounds for retests. For dose studies, the curves were generated with Genedata Condeseo and showed percent (%) activity for the individual doses. 1. Remove media with aspirator (Bio-Tek ELX405 plate washer). 2. Add 30 uL Ham's F12/0.5% Bovine Serum Albumin (fatty acid-free) /25 mM HEPES pH 7.4 (Invitrogen) + 10 ug protein/ml DiI-HDL with Thermo Combi fluid handler (slow speed setting). 3. Pin transfer 100 nL compounds and positive control (1 uM BLT-1). It is recommended that low solubility compounds (such as ML278) be sonicated in DMSO to ensure complete dissolution prior to preparing stock solutions. 4. Incubate 3 hours @ 37°C in humidified cell culture incubator. 5. Remove media with aspirator. 6. Wash twice with 30 uL PBS (+Ca and Mg) using the Thermo Combi on slow speed setting. 7. Analyze DiI-HDL uptake with Perkin-Elmer EnVision plate reader (Bodipy TMR mirror #405, excitation filter is Photometric 550 (#312) and emission filter is Cy3 595 (#229) with bottom read.

b) HDL Binding Assay
HDL binding was assessed using Alexa Fluor 488-labeled HDL particles. For this assay, the Alexa 488 dye was covalently bound to apolipoprotein components of the HDL particle via primary amines; thus, no transfer of the fluorophore to cell membranes occurs. In this manner, direct binding of the HDL particles to SR-BI can be measured. As a positive control, BLT-1 was used at 1 µM, which is known to increase binding of HDL to SR-BI. It is possible that a compound can reduce binding of HDL to the receptor, and this would lead to a decrease in signal. This assay is used to characterize the mechanism of action of a particular compound; therefore, any outcome in the assay is acceptable. Data were normalized against DMSO and BLT-1 (positive control) wells in Genedata Assay Analyzer. Curves were generated with Genedata Condeseo and showed percent (%) activity for the individual doses.

c) Cholesterol Ester Uptake Assay
The goal of this assay is to verify compounds that disrupt the binding of HDL particles to SR-BI scavenger receptor using an alternative means of labeling cholesteryl esters and avoiding any type of fluorescence measurement. To measure this binding event, HDL particles are loaded with [ 3 H]cholesteryl oleate ester and added to mSR-BI cells. Cells take up HDL and the [ 3 H]cholesteryl ester 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 [ 3 H]cholesteryl ester uptake. The uptake can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The ldlA[mSR-BI] cells utilized in the assay are a CHO cell line lacking expression of the LDL receptors and overexpressing the scavenger receptor, SR-BI. Inhibitors of SR-BI and HDL uptake will have a reduction in liquid scintillation counts.

Detailed protocol
On of 10 ug/mL and incubate 2 hours at 37°C in a humidified cell culture incubator 5. Remove the medium at the end of incubation at 4C 6. Wash the plates 2X with Tris-BSA buffer and 1X Tris-HCl buffer. 7. Add 1 ml isopropanol, and incubate for 20 min at 4C. 8. Collect 1 ml isopropanol/lysate into counting tube, add 4 ml liquid scintillation buffer, and prepare for counting. 9. Specific uptake is the difference between total and nonspecific activities. 10. Data were analyzed using Prism 6 (GraphPad Software).
11. All calculated errors represent standard errors of the mean.

e) HDL isolation and labeling
Human HDL was isolated from donors by density gradient ultracentrifugation and

Study Signatures
"This study was conducted according to the procedures described in this report. All data presented are authentic, accurate and correct to the best of our knowledge."

METHODS
Methods employed in this study have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions described in the accompanying "Methods" section of this report.

RESULTS
A summary of results meeting the significance criteria is presented in the following sections. Complete results are presented under the section labeled "Experimental Results". Individual responses, if requested, are presented in the section labeled "Individual Responses".

SUMMARY/CONCLUSION
Significant results are displayed in the following

Summary of Significant Results
Biochemical assay results are presented as the percent inhibition of specific binding or activity throughout the report. All other results are expressed in terms of that assay's quantitation method.
• For primary assays, only the lowest concentration with a significant response judged by the assays' criteria, is shown in this summary.
• Where applicable, either the secondary assay results with the lowest dose/concentration meeting the significance criteria or, if inactive, the highest dose/concentration that did not meet the significance criteria is shown.
• Unless otherwise requested, primary screening in duplicate with quantitative data (e.g., IC50 ± SEM, Ki ± SEM and nH) are shown where applicable for individual requested assays. In screening packages, primary screening in duplicate with semi-quantitative data (e.g., estimated IC50, Ki and nH) are shown where applicable (concentration range of 4 log units); available secondary functional assays are carried out (30 mM) and MEC or MIC determined only if active in primary assays >50% at 1 log unit below initial test concentration.
Significant responses (≥ 50% inhibition or stimulation for Biochemical assays) were noted in the primary assays listed below: No significant results noted.
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Experimental Results
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Note: Items meeting criteria for significance (≥50% stimulation or inhibition) are highlighted. * Batch: Represents compounds tested concurrently in the same assay(s