Overview

The primary goal of this project is to identify chemical probes that either increase or decrease the lipid content of cells. Most cells are capable of storing energy rich lipids (mainly triacylglycerols), which are generated on basis of de novo synthesized fatty acids or non-esterified free fatty acids (NEFA) taken up from the environment. There are only few drugs for treating metabolic diseases and a very limited number of chemical probes to study lipid storage in vitro. We have found that embryonic Drosophila S3 and Kc167 cells are capable of depositing LDs, and we developed an assay to measure lipid storage amounts by fluorescent staining of LDs in S3 cells, using laser-scanning microplate cytometry for detection. Importantly, most results obtained in the Drosophila cell systems could be successfully translated into different mammalian cell culture models, opening up the possibility to test for evolutionary conservation of mechanism.

Protocol

Detection of lipid droplets was performed using Drosophila S3 (obtained from NIDDK) cells, following 400 μM oleic acid feeding in growth media. In the assay 4 μL of S3 cells at 1.25 x 10⁶ cells/mL in growth media was dispensed into LoBase Aurora 1,536-well plates (black walled, clear bottom COP plastic; Aurora) (8) using a bottle-valve solenoid-based dispenser (9) to give 5,000 cells/well. Twenty-three nL of compound solution was transferred to the assay plates using a Kalypsys pin tool (San Diego, CA) equipped with a 1,536-pin array containing 10 nL slotted pins (FP1S10, 0.457 mm diameter, 50.8 mm long; V&P Scientific5). Next, 1 μl of oleic acid (400 μM) was added and the plates were lidded with stainless steel rubber gasket-lined lids containing pin-holes for gas exchange and incubated for 18–24 hr at 24 °C, 95% humidity. Detection was performed by the addition of the lipid-droplet specific dye BODIPY 493/503 (Molecular Probes), and CellTracker™ Red CMTPX (Invitrogen) was used to enumerate cell number. Lipid droplet accumulation was measured using the Acumen Explorer (TTP LapTech) (10) using a 488 nm laser. The total intensity in channel 1 (500–530 nm) was used to measure lipid droplet accumulation with cell objects defined using channel 3 (575–640 nm) and a 5 μm width and 100 μm depth filters. The ratio of the total intensity in PMT channel 1 over total intensity channel 3 was also calculated.

Assay principle and protocol

Figure 1

Top, distribution of the fluorescent signal in PMT 1 (lipid specific channel) without (left) and with (right) oleic acid (OA) feeding. The populations are defined using width and depth filters using the fluorescent signal from PMT 3 (cell- specific channel) to define cell-objects. Cell objects with low PMT 1 fluorescence are defined in the absence of OA feeding (“unfed population”, orange data), and a shift to brighter fluorescence is observed following OA feeding that is used to define lipid-containing cells (“fed population”, green data at top right). Unclassified objects not used in the calculation are shown as red. Middle pictures show false color (same color code as in histograms) plate well images from the Acumen for unfed (left) and fed (right) cells. The bottom two panels represent images from the InCell1000 using the same cell stains. Cells are treated with 400 μM OA in the presence (top) or absence (bottom) of the inhibitor Triacsin C. In the fused image, the InCell software swaps the colors so that the red color represent the lipid stain and the green represent the cell stain.

The optimized 1536-well protocol is given in Table 1.

Table 1

Final 1536-well assay protocol.

Following adaptation of the lipid droplet assay to 1536-well plates, we used the assay to screen several libraries. The libraries were screened at between seven and fifteen concentrations using quantitative HTS (qHTS) (11). A total of 98 1536-well plates were screened (Figure 2). This included 27 DMSO blanks, a 15 point titration of the LOPAC library (Sigma), a 13 point titration of the Prestwick library, seven point titrations of two combinatorial libraries targeted to GPCRs and kinases (PTL1 and PTL2), seven point titrations of two libraries from Spectrum/Micorsource and a fifteen point titration of the Tocris library. Overall the assay showed excellent performance with a Z′ = 0.77 ± 0.23, S/B = 25 ± 68, CV = 3 ± 3%. Three plates failed QC (arrows and low Z-factor plates in Figure 2c,d) due to a clogged tip.

Figure 2

Validation of the 1536-well Acumen-based lipid droplet assay in Drosophila cells. a) heatmap representation of fed cell objects. Four control columns are present at left containing untreated cells (column 1, no OA), a titration of Triacsin C (column 2), (more...)

Identification of Exo1

Following the qHTS, the CRC data was subjected to a classification scheme to rank the quality of the CRCs as described by Inglese and co-workers (11) (see scheme 1). Briefly, CRCs are placed into four classes. Class 1 contains complete CRCs showing both upper and lower asymptotes and r² values > 0.9. Class 2 contains incomplete CRCs lacking the lower asymptote and shows r² values greater than 0.9. Class 3 curves are of the lowest confidence because they are defined by a single concentration point where the minimal acceptable activity is set at 3 SD of the mean activity calculated from the lowest tested concentration. Finally, class 4 contains compounds that do not show any CRCs and are therefore classified as inactive.

Scheme 1

Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes. Top, qHTS curve-fit data from AID-361 binned into curve classifications 1–4 based classification criteria. Below, Examples of curves fitting the following (more...)

qHTS summary of assay results

Compounds showing decreasing lipid storage were generally associated with cytotoxicity, except for the positive control Triacsin C as identified in the red cell stain channel. However, we found many compounds that induced lipid overstorage (Figure 3). In the Tocris library, we noted a small molecule Arf1 modulator (Exo1) (12) that increased lipid droplet accumulation (EC₅₀ = 5 μM) in S3 cells (Figure 4). This compound is known to inhibit the vesicle-mediated Coat Protein complex I (COPI) transport complex. Further, we have identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening that included the COPI transport complex, which was found to be required for limiting lipid storage.

Figure 3

Example CRCs (fed objects) for lipids storage activators from the qHTS of S3 cells.

General procedure for the synthesis of Exo-1 (CID-310557) and its analogues

To a solution of the appropriate aryl acid (220 mg, 1.45 mmol) in DCM (20 ml) was added SOCl₂ (2.12 ml, 29.0 mmol) and several drops of DMF, and the mixture was stirred at room temperature for 0.5 h. The solvent and excess SOCl₂ were removed under reduced pressure and the resulting film was reconstituted in DCM (10 ml) followed by the addition of Et₃N (2.42 ml, 1.74 mmol, 1.2 eq.) and the appropriate analine (253 mg, 1.60 mmol, 1.1 eq). The resulting mixture was stirred for 3 h and water (10 ml) was added, and the organic layer was separated. The aqueous layer was further extracted with DCM (20 ml). The combined organic layers were washed with brine and dried over Na₂SO₄. After the removal of solvent, the residue was purified by column chromatography (EtOAc/DCM= 1/40) to afford desired product (297 mg, 75%).

¹H NMR (400 MHz, CDCl₃) δ 12.03 (brs, 1H), 8.92 (dd, J_HH= 8.6, 1.0 Hz, 1H), 8.04–8.15 (m, 3H), 7.61 (td, J_HH= 8.0, 1.6 Hz, 1H), 7.20–7.43 (m, 3H), 3.97 (s, 3H); (TOFMS) m/z 274.0878 (M+H⁺) (calculated for C₁₅H_13FNO₃+) 274.0879.

¹H NMR (400 MHz, CDCl₃) δ 8.71 (brs, 1H), 8.60 (dd, J_HH= 8.0, 1.6 Hz, 1H), 8.42 (d, J_HH= 0.4 Hz, 1H), 7.96–8.02 (m, 3H), 7.87–7.92 (m, 1H), 7.55–7.62 (m, 2H), 7.03–7.14 (m, 2H), 6.95 (dd, J_HH= 8.0 Hz, 1.6 Hz, 1H), 3.95 (s, 3H); (TOFMS) m/z (M+H⁺) 278.1196 (calculated for C₁₈H₁₆NO₂+) 278.1181

¹H NMR (400 MHz, CDCl₃) δ 12.21 (brs, 1H), 8.89 (d, J_HH= 8.2 Hz, 2H), 8.15 (d, J_HH= 8.4 Hz, 1H), 8.11 (dd, J_HH= 8.0, 1.6 Hz, 1H), 7.82 (d, J_HH= 8.2 Hz, 2H), 7.63 (td, J_HH= 7.8, 1.6 Hz, 1H), 7.17 (m, 1H), 3.98 (s, 3H); (TOFMS) m/z 281.0925 (M+H⁺) (calculated for C₁₆H₁₃N₂O₃+) 281.0926.

¹H NMR (400 MHz, CDCl₃) δ 11.82 (d, J_HF= 7.2 Hz, 1H), 8.92 (dd, J_HH= 8.4, 0.8 Hz, 1H), 8.04–8.15 (m, 2H), 7.60 (td, J_HH= 7.0, 1.6 Hz, 1H), 7.14 (td, J_HH= 7.6, 0.8 Hz, 1H), 6.90–7.06 (m, 2H), 3.94 (s, 3H); (TOFMS) m/z 292.0781 (M+H⁺) (calculated for C₁₅H₁₂F₂NO₃+) 292.0785.

Lipolysis and lipogenesis measurements in AML12 cells

Measurements of NEFA released from lipid droplets or incorporated into the TG fraction were performed as previously described (13). Briefly, AML12 cells treated with or without specific siRNAs (10 nM) for 4 d were incubated overnight with growth medium, supplemented with 400 μM oleic acid complexed to 0.4% bovine serum albumin, to promote triacylglycerol deposition and [3H] oleic acid, at 1 x 10⁶ dpm/well, was included as a tracer. In lipolysis experiments, re-esterification of fatty acids in AML12 cells was prevented by including 10 μM Triacsin C (Biomol), an inhibitor of acyl coenzyme A synthetase, in the medium. Quadruplicate wells were tested for each condition. Lipolysis was determined by measuring radioactivity released into the media in 1 h. For the lipid extraction and thin layer chromatography, the cell monolayer was washed with ice-cold PBS and scraped into 1 ml of PBS. Lipids were extracted by the Bligh-Dyer method [84], and 10% of the total lipid was analyzed by thin layer chromatography. AML12 cells treated with or without specific siRNAs were additionally incubated with either vehicle (DMSO), 5 μM of Exo1 (12.5 mg/ml DMSO), or BFA (10 mg/ml DMSO) during the time of radioactivity release into the media (2 h). NEFA incorporation into the TG fraction and NEFA release are calculated as nanomoles/milligram protein. Protein measurements were performed using a commercial BCA assay kit (Pierce Biotechnology) according to the manufacturer’s instructions. Statistical significance was tested by impaired Student t test (GraphPad software).