Compounds and cell lines
BAY-3827 and BAY-974 were synthesized in-house  and are available from the Structural Genomics Consortium as donated chemical probes. The androgen R1881 (methyltrienolone) was synthesized in-house. Enzalutamide was purchased from Selleck Chemicals (Houston, TX, USA).
Cell lines were from the American Type Culture Collection (ATCC, Manassas, VA, USA) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany), and were routinely grown in the recommended medium at 37 °C and in 5% CO2 atmosphere. Authentication was performed by the DSMZ using short tandem repeat DNA typing analysis. Cell lines were confirmed to be free of mycoplasma using the MycoAlert Mycoplasma Detection Assay (Lonza, Basel, Switzerland).
Kinase activity assays
A time-resolved fluorescence energy transfer (TR-FRET)-based AMPK activity inhibition assay was established. Full-length human AMPKα2 N-terminally fused to glutathione-S-transferase (GST) was coexpressed with GST-PRKAB1 and PRKAG1 using a baculovirus expression system (#102–114, lot 10CBS-1133D; Carna Biosciences, Kobe, Japan). Purification of the GST-AMPKα2/ß1/γ1 complex was performed using glutathione sepharose chromatography. The biotinylated peptide biotin-Ahx-HMRSAMSFAEPG (C-terminus in amide form) was used as substrate for the kinase reaction (Biosyntan, Berlin, Germany).
For the kinase assay, 50 nl of a 100-fold concentrated solution of the test compound in DMSO were pipetted into either a black low-volume 384-well microtiter plate or a black 1536-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). Then, 2 µl of a solution of GST-AMPKα2/ß1/γ1 in aqueous assay buffer (50 mM Hepes pH 7.5, 10 mM MgCl2, 5 mM β-glycerophosphate, 2.5 mM dithiothreitol, 0.5 mM EGTA, 0.01% (w/v) bovine γ-globulin, 0.01% (v/v) Triton X-100) were added and the mixture was incubated for 15 minutes at 22 °C to allow pre-binding of the test compounds to the enzyme. For the kinase reaction with the low adenosine-triphosphate (ATP) condition, ATP was added at a final concentration of 10 µM, adenosine-monophosphate (AMP) at a final concentration of 2 µM and substrate at a final concentration of 0.5 µM in assay buffer, and the resulting mixture was incubated for 90 minutes at 22 °C. For the high ATP condition, ATP was added at a final concentration of 1 mM, AMP at a final concentration of 2 µM and substrate at a final concentration of 0.5 µM in assay buffer, and the resulting mixture was incubated for 30 minutes at 22 °C. The concentration of GST-AMPKα2/ß1/γ1 was adjusted depending on the activity of the preparation batch and was chosen to have the assay in the linear range, a typical concentration being 0.05 nM. The reaction was stopped by addition of 3 µl of a solution of TR-FRET detection reagents (0.2 µM streptavidine-XL665 [Cisbio Bioassays, Codolet, France], 3.33 nM anti-phospho-serine antibody STK [Merck Millipore, Burlington, MA, USA] and 3.33 nM anti-mouse IgG-Tb cryptate, a Terbium-cryptate labelled anti-mouse IgG antibody [Cisbio Bioassays] in an aqueous EDTA solution (166.7 mM EDTA, 0.06% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5). The resulting mixture was incubated for 1 hour at 22 °C to allow the formation of a complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Tb-cryptate to the streptavidine-XL665. For that the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader (Pherastar FS, BMG Labtechnologies, Ortenberg, Germany or ViewLux, PerkinElmer, Waltham, MA, USA). The ratio of the emissions was taken as the measure of the amount of phosphorylated substrate. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). As a rule, the test compounds were analyzed on the same microtiter plate in 11 different concentrations ranging from 20 µM to 0.07 nM in duplicate values. IC50 values were calculated using the Genedata Screener™ software (Basel, Switzerland).
Aurora A kinase activity was determined using N-terminally His-tagged, recombinant protein (Merck Millipore, Burlington, MA, USA) and the biotinylated FMRLRRLSTKYRT peptide (Jerini AG, Berlin, Germany). An anti-phospho-Akt antibody was used for detection, and europium cryptate-labeled protein-A and streptavidin-Xlent! for signal measurement (Cisbio Bioassays). Flt3 kinase activity was determined with N-terminally GST-tagged recombinant protein (Merck Millipore) and the biotinylated GGEEEEYFELVKKKK peptide (Biosyntan). Signal was determined following treatment with PT66-K and streptavidin-Xlent! (Cisbio Bioassays). c-Met kinase activity was determined with in-house produced protein and PolyGT-Biotin (Cisbio Bioassays) as substrate. Signal was measured with Eu-W1024 PT66 (PerkinElmer) and streptavidin-Xlent! (Cisbio Bioassays). Rsk4 activity was determined with 6His-tagged recombinant protein (Eurofins, Brussels, Belgium) and the biotinylated KKLNRTLSFAEPG peptide (Biosyntan). Detection was with an anti-phosphoserine antibody (Amersham Bioscience), and the signal was measured after treatment with Eu-W1024 PT66 (PerkinElmer) and Dy648-conjugated streptavidin (Dyomics GmbH, Jena, Germany). Low ATP concentration (10 µM) was used in these assays.
Selectivity profiling was performed at Eurofins by testing 331 different wild-type human kinases (Additional file 1: Table S1).
Cellular mechanistic assay
The phospho-ACC1 (Ser79) HTRF assay (Cisbio Bioassays) was used to determine the levels of ACC1 phosphorylation in cell lysates. Detection was performed in a sandwich TR-FRET assay using an anti-total ACC1 antibody labeled with the acceptor d2 and an anti-phospho-ACC1 antibody labeled with the donor europium cryptate. On day 1, the cells were seeded in a 384-well Small Volume™ plate (Greiner) in the appropriate culture medium. They were then treated with the inhibitor or DMSO and incubated for 1 hour at 37 °C. Following incubation, the cells were lyzed in 4 µl of lysis buffer for 1 hour on ice. Then 4 µl of antibody solution containing equal amounts of total ACC1 and phospho-ACC1 were added and the samples were incubated overnight at 4 °C. The plate was measured the next day using a PHERAstar FS reader (BMG Labtech, Ortenberg, Germany). IC50 values were calculated using the DRC Master Spreadsheet (in-house Bella software) and setting DMSO-treated cells as the minimum inhibition (C0) and staurosporine-treated cells (1 µM of staurosporine) as the maximum inhibition (Ci).
Cell proliferation assays
LNCaP, VCaP, 22Rv1, C4-2B, PC-3 and DU-145 prostate cancer cells were plated at a number of 600, 2,400, 1,200, 600, 600 and 1,000 per well in 384-well white plates in RPMI 1640 medium without phenol red supplemented with 10% charcoal-stripped FBS, except in the case of DU-145 where DMEM/Ham´s F12 medium supplemented with stable glutamine and 10% FBS was used. Sister wells were plated on a separate plate for time zero determination. All plates were incubated overnight at 37 °C. On the next day, the androgen R1881 was added at a final concentration of 1, 0.1, 0.1, 1 and 0.1 nM, except in the case of DU-145 cells. This was followed by addition of the inhibitor in serial dilutions. The plates were incubated at 37 °C for 6 days, except for PC-3 and DU-145 cells where incubation was for 4 and 3 days, respectively. Cell viability was determined with the CellTiter-Glo® assay (Promega, Madison, WI, USA). Luminescence was measured in a Victor X3 reader. Background values measured in wells containing only medium were subtracted from all other values. Control wells containing cells with culture medium, DMSO and R1881 were used to determine the control cell growth at the end of the experiment compared to cells treated only with DMSO.
The following additional cancer cell lines were tested by plating the indicated numbers in 384-well black plates: Oci-Ly-7 (1,000 cells), Ramos (1,000 cells), REC-1 (5,000 cells), RAJI (4,000 cells), SU-DHL-10 (1,000 cells), L-363 (4,000 cells), JJN-3 (1,000 cells), AMO-1 (2,000 cells), SK-N-F1 (1,600 cells), LP-1 (4,000 cells), OPM-2 (4,000 cells), SW780 (2,000), IMR32 (1,600 cells), U251 (2,500 cells), IMR-5/75 (800 cells), MDA-MB-231 (800 cells), MDA-MB-453 (4,000 cells), HeLa (800 cells), Colo201 (800 cells), Colo320 (800 cells), LS-174T (800 cells), SW480 (800 cells), A549 (300 cells), NCI-H292 (500 cells), NCI-H460 (1,000 cells), NCI-H520 (2,000 cells), A2780 (800 cells), BxPC3 (2,500 cells), PANC-1 (900 cells), A375 (4,000 cells), Snu16 (1,000 cells). Alternatively, 96-well plates were used for Daudi (20,000 cells), U2-OS (3,000 cells) and Snu398 (3000 cells) cell proliferation assays. Culture conditions recommended by the provider were used. Sister wells were plated on a separate plate for time zero determination. All plates were incubated overnight at 37 °C. On the next day, the inhibitor was added in serial dilutions and the plates were incubated at 37 °C for 72 to 144 hours, as detailed (Additional file 2: Table S2). The time zero plate was measured by adding 25 ul/well of CellTiter-Fluor solution (Promega), followed by incubation for 30 minutes at 37 °C and measurement of fluorescence on a PHERAStar reader. After the required incubation time, the plates were measured as described above. Background values measured in wells containing only medium were subtracted from all other values. Control wells containing cells with culture medium and DMSO were used to determine the control cell growth, compared to the initial number of cells (time zero value). To distinguish between cell growth inhibition and cell killing, the fluorescence values were corrected for the mean fluorescence observed for the time zero wells at the day of drug treatment start (time zero value). IC50 values were calculated as above.
LNCaP cells were grown in 10-cm dishes and treated with 1 nM R1881 for the indicated timepoints. Protein extracts (40 µg) were separated on a NuPAGE 3–8% tris acetate gel (ThermoFisher Scientific, Waltham, MA, USA) and blotted onto a polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA, USA). Analysis was performed using monoclonal antibodies against ACC1 (#3676), pSer79-ACC1 (#3661), AMPKα (#2532), pThr172-AMPKα (#2535) or HSP90 (#4874) (Cell Signaling Technology, Danvers, MA, USA), or against CaMKK2 (H00010645-M01; Abnova, Taipei City, Taiwan). Detection was performed using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Gene expression analysis
LNCaP and VCaP cells were treated with 1 nM R1881, enzalutamide or BAY-3827, as indicated for 24 or 48 hours. RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Synthesis of cDNA was performed with the SuperScript® III First Strand Synthesis SuperMix for qRT-PCR (ThermoFisher). Analysis was performed using the RT2 Profiler™ PCR Array Human AMPK Signaling 330231 (Qiagen).
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) and bioinformatics analysis
VCaP cells were treated with 1 nM R1881 and 2 µM enzalutamide, as indicated, for 22 hours. Cells were frozen and sent to Diagenode (Liège, Belgium) for ChIP reactions, as described . Approximately 300 base pair-long DNA fragments were prepared using the Bioruptor Pico with sonication beads (Diagenode). Immunoprecipitation was performed using antibodies specific for the AR (06-680, Merck Millipore). Sequencing reads were mapped to human genome hg19 using the Burrows-Wheeler alignment tool with default settings . Additional steps were performed as recently described .
Lipid droplet determination by confocal microscopy and image analysis
Lipid droplet formation was determined using a fluorescent synthetic fatty acid precursor (Red fluorescent BODIPY 558/568 C12, D3835, ThermoFisher) . Briefly, LNCaP cells were cultured in 96-well plates in RPMI1640 supplemented with 10% cFBS and treated with the indicated compounds for 2 or 4 days. Following treatment, they were first incubated for 24 hours with 0.1 µM BODIPY 558/568 C12, then washed with medium and treated for 30 minutes with 4 µM Hoechst 33342 (ThermoFisher) to stain the nuclei, and then fixed with 1% formalin. In the case of VCaP cells, DMEM supplemented with 10% cFBS was used for culture and 1 µM C12 as precursor. This was followed by fixation and washing with 1% cFBS before staining with Hoechst 33342. Imaging was performed with a laser scanning microscope (LSM 700, Carl Zeiss Microscopy, Jena, Germany) using the predefined BTMR (C12 signal) or H342 (Hoechst 33342 signal) channels. Data were analyzed with the ImageJ (National Institute of Health, Bethesda, MD, USA) software and the ratio of lipid droplet to nuclei determined for each experimental condition.