Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay.

Reagents and Cell Lines BODIPY- FL- GTP 2′-(or-3′)-O-(N-(2-aminoethyl) urethane, G-12411 from Invitrogen Molecular Probes (Eugene, OR). Colorimetric G-LISA assay kit for quantifying Rac1/2/3 activation, rhodamine phalloidin, anti-Rac1 mAb and GST-GTPases (wild type (wt): Cdc42, Rac1, RhoA, H-Ras and constitutively active mutants: Cdc42Q61L, Rac1Q61L, RhoAQ63L, H-RasG12V were purchased from Cytoskeleton, Inc. (Denver, CO). GST-Rab2, GST-Rab7 were purified as described. 14 GST-PAK-PBD and plasmids for GST-Rac1 and Rac2 were generously provided by Dr. G. Bokoch (Scripps Research Institute). Mouse TruBlort ™ Ultra: Horseradish Peroxidase anti-mouse IgG was from eBioscience Inc. (San Diego, CA). Rac inhibitor NSC23766 was obtained from Tocris Bioscience (Ellisville, MO) and EHT1864 was provided by Dr. A. Kornienko (New Mexico Institute of Mining & Technology). Bead sets for multiplex assays were from Duke Scientific Corp. (Fremont, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Rat Basophilic Leukemia 2H3 (RBL) and Swiss 3T3 cells and IgE were provided by Dr. B. Wilson (University New Mexico). Multiplexed Primary Screens For multiplex analysis of small GTPases, we used 4 μm diameter glutathione-beads (GSH-beads) distinguished by seven different intensities of red color (various magnitude of emission at 665 +/−10 nm with excitation at 635 nm). Each polystyrene bead set is supplied at 1.4×10 5 beads/μl with about 1.2×10 6 glutathione sites per bead (determined by using GST- GFP). In preparation for protein binding, 240–250μl of each bead set was individually blocked with 0.1% BSA in buffer NP-HPS (0.01% (vol/vol) NP-40, 30 mM HEPES pH 7.5, 100 mM KCl, 20 mM NaCl) containing 1 mM EDTA (NP-HPSE) for 30 min at room temperature. Bead sets were collected by centrifugation, resuspended in 100 μl of NP-HPSE and individually incubated with 1 μM of a given GST-GTPase (Rab2 wt, Rab7 wt, Cdc42 wt, H-Ras wt, Rac1 wt and Rac1Q61L mutant) overnight at 4°C. Individual GTPase-coupled beads were washed twice with 100 μl ice cold NP-HPSE buffer supplemented with 0.1% BSA and 1 mM DTT and kept in separate tubes on ice. GTPase-coupled beads were pooled together immediately prior to loading of 5 μl of this mixture in each well of the assay plates. Next 0.1μl of test compounds (1 mM stock in DMSO) were added to individual wells to give a final concentration of 10 μM compound and 1% DMSO, after which 5 μl BODIPY- FL-GTP (200 nM stock in NP-HPSE) was added to each well. Positive controls, containing the bead mixture, 0.1 μl DMSO (1% final) and fluorescent GTP, were included in columns 1 and 2 on each plate. Negative controls, containing the bead mixture with fluorescent GTP, 0.5 mM unlabeled GTP as a competitor, and 1% DMSO were assayed separately. Each well contained approximately 2000 beads, coupled with individual GTPases. Plates were placed on rotators and incubated for 40–45 min at 4°C. Sample analysis was conducted with a HyperCyt ® high throughput flow cytometry platform as described previously. 15 Flow cytometric light scatter and fluorescence emission at 530 +/− 20 nm (FL1) and 665 +/− 10 nm (FL8) were collected on a Cyan ADP flow cytometer (Beckman Coulter, Fullerton, CA). Screening of one, 384 well, plate takes ~15min and complete library screening can be performed in 2–3 weeks. The resulting time-dependent data (one file per plate) were analyzed using IDLQuery software to determine the compound activity in each well. Gating based on forward scatter (FS) and side scatter (SS) parameters was used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the median fluorescence per bead population was calculated. A compound was considered a “potential active” if the change in activity was greater then 20% from baseline. Baseline was calculated as described in PubChem. 16 Dose Response Measurements Test compounds identified for further analysis after the primary screen were cherry-picked from compound storage plates, then serially diluted 1:3 a total of eight times from a starting concentration of 10 mM giving a 9-point dilution series in DMSO. The final concentrations in the assay ranged from 10 nM to 100 μM. Beads were coated with proteins as described under Multiplexed Primary Screens. For dose–response analyses, we used one multiplex (Rab7 wt, Rab2 wt, H-Ras wt, H-RasG12V, Cdc42 wt, and Cdc42Q61L) and 3 single-plexes (for Rac1 wt, Rac1Q61L and GST-GFP). In experiments including magnesium, we used NP-HPS buffer containing 1 mM MgCl 2 . Eight GST-GTPases were assayed simultaneously in a single multiplex (Rac1 wt, Rac1Q61L, Rac2 wt, RhoA wt and RhoAL63, Cdc42 wt, Cdc42Q61L and Ral wt) and 100 nM BODIPY-FL-GTP binding was measured in the presence or absence of the serial drug dilution series. Each dose response series was run in triplicate. Kinetic Assays Wild-type GST-Cdc42 (4 μM) was bound to GSH-beads overnight at 4°C. Cdc42 on GSH-beads was depleted of nucleotide by incubating with 10 mM EDTA containing buffer for 20 min at 30°C, washing twice with NP- HPS buffer, then resuspending in the same buffer containing 1 mM EDTA, 1 mM DTT and 0.1% BSA. Kinetic assays were performed by incubating 50 μl of GST-Cdc42-GSH-bead suspension for 2 min with either DMSO (1% final concentration), or 10 μM MLS000532223 and subsequently adding 50 μl of various concentrations of ice cold BODIPY-FL-GTP. Association of the fluorescent nucleotide was measured using a FacSCAN flow cytometer in the kinetic mode. The average number of events was 150 per sec. Data were converted to ASCII format using IDLQuery (software available free from the authors). Raw data were exported and plotted using GraphPad Prism software. Rac Activation Assay Swiss 3T3 cells were used to monitor Rac1 inhibition by MLS000532223 in cell based assay. Cells were serum starved overnight and treated with 1% DMSO (negative control) or 10 μM compound in DMSO (1% final concentration) for 20–30 min. As a positive control, cells were treated with 10 ng/ml EGF for 2 min. Cell lysis, immunoprecipitation of active Rac1 with GST-PAK-PBD immobilized on GSH beads, SDS-PAGE and immunoblotting were performed as described. 17 G-LISA Assay for Active Rac Swiss 3T3 cells were cultured and starved following standard procedures (Cytoskeleton, Inc). Individual cultures grown in 6-well dishes were incubated with MLS000532223 over a concentration range from 0.1–10 μM for 1 h and subsequently stimulated for 2 min with 10 ng/ml EGF. Cells were washed with ice cold PBS containing calcium and magnesium and further processed for protein and G-LISA assays. Positive controls included Rac1-GTP provided in kit and cell lysate prepared from control cells stimulated only with EGF. Negative controls included buffer only controls and cell lysates prepared from control cells after serum starvation.

Live Cell Microscopy

Live cell microscopy was carried out on RBL-2H3 cells. Cells were grown on coverslips overnight, washed and overlaid with Tyrode’s buffer (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose and 0.1% BSA). Time lapse images were taken after addition of 10 μM MLS000532223 (final concentration) at 60 s intervals for up to 100 min at 37°C. For ligand stimulation, IgE primed RBL cells were treated with 1μg/ml DNP-BSA. Imaging was performed using a Bio-Rad Radiance 2100 confocal microscope equipped with a 60x 1.4 NA oil immersion objective equipped with Lasersharp3000 software.

Immunofluorescence Staining and Microscopy

RBL-2H3 cells were grown on coverslips and cultured overnight in the presence or absence of 10 μM MLS000532223. As a positive control, cells were stimulated with 1 μg/ml DNP-BSA for 30 min as previously described. 18 Cells were washed with phosphate buffered saline, fixed with 3% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100 in Tyrode’s buffer, blocked for 1 h with 1% BSA in Tyrode’s buffer, and stained for 1 h with rhodamine-phalloidin. All incubations were at room temperature. For imaging, samples were mounted on glass slides using ProLong® Gold antifade reagent (Invitrogen). A Zeiss LSM 510 microscope, 40x objective was used to collect images. β-Hexosaminidase Secretion Measurements For measurement of β-hexosaminidase release, cells were cultured overnight with IgE in 24-well culture plates. IgE primed cells were washed and incubated for 1 h, with indicated concentrations of inhibitor in Tyrode’s buffer. Aliquots (100 μl) of the cell culture supernatants were analyzed for the spontaneous release of β-hexosaminidase. Cells were activated by exposure to DNP/BSA (0.1 μg/ml) in Tyrode’s buffer for 30 min at 37°C. Release of β-hexosaminidase was determined as described in Ortega et al. 19 The values were expressed as percent of total amount of β-hexosaminidase, determined using 1% Triton X-100 in Tyrode’s buffer.