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Assay Drug Dev Technol. Feb 2012; 10(1): 46–60.
PMCID: PMC3277738
Characterization of Inhibitors of Glucocorticoid Receptor Nuclear Translocation: A Model of Cytoplasmic Dynein-Mediated Cargo Transport
Hikmat N. Daghestani,1 Guangyu Zhu,2 Paul A. Johnston,corresponding author3,4 Sunita N. Shinde,3 Jeffrey L. Brodsky,5 and Billy W. Daycorresponding author2,3,4
1Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.
2Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania.
3Drug Discovery Institute, University of Pittsburgh, Pittsburgh, Pennsylvania.
4Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania.
5Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania.
corresponding authorCorresponding author.
Address correspondence to: Paul A. Johnston, PhD, Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260. E-mail:paj18/at/
Billy W. Day, PhD, Department of Pharmaceutical Sciences, University of Pittsburgh, 721 Salk Hall, 3501 Terrace St., Pittsburgh, PA 15261. E-mail:bday/at/
Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators.
To promote cell survival, essential cellular components must be organized and transported to specific locations within the cell when needed. When some steroid nuclear receptors (glucocorticoid receptor [GR], estrogen receptor [ER], and androgen receptor [AR]) become activated by agonist binding, they translocate from the cytoplasm to the nucleus, where they initiate target gene transcription leading to a cascade of important cellular events necessary for cell function and survival. For example, the GR is a nuclear receptor that, when bound to a glucocorticoid, is transported to the nucleus to activate transcription of target genes involved in the regulation of inflammatory and immune responses.1
The cell's cytoskeleton provides a highly sophisticated network of railways for motor proteins to transport macromolecules to their desired locations, including the retrograde translocation of cytoplasmic GR to the nucleus by the motor protein cytoplasmic dynein.2 Cytoplasmic dynein is a large multi-protein complex that consists of homodimers each of heavy, intermediate, light-intermediate, and light chains.3 The dynein heavy chain is a member of the adenosine triphosphatases (ATPases) associated with diverse activities (AAA+) family and the motor domain contains six AAA domains, although only the first four are capable of binding ATP.4 The first AAA domain is largely responsible for providing the force needed for cytoplasmic dynein movement, whereas the remaining AAA domains are believed to have regulatory functions (e.g., increasing the rate of microtubule [MT] movement driven by dynein).5 A coiled-coil stalk that protrudes from the dynein heavy chain contains an MT-binding domain, allowing cytoplasmic dynein to interact with and bind to MTs.6 The remaining chains connect the motor domain to a multi-protein complex that connects with the cargo. Protein cargos transported by cytoplasmic dynein are often linked through dynactin and immunophilin-heat-shock protein complexes.7 In addition to transporting GR, cytoplasmic dynein carries cargos that vary from other proteins, including ER8 and p53,9 to much larger viruses10,11 and Golgi vesicles.12 Cytoplasmic dynein has also been shown to provide force in nuclear envelope breakdown,13 chromosome segregation,14 mitotic spindle formation,15 and cytokinesis.16
Because cytoplasmic dynein contributes to many important cellular processes, small molecule modulators with specificity for dynein would provide useful chemical and cell biology tools to further investigate the critical roles of cytoplasmic dynein in the cell. To date, there are only a handful of small molecules that target cytoplasmic dynein, most of which are either ATP/transition state mimics (e.g., erythro-9-(2-hydroxy-3-nonyl)adenine, adenylyl imidodiphosphate, and orthovanadate),17,18 or sulfhydryl-reactive agents (e.g., thiourea and N-ethylmaleimide [NEM]).18,19 We recently examined purealin, a natural product derived from the sea sponge Psammaplysilla purea, that has been shown to enhance the ATPase activity of myosin20 while also inhibiting the ATPase activity of axonemal dynein.21 Synthetic analogs of purealin inhibited the ATPase activity of rat cytoplasmic dynein heavy chain 1.22 Although purealin itself was a potent inhibitor of cytoplasmic dynein heavy chain in vitro, the compound was unable to do so in cells.
We therefore chose a new method to screen for dynein modulators, one that would include cellular activity. Using a cell-based high-content screen (HCS) assay to quantify the agonist-induced translocation of GR from the cytoplasm to the nucleus as a cellular model of dynein-mediated cargo transport, we identified several concentration-dependent inhibitors in a screen of the 1280-member library of pharmacologically active compound (LOPAC) collection. In the current report, we present these results as well as those from a panel of secondary assays that we developed to characterize the cell-based screen hits and to investigate the potential mechanisms of action of these inhibitors of cytoplasmic dynein-mediated cargo transport. The distribution of GR-green fluorescent protein (GFP) present in the cytoplasm and nuclei of cells was first examined and compared to determine which compounds inhibited agonist-induced GR translocation. From the same cell images, the cellular toxicity of compounds, defined by a loss of cells, was evaluated, as were the compound-induced changes in the morphology of α-tubulin antibody staining along with the intensity and the overall MT organization of treated cells. Competitive displacement GR binding assays and in vitro MT polymerization assays were performed to identify potential GR binding antagonists and MT perturbing agents, respectively. Compounds were then tested biochemically against the ATPase activity of heat-shock protein (Hsp)90 and Hsp70 molecular chaperones, which are both important components of the GR cargo transport process.7 The ability of the GR translocation inhibitors to inhibit both the basal and the MT-stimulated ATPase activity of the recombinant dynein motor domain was then tested. Finally, to evaluate the ATPase selectivity of the hits, compounds were screened against the ATPase activity of the myosin molecular motor since the only known inhibitor of dynein (purealin) has also been shown to perturb myosin activity.
ATP, guanosine triphosphate (GTP), 2-(N-morpholino)ethanesulfonic acid (MES), monosodium glutamate (MSG), and piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) were from USB, Affymetrix, Inc. The LOPAC®1280 library, CaCl2, dimethylsulfoxide (DMSO), ethylene glycol tetraacetic acid (EGTA), HEPES, Hoechst 33342, imidazole, KCl, 2-mercaptoethanol, MgCl2, NEM, phosphate-buffered saline (PBS), Protease Inhibitor Cocktail, and tris(hydroxymethyl)aminomethane hydrochloric acid (TRIS-HCl), were from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) was from Boehringer Mannheim. Alexa-595, Dulbecco's modified Eagle's medium (DMEM), G418, L-glutamine, Hi5™ Cells, Express Five® Serum Free Medium, nonessential amino acids, penicillin and streptomycin, Green Polar Screen™ Glucocorticoid Receptor Competitor Assay Kit, sodium pyruvate, and trypan blue stain were from Invitrogen, Inc. Fetal bovine serum (FBS) was from Gemini Bio-Products. The BCA™ Protein Assay Kit and bovine serum albumin (BSA) were from Pierce. PiColorLock™ Gold was from Innova Biosciences. Myosin II from rabbit skeletal muscle was from Cytoskeleton, Inc. Nickel nitrilotriacetic acid (Ni-NTA) Superflow was from Qiagen. PD-10 Desalting and Q-Sepharose™ columns were from GE Healthcare Life Sciences. The baculovirus expression vector pVL1393 was from BD Biosciences. The 3617.4 mouse mammary adenocarcinoma cell line stably expressing GR-GFP under the control of a tetracycline (Tet) regulated promoter was kindly provided by Dr. Gordon Hager from the Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD. Triton X-100 and Tween 20 were from BioRad. Mouse monoclonal anti-α-tubulin antibody was obtained from NeoMarkers/Lab Vision Inc. Cy3-labeled donkey anti-mouse was obtained from Jackson ImmunoResearch Laboratories, Inc. Paclitaxel (PTX) was obtained from the NIH Drug Synthesis Branch. Formaldehyde was obtained from Fisher Scientific.
Dexamethasone-induced GR translocation HCS assay protocol
The 3617.4 cell line23 was maintained in complete culture medium containing 10 μg/mL Tet (Tet-on) in a humidified incubator at 37°C, 5% CO2, and 95% humidity to keep the expression of GR-GFP repressed; DMEM with 2 mM L-glutamine supplemented with 10% FBS, 100 μM nonessential amino acids, 100 μM sodium pyruvate, 100 U/mL penicillin and streptomycin, and containing 0.96 mg/mL G418. To induce GR-GFP expression in the 3617.4 cell line, cells were cultured for 48 h in the same tissue culture medium without the Tet (Tet-off). 3617.4 cells that had been cultured in Tet-containing complete culture medium were detached from tissue culture flasks by trypsinization; cells were centrifuged at 500 g for 5 min and re-suspended in 10 mL of Tet-free induction medium; and viable cells that excluded trypan blue were counted in a haemocytometer. 3617.4 cells were adjusted to 4.2×104 cells/mL in Tet-free induction medium and then 60 μL of cell suspension per well was dispensed into the wells of 384-well black-walled clear-bottom plates using the Zoom liquid handler (Titertek) to give a final seeding density of 2,500 cells/well. Assay plates were incubated under Tet-off conditions for 48 h at 37°C, 5% CO2 in a humidified incubator and then diluted compounds (20 μL) were added to wells in columns 3 through 22 using an Evolution P3 (Perkin-Elmer) outfitted with a 384-well transfer head for a final screening concentration after dexamethasone (DEX) addition of 20 μM. Compound-treated plates were incubated at 37°C, 5% CO2 in a humidified incubator for 60 min and then 20 μL of 5.0 μM DEX (1.0 μM final in well) was transferred to assay plates using the Evolution P3 liquid handler outfitted with a 384-well transfer head. The plate control wells were located in columns 1, 2, 23, and 24 and the minimum controls (n=24) were treated with 0.5% DMSO and the maximum control wells (n=32) were exposed to 1.0 μM DEX and 0.5% DMSO. Assay plates were incubated for 30 min at 37°C, 5% CO2, and 95% humidity, and then the contents of the wells were aspirated and replaced with 50 μL of 3.7% formaldehyde containing 2 μg/mL Hoechst 33342 in PBS without Ca2+ and Mg2+, prewarmed to 37°C, using a BioTek ELx405 (BioTek) plate washer, and cells were fixed for 10–30 min at ambient temperature. The fixative was then aspirated and plates were then washed twice with 50 μL PBS using the BioTek ELx405 (BioTek) plate washer and sealed with adhesive aluminum plate seals using the Abgene Seal-IT 100 plate sealer (Abgene) with the last 50 μL wash of PBS in place.
Image acquisition
Images of two fluorescent channels (Hoechst and fluorescein isothiocyanate [FITC]) were sequentially acquired on the ArrayScan VTI (AS-VTI) automated imaging platform (Thermo Fisher Scientific) using a 10×0.3NA objective and the XF100 excitation and emission filter set to obtain images of stained nuclei and GR-GFP. Excitation was provided by an X-CITEÒ 120 W high-pressure metal halide arc lamp with intelli-lamp™ technology (Photonic Solutions Inc.). Typically with the 10×0.3NA objective, the AS-VTI was set up to acquire 500 selected objects (nuclei) or two fields of view, whichever occurred first.
Image analysis
The nucleic acid dye Hoechst 33342 was used to stain and identify the nucleus, and this fluorescent signal was used to focus the instrument and to define a nuclear mask for the molecular translocation (MTL) image analysis algorithm. To quantify the DEX-induced translocation of GR-GFP from the cytoplasm to the nucleus, images were analyzed using the MTL image analysis algorithm as described previously.24 Hoechst stained objects in channel 1 (Ch1) that exhibited the appropriate fluorescent intensities above background and morphological size characteristics (width, length, and area) were identified and classified by the image segmentation as nuclei. The nuclear mask derived from Ch1 was then used to segment the GR-GFP images from Ch2 into nuclear (Circ) and cytoplasmic (Ring) regions. The nuclear mask was eroded by 1 pixel to reduce cytoplasm contamination within the nuclear area, and the reduced mask was used to quantify the amount of target Ch GR-GFP fluorescence within the nuclear region. The nuclear mask was then dilated to cover as much of the cytoplasm region as possible without going outside the cell boundary. Removal of the original nuclear region from this dilated mask created a ring mask in the cytoplasm region outside the nuclear envelope. The number of pixels away from the nuclear mask (1 pixel) and the number of pixels (width=3 pixels) between the inner and outer ring masks were selectable within the MTL bio-application software. The ring masks were then used to quantify the amount of target Ch GR-GFP fluorescence within the cytoplasm region. The MTL image analysis algorithm outputs quantitative data including the total selected object or cell counts from Ch1, and the total and average fluorescent intensities of the GR-GFP in the nuclear (Circ) or cytoplasm (Ring) compartments of the cell. To quantify the relative distribution of the GR-GFP within the nucleus and the cytoplasm regions of the 3617.4 cells, the MTL image analysis algorithm provides a mean average intensity difference calculated by subtracting the average GR-GFP intensity in the Cytoplasm (Ring) region from the average GR-GFP intensity in the Nuclear (Circ) region of Ch2 to produce a mean circ-ring average intensity difference.
Indirect immunofluorescent staining of 3617.4 cell MTs
To observe the organization of MTs in images of 3617.4 cells acquired on the AS-VTI, we adapted previously described indirect immunofluorescence sample preparation methods.24 Briefly, fixed, Hoechst-stained 3617.4 cells prepared as described above for the GR-GFP translocation HCS assay were permeabilized in 0.5% (v/v) Triton X-100 in PBS (without Ca2+ and Mg2+) for 5 min at ambient temperature; cells were washed once in PBS (without Ca2+ and Mg2+) and then incubated for 15 min in 0.1% (v/v) Tween 20 in PBS (without Ca2+ and Mg2+) blocking buffer; cells were incubated for 1 h with a 1:2,000 dilution of a mouse monoclonal anti-α-tubulin antibody in PBS (without Ca2+ and Mg2+). Cells were then washed once in PBS (without Ca2+ and Mg2+) and then incubated for 15 min in Tween 20 blocking buffer and then incubated for 45 min with a 1:1,000 dilution of goat anti-mouse immunoglobulin G antibody conjugated to Alexa-595 in PBS (without Ca2+ and Mg2+). Cells were then washed twice with 50 μL PBS (without Ca2+ and Mg2+) and sealed with the last 50 μL wash of PBS in place. Fluorescent images from three fluorescent Chs were then sequentially acquired on the AS-VTI using a 20 × 0.4NA objective with the XF53 (Hoechst and FITC) and the XF32 (tetramethylrhodamine-5-(and 6)-isothiocyanate) excitation and emission filter sets to obtain images of stained nuclei, GR-GFP, and anti-α-tubulin antibody-stained MTs. The protocol used for this HCS assay has been summarized in Table 1.
Table 1.
Table 1.
Summary of Dexamethasone-Induced Glucocorticoid Receptor Translocation High-Content Screen Assay and Microtubule Staining Protocol
Protein expression and isolation
Tubulin: Tubulin was isolated from three freshly obtained bovine brains according to a modified procedure of that reported by Hamel and Lin.25 Brains were homogenized in a blender containing 0.75 mL/g of 0.1 M MES, pH 6.4, containing 1 mM EGTA, 4 M glycerol, 1 mM MgCl2, 1 mM 2-mercaptoethanol, 0.1 mM EDTA, and 0.1 mM GTP (Solution A). The homogenate was clarified by centrifuging at 6,400 g for 15 min at 0°C with a JA-10 Beckman-Coulter rotor. All remaining centrifugations listed were performed using a Ti-45 Beckman-Coulter rotor. The supernatant was centrifuged at 90,000 g for 44 min at 0°C. ATP and GTP were added to the supernatant to give final concentrations of 1 and 0.3 mM, respectively, and the solution was incubated for 40 min at 37°C. The solution was centrifuged at 90,000 g for 44 min at 37°C. The supernatant was discarded and the pellet was resuspended in 10 mL/brain of the above-described buffer without glycerol (Solution B) and incubated on ice for 30 min. For each 1 mL of solution, 0.553 g of glycerol was added to give a final concentration of 4 M. ATP and GTP were then added to give final concentrations of 1 and 0.3 mM, respectively, and the mixture was incubated for 1 h at 37°C. The solution was centrifuged at 90,000 g for 44 min at 37°C and the pellet was resuspended in 6.25 mL/brain of Solution B. The resuspended pellet was incubated on ice for 30 min and centrifuged at 67,000 g for 30 min at 0°C. Lyophilized MES (2M, pH 6.9) was added to the solution to give a final concentration of 1.6 M followed by the addition of 1 mM GTP and 2 mM dithiothreitol and the mixture was incubated for 45 min at 37°C. The mixture was centrifuged at 90,000 g for 44 min at 37°C and the pellet was resuspended in 10 mL cold 1 M MSG, pH 6.6. The mixture was incubated on ice for 2 h and then centrifuged at 90,000 g for 30 min at 0°C. To the supernatant, 1 mM GTP was added and the solution was incubated for 1 h at 37°C and the mixture was then centrifuged at 90,000 g for 1.5 h at 37°C. The pellet was resuspended in 10 mL of cold 1 M MSG, pH 6.6 and incubated on ice for 1 h. The solution was centrifuged at 90,000 g for 1 h at 0°C and the supernatant contained soluble tubulin. The electrophoretic homogeneity of the protein was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the concentration determined by a bicinchoninic acid (BCA) Protein Assay kit using BSA as a standard.
Hsp70: Yeast Hsp70 (Ssa1p) was purified as described by McClellan and Brodsky.26 In brief, yeast expressing an active, hexahistidine-tagged form of Ssa1p on a galactose-regulated promoter were grown to log-phase and harvested, and the cell walls were digested. The resulting spheroplasts were lysed with glass beads and the lysate was chromatographed on an ATP-affinity column. The bound protein was released with ATP, and then analyzed on a Q-Sepharose column. Ssa1p was eluted through the use of a KCl gradient and then further purified by using nickel ion affinity chromatography. The protein, which was eluted from this column with imidazole, was dialyzed, and aliquots were retained at −80°C until use.
Hsp90: Yeast Hsp90 (Hsc82p) was purified according to the protocol outlined by Youker et al.27 Yeast containing a vector engineered for the overexpression of Hsc82p were propagated, harvested, and then lysed with glass beads, and a crude Hsc82p-containing fraction was obtained by running a KCl gradient on a DE52 anion exchange column. After dialysis to reduce the salt concentration, the chaperone was highly enriched by chromatography on a Q-Sepharose column that was similarly treated with a KCl gradient. Peak fractions containing Hsc82p were pooled and again dialyzed, and the protein was aliquoted and stored at −80°C.
Cytoplasmic dynein: The 380 kDa motor domain fragment was expressed as described by Hook et al.28 Briefly, the gene encoding rat cytoplasmic dynein motor domain (Gly1286-Glu4644) with a C-terminal in-frame hexahistidine tag was inserted into the baculovirus expression vector pVL1393. Hi5™ cells plated with Express Five™ Serum Free Medium were infected with the virus for 40 h. The cells were then washed and resuspended in PBS buffer, and the recombinant motor domain fragments were extracted from the cells by homogenization. Extracts were centrifuged at 5,000 g for 10 min and then 100,000 g for 30 min. The supernatant was applied to an Ni-NTA Superflow column pre-equilibrated with 50 mM PIPES, 50 mM HEPES, pH 7.2, containing 2 mM MgCl2 and 2 mM EGTA. The column was washed with 16 volumes of buffer containing 20 mM imidazole before eluting the protein with buffer supplemented with 125 mM imidazole. The imidazole was removed using a PD-10 Desalting Column and sample was resuspended in 10 mM PIPES, 10 mM HEPES, pH 7.2, containing 0.4 mM MgCl2 and 0.4 mM EGTA. Protein concentrations and purity were then determined by a BCA assay with albumin as the standard and gel electrophoresis, respectively. ATPase activity of the motor domain was determined using a modified malachite kit (PiColorlock™ Gold Assay) according to the manufacturer's protocol. Samples were then read with a SpectraMax® M5 Microplate Reader (MDS Analytical Devices Inc.) at 630 nm.
Activity assays
GR competitor assay: The Green Polar Screen™ Glucocorticoid Receptor Competitor Assay Kit was used according to the manufacturer's recommendations with the final sample volume reduced to 40 μL and is summarized in Table 2. Briefly, human recombinant GR was used at the recommended concentration of 15 nM and the Fluormone™ GS1 was used at a concentration of 1 nM. An initial screen was performed with aliquots (20 μL) of the mixture of test compounds in DMSO screening buffer with final concentrations of 12.5 and 50 μM were distributed in 384-well, black flat-bottom plates. 4×Fluormone (10 μL) followed by 4×GR (10 μL) was then added to the wells. Each compound was tested in triplicate, and 50 μM DEX was used as a positive control. The DMSO concentration was kept at 1% (v/v) throughout the experiment. After 2 h, the fluorescence polarization (FP) (485 nm excitation and 530 nm emission) was measured using a SpectraMax M5 reader. Data were then fitted to a one-site competition curve using KaleidaGraph Software V.4.03. The fit was constrained by the high polarization control, which was the GR/GS1 complex with no competition, and the low polarization control, which was the GR/GS1 complex with 50 μM DEX for 100% competition.
Table 2.
Table 2.
Summary of Glucocorticoid Receptor Competitor Assay
MT polymerization assay: Test agents at 10 μM or DMSO (vehicle control) with 10 μM bovine tubulin in 1 M MSG, pH 6.6, and 400 μM GTP were combined to give a total volume of 100 μL per well and incubated on ice for 15 min to screen for MT destabilizers. The same mixture was used without GTP to identify MT stabilizers. Positive and negative controls were performed using 10 μM PTX and colchicine (CHC), respectively. Sample mixtures were transferred to a 96-well plate preheated to 37°C and absorbance readings were taken immediately every 1 min for 30 min. MT polymerization was measured by the change in absorbance of the solution at 340 nm with a SpectraMax M5 Microplate Reader. The relative MT stabilization or destabilization was measured from the absolute change in absorbance after 20 min normalized to the PTX or CHC absorbance, respectively, also at the 20 min time point. Table 3 provides a summary of the MT polymerization assay.
Table 3.
Table 3.
Summary of Microtubule Polymerization Assay
Hsp70 ATPase assay: Hsp70 (1300 nM) was incubated on ice with test agents at 10 and 50 μM or DMSO for 15 min in buffer consisting of 50 mM HEPES, pH 7.2, containing 2 mM MgCl2. Sodium orthovanadate (100 μM) was used as the positive control. Then, 1 mM ATP was added to the sample mixtures and were incubated at 32°C for 1 h in a final volume of 30 μL per well of a 384-well clear, flat-bottom plate. The amount of free phosphate was detected using the modified malachite kit according to the manufacturer's protocol. Samples were then read with a SpectraMax M5 Microplate Reader at 630 nm. The absorbance of an ATP blank was subtracted from all sample readings. Amount of ATPase activity inhibition was calculated by the amount of absorbance normalized to DMSO control. Table 4 provides a summary of this ATPase assay.
Table 4.
Table 4.
Summary of Hsp70 Adenosine Triphosphatase Activity Assay
Hsp90 ATPase assay: Hsp90 (1,300 nM) was incubated on ice with test agents at 10 and 50 μM or DMSO for 15 min in buffer consisting of 20 mM Tris-HCl, pH 7.4, containing 2 mM MgCl2. 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) (50 μM) was used as the positive control. Then, 1 mM ATP was added to the sample mixtures and were incubated at 32°C for 1 h in a final volume of 30 μL per well of a 384-well clear, flat-bottom plate. The amount of free phosphate was detected and ATPase activity inhibition was determined as described above and the protocol summarized in Table 5.
Table 5.
Table 5.
Summary of Hsp90 Adenosine Triphosphatase Activity Assay
Cytoplasmic dynein ATPase assay: Test agents at 10 and 50 μM or DMSO were added to buffer consisting of 20 mM Tris-HCl, pH 7.6, containing 50 mM KCl, 5 mM MgCl2, and 28 nM dynein and were incubated for 15 min on ice. A positive control was performed using 100 μM sodium orthovanadate. Separately, MTs were performed by incubating 10 μM bovine tubulin with 10 μM PTX in 1 M MSG, pH 6.6, at 37°C for 30 min. The preformed MTs were then centrifuged at 16,000 g for 30 min at 37°C. The pellet was resuspended in the same buffer used for the cytoplasmic dynein with 10 μM PTX and incubated for an additional 20 min while maintaining samples at 37°C. Then, 1 mM ATP was added to the sample mixtures and were incubated at 37°C for 30 min with or without preformed MTs for a final volume of 50 μL per well of a 384-well clear, flat-bottom plate. The amount of free phosphate was detected and ATPase activity inhibition was determined as described above and the protocol summarized in Table 6.
Table 6.
Table 6.
Summary of Dynein Adenosine Triphosphatase Activity Assay
Myosin Ca2+-ATPase assay: Rabbit skeletal muscle myosin II (125 nM) and the test agents at 10 and 50 μM were premixed in 50 mM Tris-HCl, pH 7.9, containing 0.23 M KCl and 2.5 mM CaCl2 and incubated on ice for 15 min. Then, 1 mM ATP was added to give a final volume of 50 μL per well of a 384-well clear, flat-bottom plate. NEM (100 μM) was used as the positive control. The reaction mixture was incubated at room temperature for 30 min. The amount of free phosphate was detected and activity inhibition was determined as described above and the protocol summarized in Table 7.
Table 7.
Table 7.
Summary of Myosin Ca2+-Adenosine Triphosphatase Assay
The Z′-factor statistical coefficient of the cell-based GR-GFP translocation assay was calculated defined by equation M1,29 where σ is the standard deviation of the max and min fluorescence signals, and μ is the mean value of max and min signals, respectively. The Z-factor over a 3-day window to assess signal variability was reported as the mean±standard deviation (SD). Quantitation of cell images, percent GR ligand competition, MT polymerization assay, and percent ATPase inhibition were also reported as the mean±SD. The coefficient of determination (R2) was reported for the concentration-dependent curve fits.
In an effort to identify compounds that inhibited the retrograde transport of GR via cytoplasmic dynein, a HCS cell-based assay was used to screen the LOPAC-1,280 collection from Sigma. Based on these studies, we were able to identify a handful of compounds (shown in Fig. 1) that inhibited the DEX-induced translocation of GR-GFP to the nucleus mediated by cytoplasmic dynein. We further investigated the molecular targets of these compounds via a series of biochemical assays.
Fig. 1.
Fig. 1.
Chemical Structures of library of pharmacologically active compound-1280 hits. (A) 4-Phenyl-3-furoxancarbonitrile (3-cyano-4-phenyl-1,2,5-oxadiazole 2-oxide), (B) Parthenolide ((1aR,7aS,10aS,10bR,E)-1a,5-dimethyl-8-methylene-2,3,6,7,7a,8,10a,10b-octahydrooxireno[2′,3′:9,10]cyclodeca[1,2- (more ...)
Multi-parameter automated immunofluorescence microscopy was first utilized to provide high-content information on the cellular effects of the compounds. Such an analysis provided information on the location of GR-GFP within the cell, as well as effects on MT organization and mass, nuclear morphology, and cytotoxicity by surveying different components of the cell with specific labels: GR-GFP, tubulin antibodies, and Hoechst (Fig. 2A). The cellular phenotype of the DMSO vehicle control demonstrated well-organized cells with a few cells undergoing mitosis and GR-GFP located predominantly in the cytoplasm. The cells were also uniformly rounded with numerous sizeable cellular projections. Upon the addition of the GR agonist DEX, the cell morphology appeared very similar to the DMSO vehicle control with the exception that all of the GR-GFP had translocated into the nuclei as expected (Fig. 2A, B). The addition of several compounds in the LOPAC set 60 min before DEX treatment noticeably altered the GR-GFP distribution phenotype and decreased the DEX-induced nuclear GR-GFP content relative to the DEX control (Fig. 2). Compounds that caused a ≥50% decrease in GR-GFP accumulation in the nuclei were considered hits. Pretreatment with parthenolide, apomorphine, Bay 11–7085, and 4-phenyl-3-furoxancarbonitrile all inhibited DEX-induced GR nuclear translocation and led to the retention of GR-GFP in the cytoplasm (Fig. 2A, B). These five compounds also appeared to alter cell morphology in that cells exposed to these molecules exhibited fewer and shorter cellular projections protruding from the cell body when compared to the images of the DMSO- and DEX-treated control cells. With respect to the MT mass, however, only exposure to Bay 11–7085 altered the α-tubulin staining intensities of the treated cells relative to the DMSO and DEX control cells, and significantly reduced the cytoplasmic (mean ring average intensity) signal (Fig. 2C). In addition, only exposure to Bay 11–7085 significantly altered the number of cell nuclei detected in treated cells relative to the DMSO and DEX control cells (Fig. 2D). At concentrations >12.5 μM, Bay 11–7085 treatment was cytotoxic as indicated by the apparent reductions in cell counts and MT mass (Fig. 2C, D). The apparent reduction in MT mass produced in cells treated with 50 μM Bay 11–7085 (Fig. 2C) was probably secondary to its cytotoxic effects on cell number (Fig. 2D). Even though Bay 11–7085 at 12.5 μM was noncytotoxic, inhibition of GR-GFP translocation was still observed at this concentration. In contrast, the other compounds that inhibited DEX-induced GR-GFP translocation did not significantly affect either cell counts or MT mass relative to the DMSO and DEX controls. The Z′-factor for the GR-GFP translocation assay was determined to be 0.52±0.11 by measuring the max and min fluorescence signal of GR-GFP from fixed cells over a 3-day period, suggesting that the assay is well-suited and reliable for high-throughput screening.29
Fig. 2.
Fig. 2.
Multi-parameter immunofluorescence visualization and quantitation of treated cells. (A) GR-GFP (green) was used to track cytoplasmic dynein translocation, a fluorescent tubulin antibody (red) to label MTs, and Hoechst 33342 was used to label DNA (blue) (more ...)
Since ligand binding to GR is required to induce translocation from the cytoplasm to the nucleus,30 we investigated the ability of the five inhibitors to disrupt the binding of a fluorescent-labeled ligand to GR in a FP competitive binding assay. The GR agonist DEX inhibited ligand binding in the FP assay in a concentration-dependent manner (Fig. 3A) and exhibited an half maximal inhibitory concentration (IC50) value of 10.1±1.5 nM, corresponding well to previously published values.31 At both of the concentrations tested, 12.5 and 50 μM, neither parthenolide nor 4-phenyl-3-furoxancarbonitrile disrupted agonist binding to GR (Fig. 3B). In contrast, at 50 μM the other GR-translocation inhibitors appeared to compete for the GR ligand binding domain by an average of >60% (Fig. 2B). At 12.5 μM, however, the percent competition of GR-binding exhibited by these compounds was considerably lower and much more variable (Fig. 3B). Given the relatively large standard errors in the compound binding data, it is unclear whether the apparent competition of GR binding at high concentrations contributes to the inhibition of DEX-induced GR translocation observed at compound concentrations <12.5 μM. This was perhaps an indication that these compounds inhibited GR translocation through interactions with other targets within the cell.
Fig. 3.
Fig. 3.
Fluorescence polarization GR competitive binding assay. (A) Concentration-dependent evaluation of the competitive binding of the GR agonist DEX fitted by a one-site competition curve with R2=0.9976 and an half maximal inhibitory concentration value of (more ...)
Since retrograde transport requires the existence of functionally structured MTs,30 alterations in MTs can stall GR translocation by cytoplasmic dynein. Many compounds have been previously demonstrated to alter MT dynamics by either acting as MT stabilizers (PTX, discodermolide)32,33 or destabilizers (CHC, vincristine).34,35 MT perturbing agents have been shown to inhibit transport of p53 via cytoplasmic dynein to the nucleus.36 Compounds identified as MT perturbing agents may also serve as cytoplasmic dynein enhancers when used at low concentrations.37 Although only exposure to Bay 11–7085 was shown to alter MT mass in treated cells (Fig. 2C), we investigated the ability of the selected inhibitors to perturb MT dynamics in vitro relative to the MT stabilizer PTX and the MT destabilizer CHC, respectively (Table 8). Of the six compounds tested, 4-phenyl-3-furoxancarbonitrile and Bay 11–7085 both exhibited MT destabilization characteristics. 4-Phenyl-3-furoxancarbonitrile is a nitric oxide donor,38 whereas Bay 11–7085 is an nuclear factor kappa B inhibitor that induces apoptosis,39 and neither compound has been reported to alter MT dynamics. In contrast, parthenolide has been previously reported to exhibit MT stabilizing activity,40 and was the only compound tested that was found to induce MT polymerization in vitro. MT perturbation measurements by turbidity are effective for a small number of samples, but are not robust enough for high-throughput assays. We have recently examined the ability to polymerize MTs on biosensor surfaces and distinguish MT stabilizers from destabilizers,41 an assay format that may be applied to screening MT-perturbing agents at greater throughput in the future.
Table 8.
Table 8.
Inhibition of Guanosine Triphosphate-Induced Assembly and Hypernucleation/Polymer Stabilization of Bovine Brain Tubulin by Hits from the High-Content Screen Analysis of the Library of Pharmacologically Active Compound-1280 Collection
Other factors, including the stability of the dynein-cargo complex, play an important role in the transport of cytoplasmic dynein. In the case of the GR, complex formation begins with the ATP-dependent binding of Hsp70 to the GR along with the nonessential co-chaperone Hsp40 creating a “primed” GR/Hsp70 complex.42 The GR/Hsp70 complex then binds to Hsp90 followed by the binding of p23 to stabilize this newly formed complex.43 GR translocation is dependent on its association with Hsp90 and it has been suggested that the dynamics of this complex is dependent on ligand binding to the receptor.7 A number of Hsp-binding immunophilins then link the cargo complex to the light and intermediate chains of cytoplasmic dynein.2 Inhibition of Hsp90 has been demonstrated to halt GR translocation.44 Hsp90 has a unique ATP binding pocket near its N-terminus, where specific inhibitors such as geldanamycin bind.45 The use of 17-AAG, which is an ansamycin antibiotic that binds Hsp90,46 as a control (Fig. 4) exhibited expected inhibition of Hsp90 activity.47 Given the importance of HSP in stabilizing the cargo complex with cytoplasmic dynein, we investigated whether or not the targeting of these proteins inhibited GR translocation. Ssa1p and Hsc82p, the yeast versions of Hsp70 and 90, respectively, were used in this study because of the shared homology with mammalian versions48 along with the fact that the formation of the cargo complex is conserved among different species.49 Therefore, screening for compounds that alter the ATPase activity of HSPs can indicate potential targets of compounds inhibiting GR translocation. In addition, the identification of HSP inhibitors/activators could prove to be useful since Hsps are a therapeutic target in oncology50 and against neurodegenerative diseases because of their involvement in protein folding.51 Through the use of a colorimetric assay, the percent inhibition of ATPase activity was calculated by comparing the amount of inorganic phosphate released in the presence of the compounds relative to a DMSO control. At the highest concentration tested, 50 μM, none of the compounds had any significant effect on Hsc82p (Hsp90) (Fig. 4). Concentrations of test agents >10 μM caused Ssa1p to precipitate, rendering measurement of ATPase activity inaccurate.
Fig. 4.
Fig. 4.
HSP ATPase inhibition assays. (A) Percent inhibition of ATPase activity of yeast Hsp70 (Ssa1p) with test compounds compared to DMSO control and sodium orthovanadate as a control. (B) Percent inhibition of ATPase activity yeast Hsp90 (Hsc82p) with test (more ...)
The ability of the compounds to inhibit cytoplasmic dynein's ATPase activity was then investigated. Only four of the compounds exhibited a significant concentration-dependent inhibition of the ATPase activity of both basal and MT-enhanced activity of cytoplasmic dynein (Fig. 5). Both enantiomers of apomorphine inhibited cytoplasmic dynein with IC50 values of ~1.5 μM, whether in the basal or MT-stimulated state (Table 9). 6-Nitroso-1,2-benzopyrone and Bay 11–7085 also inhibited cytoplasmic dynein, but there was a more substantial change in their IC50 values between basal and MT-stimulated states. Although there was no indication that any of the compounds significantly altered the interaction of cytoplasmic dynein's MT-binding domain with the MTs, this type of comparison can be important in identifying compounds that interfere with the dynein-MT interaction in future screens. The decreased IC50 value of 6-nitroso-1,2-benzopyrone in the MT-stimulated state could indicate the targeting of the sulfhydryls on tubulin heterodimers52 since nitroso derivatives have been shown bind to protein sulfhydryls,53 subsequently destabilizing the MTs as suggested in Table 8, rather than binding to a defined active site. The double sigmoidal behavior of 6-nitroso-1,2-benzopyrone also suggested its nonspecific binding to cytoplasmic dynein (Fig. 5B, D).
Fig. 5.
Fig. 5.
Cytoplasmic dynein ATPase inhibition assay. (A) Percent inhibition of ATPase activity of basal dynein with test compounds compared to DMSO control and sodium orthovanadate as a control. (B) Concentration-dependent inhibition of basal cytoplasmic dynein (more ...)
Table 9.
Table 9.
Biochemical Half Maximal Inhibitory Concentration Values of Test Agents Against Both Basal and Microtubule-Stimulated Cytoplasmic Dynein
Finally, to rule out any compounds that might be general ATPase motor protein inhibitors, we tested the ability of compounds to inhibit the ATPase activity of myosin. At the two concentrations tested, 10 and 50 μM, only the apomorphines exhibited any significant inhibition of the ATPase activity of myosin (Fig. 6A) with concentration-dependent behavior and IC50 values of 1.4±0.4 and 3.2±0.7 μM for the R and S enantiomers, respectively (Fig. 6B). Despite being the most potent cytoplasmic dynein ATPase inhibitors, the broad spectrum of target activities that the apomorphine enantiomers inhibited in our panel of assays suggests that they were promiscuous hits.
Fig. 6.
Fig. 6.
Myosin ATPase inhibition assay. (A) Percent inhibition of ATPase activity of myosin with test compounds compared to DMSO control and NEM as a control. (B) Concentration-dependent inhibition of myosin ATPase activity by (R)-apomorphine (R2=0.9661) and (more ...)
Cell-based screening reliably revealed a number of small molecules from a LOPAC collection that stalled the agonist-induced transport of GR from the cytoplasm to the nucleus. Since there are a number of potential reasons for interruption of this dynein-mediated transport, we sought to identify the target(s) of these molecules through a number of biochemical assays applicable to the translocation process. From the five compounds tested, the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein and myosin, but also appeared to inhibit GR binding. The lack of specificity these mirror image molecules exhibit, however, made them unattractive for further study in our quest. Parthenolide is a previously recognized MT stabilizer that most likely altered the tracks needed for dynein to transport GR-GFP to the nuclei. Based on the chemical structures of the remaining cell-based screening hits, it is likely that the compounds targeted reactive sulfhydryls of dynein and MTs, both contributing to inhibition of dynein-mediated transport. The reliability of the hits from the cell-based screen was further supplemented by the concentration-dependent inhibition of activity determined from the biochemical assays and with good R2 values. Ideally, specific dynein-mediated transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, these assays demonstrated the ability to examine cell permeable inhibitors of GR translocation and attempt to identify the target(s) of these molecules through a series of readily implemented biochemical assays. We envision that screening of a larger library of compounds for inhibition of cell-based cytoplasmic dynein transport will reveal specific cytoplasmic dynein and/or HSP inhibitors. We also believe that this route of identifyifollowed by target validation is an improvement over conventional biochemical screening that suffers when hits eventually are cell impermeable and/or severely cytotoxic.
AAAATPases associated with diverse activities
ARandrogen receptor
ATPadenosine triphosphate
BCAbicinchoninic acid
BSAbovine serum albumin
DMEMDulbecco's modified Eagle's medium
EDTAethylenediaminetetraacetic acid
EGTAethylene glycol tetraacetic acid
ERestrogen receptor
FBSfetal bovine serum
FPfluorescence polarization
FITCfluorescein isothiocyanate
GFPgreen fluorescent protein
GRglucocorticoid receptor
GTPguanosine triphosphate
HCShigh-content screen
HEPES-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HSCheat-shock cognate
HSPheat-shock protein
IC50half maximal inhibitory concentration
LOPAClibrary of pharmacologically active compounds
MCRAIDmean circ-ring average intensity difference
MES2-(N-morpholino)ethanesulfonic acid
MSGmonosodium glutamate
MTLmolecular translocation
Ni-NTAnickel nitrilotriacetic acid
PBSphosphate-buffered saline
PIPESpiperazine-N,N0-bis(2-ethanesulfonic acid)
SDstandard deviation
TRIS-HCltris(hydroxymethyl)aminomethane hydrochloric acid
TRITCtetramethylrhodamine-5-(and 6)-isothiocyanate.

We thank Prof. Richard Vallee along with Drs. Peter Hook and Richard McKenney from Columbia University for generously providing the baculovirus required for dynein purification as well as their continued support and discussions throughout the project. We also thank Robert Youker and Christine Wright for supplying some of the purified Hsc82p and Ssa1p, respectively, that was used in this study and Dr. Annette Chiang for her technical assistance. This work was funded by National Institutes of Health grants NS057026 to B.W.D. and DK079307 (the University of Pittsburgh George O'Brien Kidney Research Core Center) to J.L.B.
Disclosure Statement
No competing financial interests exist.
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