HCS to Identify Inhibitors of Dynein Mediated Cargo Transport on Microtubules.
Cytoplasmic dynein is the molecular motor that carries cargo to the minus ends of microtubules (MTs) (e.g., from the cytoplasm to the nucleus), and provides the mechancial force for many other important fuctions, including nuclear envelope breakdown and sister chromatid exchange at mitosis. Unlike the numerous MT plus end-directed molecular motors, the kinesins, no specific small molecule more ..
BioActive Compounds: 1035
Excerpts from the R21NS057026 Application - Dr. Billy Day University of Pittsburgh.
Cytoplasmic dynein is the molecular motor that carries cargo to the minus ends of microtubules (MTs) (e.g., from the cytoplasm to the nucleus), and provides the mechancial force for many other important fuctions, including nuclear envelope breakdown and sister chromatid exchange at mitosis. Unlike the numerous MT plus end-directed molecular motors, the kinesins, no specific small molecule inhibitors of dynein are known. Weak and nonspecific redox perturbers and nonspecific mimics of ATP (e.g., EHNA) or phosphate anion (e.g., vanadate) are the only known dynein inhibitors. New inhibitors with potency and specificity would be invaluable cell biology tools. This void in our chemical biology toolbox is due in large part to both the large size of the work-performing component of the complex, dynein heavy chain (DYNC1H1) and the working complex itself, as well as the relative difficulty of working with functional dynein from biological systems. The overall goal of this work is to develop a refined suite of high throughput cell and biochemical assays for screening of chemical libraries to find experimentally useful inhibitors of cellular cytoplasmic dynein.
The bidirectionality of MTs provides for intracellular shuttling of intracellular components. Two major types of ATP-driven motor proteins deliver proteins, vesicles and organelles ("cargos") between the cell membrane, the endoplasmic reticulum and the nucleus:
dyneins and kinesins. Dyneins are molecular motor complexes (dynein + dynactin + adaptor proteins) that generate force towards the minus ends of MTs. Cytoplasmic dynein is a multisubunit, ca. 2 MDa protein complex made up of two or three, enormous (4600 amino acid, ~500 kDa) heavy chains containing the motor domain (DYNC1H1) complexed with intermediate and light chains responsible for subcellular localization and recognizing and binding the various forms of cargo carried by dynein. Molecular motors contain a motor, cargo-binding and regulatory components, and utilize the hydrolysis of ATP to ADP + Pi to power their steps along MTs. DYNC1H1 consists of an N-terminal stem, which binds cargo and interacts with other dynein components, and a head or motor domain. The motor contains six tandemly-linked AAA domains in the head, which form a ring. A stalk-like structure (formed by two of the coiled coil domains) protrudes between AAA 4 and AAA 5 and terminates in a MT-binding site. There are four well-conserved and two non-conserved ATPase sites, one per AAA domain. Probably only one of these (within AAA 1) actually hydrolyzes ATP, with the others likely serving a regulatory function].
Cytoplasmic dynein is involved in vesicular return to the MT organizing center, as well as the retention of the vesicles at this subcellular locale. Cytoplasmic dynein is intricately involved in the maintenance of the Golgi apparatus and the trafficking of membrane-encapsulated vesicles and proteins (e.g., membrane-bound receptors). It is also involved in mitosis, including nuclear envelope breakdown at late prometaphase, several aspects of chromosome segregation and helping to form the mitotic spindle. Examples of important known cargo and interacting proteins for the dyneins include TP53 (a.k.a. p53) , several nuclear hormone receptors (e.g., glucocorticoid and estrogen receptors), the proapoptotic Bcl-2 family member Bim, dynactin, which links dynein to its cargo, and Cdc2 kinase, which phosphorylates a light intermediate chain of dynein during mitosis. There are likely many, many other as yet undiscovered dynein cargo. Interactions between MT and actin networks are thought to be crucial for mechanical and signaling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions more prominently than merely through the localization of interacting proteins. Beyond those listed above, several important known or potential drug targets interact with the Cytoplasmic dynein complex at one or more points in their cellular lifetimes. Some examples include the tubulin deactylase HDAC6 and HSP90. The latter may well be an important partner in dynein's translocation of both hormone receptors and p53 protein to the MT organizing center at the nucleus. Galigniana et al. in collaboration with co-investigator DeFranco have recently presented compelling models of the interactions between p53 as well as the glucocoticoid receptor with dynein via linkages provided by HSP90, in concert with HSP70, to immunophilins (e.g., FKBP52, CyP-40, or PP5) bound to region of the dynactin-associated portion of the dynein complex that binds the inhibitor dynamitin.
Microarray analyses have shown that dynein light intermediate chains 1 and 2 are significantly overexpressed genes in nasopharyngeal and prostate cancers. Perturbation of dynein-dynactin interaction blocks mitotic spindle assembly, and treatment of drosophila cells with siRNA to Cytoplasmic dynein heavy chain causes a block in the progression to anaphase; in interphase cells, perturbation of the dynein-dynactin complex is correlated with an inhibition of endoplasmic reticulum-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system. LIS1, a component of the dynein/dynactin motor complex, is encoded by the gene mutated in patients with type I lissencephaly ("smooth brain"), thereby causing deregulation of endoplasmatic reticilum-to-Golgi vesicular transport, resulting in rapid disappearance of short-living receptors from the plasma membrane and loss of cell sensitivity to TNF and interferon. It thus seems reasonable that a small molecule inhibitor of Cytoplasmic dynein could serve as a valuable cell biology tool, and perhaps provide useful clues into the therapy of immune and neurological disorders or against cancers.
One of the specific aims of the R21 proposal was to develop a cell-, micotiter plate- and robotic liquid handling based phenotypic multi-parameter fluorescence screen to detect dynein inhibition in interphase cells, as well as to detect possible mitotic block due to inhibition of dynein. Three experimental systems to measure dynein-mediated transport from the cytoplasm to the nucleus were proposed as potential assays that might be suitable for HCS: (i) p53 translocation after mild DNA damage; (ii) translocation of stably expressed green fluorescent protein-labeled glucocorticoid receptor after binding with an agonist; and (iii) translocation of a fluorescently-labeled adenovirus. We describe here an HCS assay to measure agonist induced Glucocorticoid Nuclear Hormone Receptor-EGFP (GNHR-EGFP) translocation from the cytoplasm to the nucleus as a suitable assay to screen for inhibitors of cytoplasmic dynein.
Glucocorticoid Nuclear Hormone Receptor-EGFP (GNHR-EGFP) Translocation HCS Assay Protocol.
The Mouse mammary adenocarcinoma cell line (3617.4) stably expressing a rat glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GNHR-EGFP) under the control of a tetracycline (tet)-regulated promoter was utilized for the HCS assay (Elbi et al, 2004, Sci STKE. 238:pl10.).
Automated Imaging Platform.
The ArrayScan VTi houses a Zeiss 200M inverted microscope outfitted with 5X/0.25 NA, 10X/0.3NA, 20X/0.4 NA and 40X 0.5 NA Zeiss objectives. Illumination is provided by a full spectrum (300-2000 nM) Hg-halide arc lamp source (EXFO, Quebec, Canada) and fluorescence is detected by a high sensitivity cooled Orca CCD Camera (Photometrics Quantix). The ArrayScan VTi uses an image-based auto-focus system, and has the capability of imaging multi-wavelength fluorescence, up to six excitation and emission channels excited and acquired sequentially. Channel selection is accomplished using a fast excitation filter wheel combined with a multi-band emission filter.
Images were acquired using a 10 x 0.3NA objective in two fluorescent channels using the XF100 filter set for Hoechst in Channel 1 and GFP (FITC) in Channel 2. The ArrayScan VTI was set up to acquire 2 fields of view or 100 valid objects (nuclei) which ever came first.
Image Analysis Algorithm.
The Molecular Translocation algorithm was used to quantify the relative distribution of the GNHR-EGFP between two cellular compartments, the cytoplasm and the nucleus. 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. The mask is eroded to reduce cytoplasmic contamination within the nuclear area, and the reduced mask is used to quantify the amount of target channel GNHR-EGFP fluorescence within the nucleus. The nuclear mask was then dilated to cover as much of the cytoplasmic region as possible without going outside the cell boundary. Removal of the original nuclear region from this dilated mask creates a ring mask that covers the cytoplasmic region outside the nuclear envelope. The image analysis algorithm outputs quantitative data such as the total or average fluorescent intensities of the GNHR-EGFP signal in the nucleus (Circ) or cytoplasm (Ring) on a per cell basis, that may also be reported as an overall well averaged value.
Detailed Protocol of the GNHR-translocation Assay.
A. Mouse mammary adenocarcinoma cell line 3617.4 Culture Media:
DMEM + 10% FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL + Tetracycline 5 mg/mL.
B. Mouse mammary adenocarcinoma cell line 3617.4 Induction Media
DMEM + 10% charcoal stripped FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL.
GNHR-EGFP Translocation Protocol
1. Passage and maintain 3617.4 GNHR-EGFP-GR cells in culture medium with Tetracycline and Geneticin.
2. Aspirate culture media and incubate in Induction medium (no Tet) for 15 min at 37 C, 5% CO2 and 95% humidity
3. Wash cells three times with HBSS and harvest cells by trypsinization.
4. Centrifuge cells at 500 x g for 5 min and re-suspend in 10 mL induction medium
5. Count viable cells via trypan exclusion using a haemocytometer
6. Re-suspend cells to 41,666 cells/mL in GNHR-EGFPInduction medium
7. Add 60 uL of cell suspension per well to the 384-well black walled clear bottom plates (Greiner Bio-one Cat # 781091) for a final density of 2500 cells/well using the Zoom dispenser.
8. Incubate plates for 48 hrs at 37 C, 5% CO2 and 95% humidity
9. Transfer 20 uL of pre-diluted compounds to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head
10. Incubate plates for 1.0 hrs at 37 C, 5% CO2 and 95% humidity
11. Add 20 uL of 5.0 uM Dexamethasone (final in well 1.0 uM) to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head.
12. Incubate for 30 min at 37 C, 5% CO2 and 95% humidity.
13. Aspirate media and fix cells for 10 min with 50 uL 3.7% Formaldehyde, 2 ug/mL (final) Hoechest 33342 in PBS without Ca2+ and Mg2+, pre-warmed to 37 C.
14. Aspirate fixation solution and wash cells 1x with 50 uL PBS.
15. Aspirate PBS, add 50 uL PBS and seal plate on Abgene plate sealer.
16. Acquire images on the ArrayScan VTI and perform image analysis using the established Molecular Translocation algorithm.
HCS Parameters Reported:
1. MCRAID-CH2: Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity Difference in Channel 2
2. Z-score_MCRAID-CH2:Z-score of the Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity Difference in Channel 2
3. HCS_cmpd_conc: HCS compound concentration in uM
4. Max_MACRAID: Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2 of the maximum assay signal window plate controls n=32
5. Min_MCRAID-CH2: Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2 of the minimum assay signal window plate controls n=24
6. MRAI-CH2: Mean Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2
7. Z-score_MRAI-CH2: Z-score of the Mean Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2
8. MRAI Outcome: 1 = fluorescence intensity within the normal range & 2 = fluorescence intensity > 4 x SD above the mean fluorescent intensity
9. MCAI-CH2: Mean Circ (Nucleus) Average GNHR-EGFP Intensity in Channel 2
10. Z-score_MCAI-CH2: Z-score of the Mean Circ (Nucleus) Average GNHR-EGFP Intensity in Channel 2
11. MCAI Outcome: 1 = fluorescence intensity within the normal range & 2 = fluorescence intensity > 4 x SD above the mean fluorescent intensity
12. SCCPVF: Selected cell counts per valid field of view
13. Z-score_SCCPVF: Z-score of Selected cell counts per valid field of view
14. SCCVF Outcome: 1 = cell counts within normal range, & 2 = cell counts > 4 SD below the mean cell count per well
14. Assay Date: Date the HCS assay was performed
The GNHR-EGFP translocation HCS conducted by the PMLSC utilized a Z score statistical scoring method to identify active compounds (Brideau et al, 2003 J. Biomolecular Screening 8(6): p634-637.
Target Activity Score (MCRAID-CH2).
The Z score for a compound is computed on a plate-by-plate basis. The Z score for the Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity Difference in Channel 2 (MCRAID-CH2) Xi is defined as Zi = (Xi-Xm)/Sm, where Xm is the mean of all the raw MCRAID-CH2 values of the compounds on a plate (n=320), and Sm is the standard deviation of all these values. A cut off Z score of -3.5 was selected as the active criterion for the GNHR-EGFP translocation HCS.
Fluorescence Intensity Outliers
Cytoplasm (MRAI-CH2). The Z score for a compound is computed on a screening run basis, well data from all plates run on the date the HCS assay screening operations were performed. The Z score for the Mean Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2 (MRAI-CH2) Xi is defined as Zi = (Xi-Xm)/Sm, where Xm is the mean of all the raw MRAI-CH2 values of the wells on the multiple plates run on that date, and Sm is the standard deviation of all these values. A cut off Z score of > 4.0 was selected as the definition of a fluorescent outlier for the GNHR-EGFP translocation HCS.
Nucleus (MCAI-CH2). The Z score for a compound is computed on a screening run basis, well data from all plates run on the date the HCS assay screening operations were performed. The Z score for the Mean Circ (Nucleus) Average GNHR-EGFP Intensity in Channel 2 (MCAI-CH2) Xi is defined as Zi = (Xi-Xm)/Sm, where Xm is the mean of all the raw MCAI-CH2 values of the wells on the multiple plates run on that date, and Sm is the standard deviation of all these values. A cut off Z score of > 4.0 was selected as the definition of a fluorescent outlier for the GNHR-EGFP translocation HCS.
Cell Counts-CH1 (SCCPVF). The Z score for a compound is computed on a screening run basis, well data from all plates run on the date the HCS assay screening operations were performed. The Z score for the Selected cell counts per valid field of view (SCCPVF) Xi is defined as Zi = (Xi-Xm)/Sm, where Xm is the mean of all the raw SCCPVF values of the wells on the multiple plates run on that date, and Sm is the standard deviation of all these values. A cut off Z score of < -4.0 was selected as the definition of a cytotoxic outlier for the GNHR-EGFP translocation HCS.
Definition of an active compound:
Z-score MCRAID < -3.5, Z-score MRAI < 4.0, Z-score MCAI < 4.0, and Z-score SCCPVF > -4.0.
1 - Substance is considered inactive when the Z-score MCRAID > -3.5, Z-score MRAI > 4.0, Z-score MCAI > 4.0, and Z-score SCCPVF < -4.0.
2 - Substance is considered active when the Z-score MCRAID < -3.5, Z-score MRAI < 4.0, Z-score MCAI < 4.0, and Z-score SCCPVF > -4.0.
3 - Substance activity outcome is inconclusive
0-40 scoring range is reserved for primary HTS data
a) if the substance is considered active the score is 40.
b) if the substance is considered in active the score is 0.
Definition of a Hit:
Rapid HCS screen substance is considered active when Z-score MCRAID < -3.5, Z-score MRAI < 4.0, Z-score MCAI < 4.0, and Z-score SCCPVF > -4.0, at 20 uM.
Confirmation of inhibition of GNHR-EGFP translocation in 2 independent tests at 20 uM.
Concentration Response IC50 < 20 uM in the GNHR-EGFP translocation assay.
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.
Secondary Testing Paradigm.
Confirmed concentration dependent actives in the GNHR-EGFP translocation assay would be counter screened in the p53 translocation after mild DNA damage assay to confirm that they inhibited dynein-mediated transport events.
Dr Day plans to implement the necessary biochemical screens to confirm that a small molecule's molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin.
Data Table (Concise)