uHTS identification of small molecule activators of alpha dystroglycan glycosylation
A number of muscular dystrophies (MD) are associated with abnormal O-mannosylation of alpha dystroglycan (alpha-DG) that plays a central role in linking the extracellular matrix (ECM) to the actin-based cytoskeleton in muscle. Hypoglycosylation of alpha-DG impairs its ability to bind to the ECM proteins such as laminins and results in pathogenesis in the muscle and nerve systems and often causes more ..
BioActive Compounds: 805
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, La Jolla, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH085693-01A1
Assay Provider: Xiaohua Wu, M.D., Ph.D., Carolinas Medical Center, Charlotte, NC
A number of muscular dystrophies (MD) are associated with abnormal O-mannosylation of alpha dystroglycan (alpha-DG) that plays a central role in linking the extracellular matrix (ECM) to the actin-based cytoskeleton in muscle. Hypoglycosylation of alpha-DG impairs its ability to bind to the ECM proteins such as laminins and results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. Increasing evidence suggests that over-expression of glycosyltransferases enhancing the glycosylation of alpha-DG can restore the functions of alpha-DG in MD mouse models. We hypothesize that cells can enhance glycosylation of alpha-DG in response to certain chemical stimuli. The main goal for this project will be to screen the MLPCN library and identify small molecule compounds that enhance glycosylation of alpha-DG. The assay described herein is a fluorescent, alphascreen-based assay that utilizes an alpha-DG expressing Pro5 cell line to achieve this goal.
A. Brief Description of the Assay:
This assay identifies compounds that may potentially enhance the glycosylation of alpha-dystroglycan in Pro5-aDG cells. It is a fluorescent, alphascreen-based assay.
Item, Source, Cat #
Pro5-aDG(HIG) (nonglycosylated a-DG), #Assay Provider,#N/A
B10-LARGE-aDG(HIG) (glycosylated a-DG), Assay Provider, N/A
Ham's F12 Modified Media, Mediatech, #10-025-CV
DMEM:F12 Media Phenol-red Free, Gibco, 21041
Heat Inactivated Fetal Bovine Serum, Gibco, 10437
Penicillin Streptomycin solution, Omega Scientific, PS-20
L-Glutamine, Mediatech, 25-005-CL
Zeocin, Invitrogen, R25005
Geneticin (G418), MP Biomedicals, 1672548
Trypsin, Mediatech, 25-050-Cl
DPBS with Calcium and Magnesium, Mediatech, 21-030-CV
Streptavidin Donor Beads, Perkin Elmer, 6760002S
Anti-Mouse IgM AlphaLISA Acceptor Beads, Perkin Elmer, AL130C
IIH6 antibody, Santa Cruz, SC-53987
Goat Biotin-anti-HIG, Thermo Scientific, 31770
HEPES, Fisher, BP310-500
Bovine Serum Albumin (fatty acid free), Sigma, A6003
NaCl, Fisher, BP358-212
Tween 20, Sigma, P1379
Molecular Grade Water, Mediatech, Inc., 46-000-CM
T225 TC Flask, Nunc, 159934
1536-Well, White, Solid bottom, TC-treated, sterile plate, Corning, 3727
C. Cell Culture Procedures:
1. Aspirate media off flask.
2. Wash flask with 5 ml DPBS.
3. Aspirate DPBS off flask.
4. Add 5ml of 0.05% Trypsin to each flask to detach cells.
5. Incubate flasks in incubator for 5 mins to allow cells to detach.
6. Tap flask gently to detach remaining cells. Examine under the microscope to assure detachment and if necessary, incubate longer.
7. Once cells are detached, wash flask with 5 ml of Assay Media.
8. Collect all 10 ml of cell suspension into a conical.
9. Spin cells down at 1200 rpm for 4 minutes.
10. Aspirate the supernatant.
11. Resuspend the cell pellet in 10ml of Assay Media.
11a. For maintenance: With a 75-80% confluent flask, the split ratio should be 1/25 for 2-3 days, and 1/50 for up to 4 days.
11b. For cell-plating: Perform a cell count using the Cellometer. Bring the cell concentration to 60,000 cells/ml using the Assay Media.
D. Assay Procedures:
1. Using LabCyte Echo, transfer 25 nL from a 2 mM Echo qualified plate containing test compounds into Col. 5 - 48 of assay plate. Transfer 25 nL of DMSO to col. 1-4 for positive and negative control wells.
2. Using the Combi, dispense 300 cells/well, 5 ul/well of PRO5 cells to columns 3-48.
3. Using the Combi, dispense 300 cells/well, 5 ul/well of B10 cells to columns 1-2.
4. Spin plates at 500 rpm for 1 minute in centrifuge.
5. Stack the assay plates in between dummy plates filled with water, and saran wrap the entire stack.
6. Spin plates at 500 rpm for 1 minute in centrifuge.
7. Incubate plates for 2 days in 37 degrees, 5% CO2 incubator.
7. Using the Combi, dispense 1.5 ul/well of Antibody Mix to col 1-48.
8. Spin plates at 500 rpm for 1 minute in centrifuge.
9. Incubate plates for 10mins at Room Temperature in the dark.
10. Using the Combi, dispense 1.5 ul/well of Donor/Acceptor Bead Mix to col 1-48. (light sensitive)
11. Spin plates at 500 rpm for 1 minute in centrifuge.
12. Incubate plates for 6 hours at Room Temperature in the dark.
13. Read plates on the Envision using an Alphascreen Protocol.
E. Plate Map:
Positive (High) control in columns 1 and 2, DMSO with B10 cells
Negative (Low) control in columns 3 and 4, DMSO with PRO5 cells
Test compound in columns 5-48, compound test concentration = 10 uM
PRO5 Growth Media
500 ml Ham's F12 Modified
200 ug/ml Zeocin
B10 Growth Media
500 ml Ham's F12 Modified
200 ug/ml Geneticin (G418)
Assay Media (for cells):
500 ml Phenol-Red Free DMEM: F12 media
Assay Buffer (for dilution of beads and antibodies)
50 mM HEPES pH 7.4
100 mM NaCl
0.01% Tween 20
Antibody Mix (5.33X final concentration)
10.66 nM IIH6 antibody
5.33 nM Goat Biotinylated anti-HIG antibody
Bead Mix (5.33X final concentration)
53.3 ug/ml Anti-Mouse IgM AlphaLISA Acceptor Beads
53.3 ug/ml Streptavidin Donor Beads
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Store antibodies and beads at 4 degrees.
3. Protect the Goat Biotin-anti-HIG and the AlphaScreen beads from light. The AlphaScreen beads are very sensitive to light.
Compounds that demonstrated a corrected % activity >= 8% compared to the controls or that have a z-score >= 8 relative to the rest of the assay plate are defined as active in the assay.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)