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BioAssay: AID 651748

Dose responses of compounds that activate the Choline Transporter (CHT) in a 3H-choline uptake radioactive assay

Assay Implementation: Meng Wu Ph.D., Xiaofang Huang M.S., Zhihong Lin Ph.D., Kaiping Xu M.S., Shunyou Long M.S., and Owen McManus Ph.D. ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Inactive(14)
 
 
AID: 651748
Data Source: Johns Hopkins Ion Channel Center (JHICC_CHT_Act_3H uptake_CRC2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-11-05

Data Table ( Complete ):           View All Data
Target
Sequence: high affinity choline transporter 1 [Homo sapiens]
Description ..   
Protein Family: Na(+)- and Cl(-)-dependent choline cotransporter CHT and related proteins; solute-binding domain

Gene:SLC5A7     Related Protein 3D Structures     More BioActivity Data..
Tested Compounds:
Related Experiments
AIDNameTypeComment
488977Primary cell-based screen for identification of compounds that allosterically activate the Choline Transporter (CHT)Screeningdepositor-specified cross reference
488996Summary of probe development for activators of the Choline Transporter (CHT)Summarydepositor-specified cross reference
504833Confirmatory screen for compounds that activate the Choline Transporter (CHT)Screeningdepositor-specified cross reference
602376Dose responses of compounds that activate the Choline Transporter (CHT) - 10 point CRCConfirmatorydepositor-specified cross reference
623908Counter screen assay of the parental HEK293 cells for compounds that activate the Choline Transporter (CHT)Screeningdepositor-specified cross reference
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC_CHT_Act_3H uptake_CRC2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network

Data Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Alicia Ruggiero, Ph.D., Vanderbilt University Medical Center
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R03DA028852-01
Grant Proposal PI: Alicia Ruggiero, Ph.D., Vanderbilt University Medical Center
Assay Implementation: Meng Wu Ph.D., Xiaofang Huang M.S., Zhihong Lin Ph.D., Kaiping Xu M.S., Shunyou Long M.S., and Owen McManus Ph.D.

Description:
In the brain, the chemical acetylcholine (ACh) exerts powerful modulatory control over arousal, motor and cognitive circuits, and has been found to be deficient in Alzheimer's Disease (AD). The current drugs available to positively impact cognitive deficits in Alzheimer's Disease (AD) and other dementias are the cholinesterase inhibitors. These prevent the breakdown of the neurotransmitter acetylcholine (ACh), and thus augment Ach function. Due to the limited utility of the cholinesterase inhibitors, alternative therapies to augment ACh deficits are critical in our aging population.

Another vital protein, the hemicholinium-3 sensitive choline transporter (CHT) is believed to be responsible for the efficient uptake of choline by neurons to allow for ACh synthesis. An assay system for high throughput screening has been developed to identify compounds with high selectivity for CHT. It is anticipated that these compounds may lead to future cholinergic therapies in AD, and multiple other CNS diseases regulated by cholinergic signaling. These compounds may be able to modulate choline uptake and the levels of ACh produced in the neuron by impacting the kinetics of neurotransmitter synthesis. Such reagents would provide useful probes for the role of this transporter in normal and diseased states.

Keywords:
Choline transporter, CHT, Choline, Validation, Confirmatory, Hemicholinium 3, Acetylcholine, HTS assay, 384, Primary, Allosteric Activator, FDSS, Membrane potential, Fluorescence, Kinetic, MPD, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Principle of the assay:
This 3H -choline uptake radioactive assay is considered as the orthogonal assay for CHT. The effect of the CHT activators can be monitored by uptaking of 3H labeled choline by CHT. Compounds that decrease the signal of the 3H choline uptake assay at given choline concentration will be selected for further validation.

Protocol for the CHT project:
1. Cell culture: Cells are routinely cultured in MEM Earles medium, supplemented with Fetal Bovine Serum (FBS), penicillin, streptomycin, glutamine, non-essential amino acids and 250 ug/mL G418.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in MEM full medium without G418.
3. Incubate at 37 degrees C and 5% CO2 for 2 days.
4. Remove medium and wash wells once with 20ul 1x HBSS-20 mM HEPES buffer (pH 7.4) using multidrop. Add 20 uL/well HBSS-HEPES.
5. Drug plates were prepared with 5x concentration in HBSS-HEPES buffer. HC-3 were added into Columns 23-24 as controls.
6. Add 4 uL/well from the drug plates prepared in HBSS-HEPES.
7. Pre-Incubate cells with compounds and/or HC-3 for 15 min in cell incubator at 37 degrees C.
8. Add 6ul of 3H Choline Chloride mix: Final 50 nM total Choline (5X = 250 nM), 10% of hot 3H Choline Chloride in HBSS-HEPES buffer.
9. Incubate for 15 min in cell incubator at 37 degrees C.
10. Wash once with ice-cold HBSS-HEPES buffer (collect wash according to your radiation policy).
11. Add 30ul of liquid scintilant per well and seal with sealing film.
12. Incubate plate with shaking for 1 hour at room temperature.
13. Count on top count using tritium protocol and 384-well setting for 1 min /well.
14. Calculate the percentage of responses of tested compounds from Step 13 by setting buffer as 100%, and HC-3 as 0%.
15. Outcome assignment: If the compound has a fit for the dose response curve, and has a readout of more than 120% at 33.3uM, the compound is considered to be active (Value=2). Otherwise, it is designated as inactive (Value=1).
16. Score assignment: An active test compound is assigned a score of 100. The inactive test compounds are assigned a score of 0.

List of reagents:
1. CHT-expressing HEK293 Cells (CHT LV-AA HEK293 provided by Assay Provider)
2. MEME Earles (Mediatech, Cat #15-010-CV)
3. Fetal Bovine Serum (Gibco, Cat #26140)
4. L-Glutamine (Invitrogen, Cat #25030081)
5. 100x Penicillin-Streptomycin (Mediatech, Cat #30-001-CI)
6. CellStripper (Mediatech, #25-056-Cl)
7. G418 (Invitrogen, Cat #11811-031)
8. Hemicholimium-3 (HC-3) (Sigma, Cat #H108)
9. Choline (Acros Organics, Cat #219770500)
10. Non-essential amino acids (NEAA) (Invitrogen, Cat #11140-050)
11. HEPES (Sigma, Cat #H4034)
12. 10x HBSS (Invitrogen, Cat #14065056)
13. FLIPR Membrane Potential Blue, Bulk (Molecular Devices, Cat #R8123)
14. Triple-layer flask (VWR, Cat #62407-082)
15. White Culture Plate 96 well plates (BD plates) and TopSeal-A 96 well Microplate sealing film (PE, Cat #6005185)
16. Radiolabel: 3H-Choline 1 mCi/mL in EtOH approx 12 uM (Perkin Elmer)
17. Scintillant: Microscint 20 for TopCount (PE, Cat #6013621)
Comment
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust, in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that 3H radioactivity within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary, based upon the actual sample provided by the MLSMR.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1IC50*mean IC50 FloatμM
2Hill constantHill constantFloat
3Nnumber of repeats Integer
433.3uM-Percentage% (33.3μM**)% Percentage treated with the specified concentration Float
533.3 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
611.1uM-Percentage% (11.1μM**)% Percentage treated with the specified concentration Float
711.1 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
83.7uM-Percentage% (3.7μM**)% Percentage treated with the specified concentration Float
93.7 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
101.2uM-Percentage% (1.2μM**)% Percentage treated with the specified concentration Float
111.2 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
120.4uM-Percentage% (0.4μM**)% Percentage treated with the specified concentration Float
130.4 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
140.14uM-Percentage% (0.014μM**)% Percentage treated with the specified concentration Float
150.14 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
160.046uM-Percentage% (0.0046μM**)% Percentage treated with the specified concentration Float
170.046 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
180.015uM-Percentage% (0.0015μM**)% Percentage treated with the specified concentration Float
190.015 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
200.0051uM-Percentage% (0.0051μM**)% Percentage treated with the specified concentration Float
210.0051 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat
220.0017uM-Percentage% (0.0017μM**)% Percentage treated with the specified concentration Float
230.0017 uM-SDStandard Deviation of measure for % Percentage with the specified concentrationFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA028852-01

Data Table (Concise)
Data Table ( Complete ):     View All Data
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