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BioAssay: AID 651756

Dose responses of compounds that inhibit 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(29)
 
 
Active(1)
 
 
Inactive(28)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Active(1)
 
 
Inactive(28)
 
 
AID: 651756
Data Source: Johns Hopkins Ion Channel Center (JHICC_CHT_Inh_3H uptake_CRC2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-11-06

Data Table ( Complete ):           View Active Data    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..
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
488975Primary cell-based screen for identification of compounds that inhibit the Choline Transporter (CHT)Screening depositor-specified cross reference
488997Summary of probe development for inhibitors of the Choline Transporter (CHT)Summary depositor-specified cross reference
493221Confirmatory screen for compounds that inhibit the Choline Transporter (CHT)Screening depositor-specified cross reference
493222Counter screen assay of the parental HEK293 cells for compounds that inhibit the Choline Transporter (CHT)Screening depositor-specified cross reference
504840Dose responses of compounds that inhibit the Choline Transporter (CHT) - 5 point CRCConfirmatory depositor-specified cross reference
588401Dose responses of compounds that inhibit the Choline Transporter (CHT) - 10 point CRCConfirmatory depositor-specified cross reference
602208JHICC_CHT_Inh_3H uptake_CRCConfirmatory depositor-specified cross reference
652177Discovery and structure-activity relationship of a novel choline transporter inhibitorConfirmatory same project related to Summary assay
652215Discovery and structure-activity relationship of a novel choline transporter inhibitor, Selectivity StudiesConfirmatory1 same project related to Summary assay
652262Discovery and structure-activity relationship of a novel choline transporter inhibitor: ML352Summary same project related to Summary assay
Description:
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.

Principle of the assay:
This 3H -choline uptake radioactive assay is considered as the orthogonal assay for CHT. The effect of the CHT inhibitors 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.

Keywords:
Choline transporter, CHT, Choline, Validation, Confirmatory, Hemicholinium 3, Acetylcholine, HTS assay, 384, Primary, Inhibitor, Antagonist, FDSS, Membrane potential, Fluorescence, Kinetic, MPD, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay overview:
The objective is to validate compounds generated from the primary screen assay (PubChem AID 488975) and their consequential re-confirmation and counter screens for the CHT specific inhibitor hits. It employs the orthogonal assay of 3H choline uptake assay to validate the CHT specific effects in a concentration-dependent mode.
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 scintillant 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 less than 40% 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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*mean IC50 FloatμM
2Hill constantHill constantFloat
3Nnumber of repeats Integer
4Av-33.3uM (33.3μM**)% Percentage treated with the specified concentration Float
5Av-11.1uM (11.1μM**)% Percentage treated with the specified concentration Float
6Av-3.7uM (3.7μM**)% Percentage treated with the specified concentration Float
7Av-1.2uM (1.2μM**)% Percentage treated with the specified concentration Float
8Av-0.4uM (0.4μM**)% Percentage treated with the specified concentration Float
9Av-0.14uM (0.14μM**)% Percentage treated with the specified concentration Float
10Av-0.046uM (0.046μM**)% Percentage treated with the specified concentration Float
11Av-0.015uM (0.015μM**)% Percentage treated with the specified concentration Float
12Av-0.0051uM (0.0051μM**)% Percentage treated with the specified concentration Float
13Av-0.0017uM (0.0017μM**)% Percentage treated with the specified concentration Float

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

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