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
BioActive Compound: 1
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.
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.
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
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)
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)