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BioAssay: AID 488975

Primary cell-based screen for identification of compounds that inhibit the Choline Transporter (CHT)

Assay Implementation: Zhihong Lin Ph. D., Xiaofang Huang M.S., Shunyou Long M.S., David Meyers Ph.D., Owen McManus Ph.D., and Meng Wu Ph.D. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 488975
Data Source: Johns Hopkins Ion Channel Center (JHICC_ CHT_Inh_Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-11-01
Modify Date: 2010-11-17

Data Table ( Complete ):           Active    All
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 Compounds: 2634
Depositor Specified Assays
602208JHICC_CHT_Inh_3H uptake_CRCconfirmatory
493222Counter screen assay of the parental HEK293 cells for compounds that inhibit the Choline Transporter (CHT)screening
488997Summary of probe development for inhibitors of the Choline Transporter (CHT)summary
504840Dose responses of compounds that inhibit the Choline Transporter (CHT) - 5 point CRCconfirmatory
651756Dose responses of compounds that inhibit the Choline Transporter (CHT) in a 3H-choline uptake radioactive assayconfirmatory
493221Confirmatory screen for compounds that inhibit the Choline Transporter (CHT)screening
1053196Confirmed inhibitors of the Choline Transporter (CHT)other
588401Dose responses of compounds that inhibit the Choline Transporter (CHT) - 10 point CRCconfirmatory
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: Zhihong Lin Ph. D., Xiaofang Huang M.S., Shunyou Long M.S., David Meyers Ph.D., Owen McManus Ph.D., and Meng Wu Ph.D.
HTS execution: Xiaofang Huang M.S., Zhihong Lin Ph. D., Shunyou Long M.S., and Meng Wu 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 HTS assay is a choline-induced membrane potential assay measuring choline coupled sodium flow through CHT. The kinetic relationship between sodium and choline in the transporter is not well established, but dephosphorylation of the transporter is associated with a decrease in the sodium current and a decrease in the km of transport. Allosteric modulation of CHT may mimic this finding and induce a higher efficiency kinetic state of CHT. Compounds that decrease the signal of the choline-induced membrane potential assay at this choline concentration will be selected to retest for choline uptake.
The objective of the current screen is to identify compounds that inhibit the choline induced membrane depolarization of cells by CHT using a HEK293 cell line which stably expresses choline transporter (CHT). Compounds selected as CHT inhibitors will later be counter-screened for specificity.
Choline transporter, CHT, Choline, 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.
1) Ferguson SM, Blakely RD. The choline transporter resurfaces: new roles for synaptic vesicles? Mol Interv. 2004 Feb;4(1):22-37. Review. PubMed PMID: 14993474.
2) Iwamoto H, Blakely RD, De Felice LJ. Na+, Cl-, and pH dependence of the human choline transporter (hCHT) in Xenopus oocytes: the proton inactivation hypothesis of hCHT in synaptic vesicles. J Neurosci. 2006 Sep 27;26(39):9851-9. PubMed PMID: 17005849.
3) Le Novere N, Corringer PJ, Changeux JP. The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J Neurobiol. 2002 Dec;53(4):447-56. Review. PubMed PMID: 12436412.
4) Laviolette SR, van der Kooy D. The neurobiology of nicotine addiction: bridging the gap from molecules to behaviour. Nat Rev Neurosci. 2004 Jan;5(1):55-65. Review. PubMed PMID: 14708004.
5) Misgeld T, Burgess RW, Lewis RM, Cunningham JM, Lichtman JW, Sanes JR. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron. 2002 Nov 14;36(4):635-48. PubMed PMID: 12441053.
6) Ohno K, Tsujino A, Brengman JM, Harper CM, Bajzer Z, Udd B, Beyring R, Robb S,Kirkham FJ, Engel AG. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):2017-22. PubMed PMID: 11172068; PubMed Central PMCID: PMC29374.
7) Winkler J, Thal LJ, Gage FH, Fisher LJ. Cholinergic strategies for Alzheimer's disease. J Mol Med. 1998 Jul;76(8):555-67. Review. PubMed PMID: 9694432.
8) Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 4(2),67-73 (1999) PMID: 10838414.
9) Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 24(2), 167-175 (2006). PMID: 16465162.
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 500,000 cells/ml re-suspended in MEM full medium without G418.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of 1x membrane potential dye solution to cells
5. Incubate 40 minutes at 37 C in the dark
6. Prepare 7.5X compound plates with controls on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and EC90 of Choline. Drug plates are incubated in 37 C for 45 mins.
7. Cell plates and drug plates are then mounted to FDSS, pre-warmed to 37 C for another 5 min.
8. Measure fluorescence for 20 seconds at 1Hz to establish baseline
9. Add 4 ul of 7.5x compound stock into the cell plates on FDSS and monitor for another 110s.
10. After an interim step of addition of EC20, Add 6 ul/well of EC90 of choline pre-heated to 37 C within FDSS at 240s and monitor for another 110s.
11. Calculate ratio readout as F(max-min)/F0 and integrated ratios (intRatio) at Step 10.
12. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [8]
13. Calculate B scores [9] for test compounds using integrated ratios (intRatio) calculated in Step 11
14. Outcome assignment: If the B score of the integrated ratio of the test compound is less than the mean minus 3 times the standard deviation (SD) of the B scores of ratios of all library compounds (BScore_intRatio <-3*SD), the ratio of initial fluorescence intensity is within 3 times the standard deviation plus the mean of the ratios of the complete library the compound is designated in the Outcome as active (value=2) as an inhibitor of the CHT target. Otherwise, it is designated as inactive (value=1).
15. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int((Lg(Abs([BScore_intRatio]))-0.66)*154.2). 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 (Sigma, H108)
9. Choline(Acros Organics, 219770500)
10. NEAA (non-essential amino acids, Invitrogen Cat#11140-050)
11. HEPES (Sigma, Cat#H4034)
12. 10XHBSS (#Invitrogen Cat#14065056)
13. FLIPR Membrane Potential Blue, Bulk (Molecular Devices, Cat #R8123)
14. Triple-layer flask (VWR, Cat #62407-082)
15. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
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 quench or emit light or fluorescence 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
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1BScore_intRatio (10μM**)B score of the integrated ratio of the test compound calculated based on the integrated ratio of the test compound of the primary screen at a compound concentration of 10 uMFloat

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

Data Table (Concise)