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BioAssay: AID 652236

Specificity screen assay against voltage-dependent chloride channel 1 (ClC-1) for identification of compounds that inhibit/block calcium-activated chloride channels (TMEM16A)

Assay Implementation: Zhihong Lin Ph.D., Kaiping Xu M.S., Alison Neal, Owen McManus Ph.D., Meng Wu Ph.D. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 652236
Data Source: Johns Hopkins Ion Channel Center (JHICC_CaCC_Inh_counterClC-1)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-29

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
588551Summary of assays for identification of compounds that inhibit/block calcium-activated chloride channels (TMEM16A)summary
652189Re-confirmation assay for identification of compounds that inhibit/block calcium-activated chloride channels (TMEM16A)screening
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: Min Li, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA031670-01
Grant Proposal PI: Min Li, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Zhihong Lin Ph.D., Kaiping Xu M.S., Alison Neal, Owen McManus Ph.D., Meng Wu Ph.D.

Name: Specificity screen assay against voltage-dependent chloride channel 1 (ClC-1) for identification of compounds that inhibit/block calcium-activated chloride channels (TMEM16A)


Recent discovery of TMEM16A which encodes a calcium-activated chloride channel (CaCC) has provided a new pathway to identify novel small molecule probes (1-9) for this important target. CaCC activity may be linked to several major diseases, including cystic fibrosis, asthma, chronic bronchitis, and hypertension. Its roles are implicated in numerous physiological processes including cardiac and neuronal excitation, sensory transduction, trans-epithelial secretion, smooth muscle contraction, and fertilization. Discovery of CaCC-specific chemical probes can provide not only useful tool molecules for pharmacological interrogation of the CaCC function and diversity, but may also provide compounds as starting points for therapeutic efforts to treat various diseases resulting from dysfunction in chloride transport.

Our objective is to find potent and specific small molecule chemical probes that modulate TMEM16A-based calcium-activated chloride channel, by performing a primary screen of the MLSMR library of 300,000 compounds to identify small molecules and structural scaffolds that modulate this calcium-activated chloride channel; and examining and identifying hits with preferential effects on TMEM16A versus other chloride channels after validation, confirmatory and counter-screens.

Despite the discovery of the molecular identity of CaCC, few CaCC/TMEM16A inhibitors/blockers are available. Only recently have CaCC (TMEM16A) modulators been reported (10-12), including 1) niflumic acid (non-specific, 17 uM) (10); 2) 9-anthracene carboxylic acid (Non-specific; 10.3uM); 3) 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, Non-specific, 22-68 uM); ;4) DIDS (Non-specific, 48/10.7 uM) (10); 5) Flufenamic acid (N-(3-Trifluoromethylphenyl)anthranilic acid, Non-specific, 28 uM); 6) Digallic acid (TMEM16A; 3.6 uM) (11-12); 7) Tannic acid (TMEM16A, 6.1uM, PMID: 20581223; 21084298); and 8) T16Ainh-A01 (TMEM16A, 1.1uM) (11-12). Most are non-specific inhibitors and were discovered without the information of the molecular identity of the CaCCs, except the last three in the table, which were reported after initiation of the current project. Still, potent and specific CaCC inhibitors are needed
ClC-1 is a voltage-dependent anion channel; selectivity against this channel is evaluated for TMEM16A inhibitor compounds to aid in defining the pharmacological profile of select compounds identified in a high throughput screen of TMEM16A
Principle of the assay:

The iodide ion, which is permeable through the ClC-1 channels, serves as a surrogate for Cl-flux. The iodide flux is detected by YFP iodide Biosensors (YFP mutant at H148Q/ I152L)(13). Any compounds which inhibit/block the ClC1 channels will result in reduced quenching and higher remaining fluorescence as detected by imaging plate reader FDSS (14).

Assay overview:

The purpose of this assay is to evaluate the selectivity of compounds that inhibit/block the calcium-activated chloride channel (TMEM16A) identified in a primary screening (AID: 588511). This assay employs a CHO cell line that stably expresses ClC-1 channels with YFP Iodide Biosensors. The cells were treated with test compounds, followed by measurement of intracellular iodide, as monitored by an iodide-sensitive fluorescent protein YFP mutant at H148Q/ I152L. ClC-1-YFP-CHO cells were plated into 384-well plates. On the following day, cells were incubated with assay buffer (replacing NaCl with equimolar sodium gluconate) after removing media. Compounds were added to the assay buffer. Cells were incubated with 10 uM compound for 20 minutes, and fluorescence changes measured upon the addition of trigger solution (34 mM iodide final in the well). The fluorescence of YFP was measured on a Hamamatsu FDSS 6000 kinetic imaging plate reader. Compound effect was evaluated by the calculated YFP fluorescence signal (excitation 514 nm, emission 527 nm), normalized with negative (buffer) and positive (iodide) controls, from the duplicates. If the compound causes an increase in fluorescent signal more than 5 times the standard deviation of the positive (iodide) controls, the compound is then considered to be active as an inhibitor/blocker of the ClC-1 channels.


Specificity assay, ClC-1, CaCC, TMEM16A, ANO-1, anoctamine-1, chloride channel, calcium, calcium activated chloride channel, HTS assay, counter, CHO, 384, primary, antagonist, inhibitor, FDSS, fluorescence, Kinetic, YFP, quenching, YFP biosensor, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN


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Protocol for the TMEM16A counter ClC-1 project:

1. Cell culture: ClC-1-YFP (H148Q/I152L)-CHO cells are routinely cultured in DMEM with 10 % Cosmic Calf Serum, 100 units penicillin and 100 mug/ml streptomycin, and 400 ug/ml for both G418 and Hygromycin.
2. Cell plating: Add 50 ul/well of 8,000/well re-suspended in media on BD PDK-pre-coated 384-well plate.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 25 ul /well of HBSS-HEPES (replacing NaCl (138mM) with equimolar sodium gluconate) (pH 7.4) to cells.
5. Prepare 7.5x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0).
6. Add 5 microl drugs/compounds and controls to cell plates by Cybi and incubate at RT for 20 min.
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Prepare 4x trigger solution containing 138mM iodide-HBSS solution (pH 7.4)
9. Measure fluorescence for 10 seconds at 1Hz to establish baseline
10. Add 5 ul of trigger solution into the cell plates on FDSS and continue measuring fluorescence for 50 seconds
11. Calculate ratio readout as F(max-min)/F0
12. Calculate the percentage of tested compounds with the following formula: Percentage (%)=100* (Ratio(cmpd)- AvgRatio(Iodide))/(AvgRatio(Buffer)-AvgRatio(Iodide)); Percentage(%): percentage change of compound readout over those of negative controls (Buffer), Ratio(cmpd): Ratio of the test compound. AvgRatio(Buffer): Ratio average of the negative controls with Buffer, Ratio(iodide): Ratio of iodide.
13. Outcome assignment: If the signal in compound (the average of the duplicates of the Percentage (%, RatioPercentage) as readout) increases more than those of positive controls (iodide) plus 5SD of positive controls (iodide), the compound is considered to be active (Value=2). Otherwise, it is designated as inactive (Value=1).
14. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation, RatioPercentage, as in the result definition. The inactive test compounds are assigned a score of 0.

List of reagents:

(1)#Cells (ClC-1-YFP (H148Q/I152L)-CHO cells) from Dr. Merritt Maduke, Stanford University
(2)#PBS: pH 7.4 (Gibco, Cat #10010)
(3)#Medium: DMEM (Cellgro, Cat #10-013-CV)
(4)#Cosmic Calf Serum (Fisher, Cat #SH3008703)
(5)#200 mM L-Glutamine (Gibco, Cat #25030)
(6)#Penicillin-Streptomycin (Mediatech, Cat #30-001-CI)
(7)#Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat #25300)
(8)#G418 (100X): 50mg/mL (filtered) (Gibco, Cat #11811-031)
(9)#HEPES (Sigma, Cat #H4034)
(10)#BD Biocoat 384-well plates (BD, Cat #356663 and Lot #7346273)
(11)#Hygromycin (Mediatech, Cat #30-240-CR)
(12)#Sodium D- gluconate (Sigma, Cat #186333)
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
1RatioPercent_ClC1 (10μM**)Average ratio-percentage of the duplicate of each test compound readout in ClC-1-YFP (H148Q/I152L)-CHO cells at a concentration of 10 uM.Float

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA031670-01

Data Table (Concise)