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BioAssay: AID 449739

Inhibitors of Cav3 T-type Calcium Channels: Primary Screen

T-type Ca2+ channels are also called low voltage-activated channels because they open at voltages near the resting membrane potential of most cells. In many types of neurons, Ca2+ influx through T-type channels triggers low-threshold spikes, which in turn trigger a burst of action potentials mediated by Na+ channels (1). Burst firing is thought to play an important role in the synchronized more ..
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 Tested Compounds
 Tested Compounds
All(104728)
 
 
Active(4230)
 
 
Inactive(100498)
 
 
 Tested Substances
 Tested Substances
All(104742)
 
 
Active(4230)
 
 
Inactive(100512)
 
 
AID: 449739
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (T Type Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-07-08
Modify Date: 2011-03-09

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4230
Depositor Specified Assays
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AIDNameTypeComment
463087Inhibitors of Cav3 T-type Calcium Channelssummary
489005Inhibitors of T-Type Calcium Channelconfirmatory
493021Inhibitors of T-Type Calcium Channelsconfirmatory
493022Inhibitors of T-Type Calcium Channels (SynthLib1)confirmatory
493023Inhibitors of T-Type Calcium Channels (SynthLib2)confirmatory
493041Inhibitors of T-Type Calcium Channels (SynthLib3)confirmatory
504425Mode of action assay-Specificity dose response assay for the identification of selective inhibitors of T-type calcium subunit Cav3.2 in the Cav3.3 expressing cell line on automated patch clampconfirmatory
504426Mode of action assay-Dose response assay for compounds that inhibit T-type calcium channel subunit Cav3.2 on automated patch clampconfirmatory
504579Inhibitors of T-Type Calcium Channels (rat DRG neuron currents)other
504584Inhibitors of T-Type Calcium Channels (Ancillary Pharm)other
504619Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell CRC)confirmatory
504628Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell)other
Description:
Assay Provider: Xinmin Xie
Assay Provider Affiliation: Bioscience Division, SRI International, Menlo Park, CA
Grant Title: HTS Assay for Cav3 T-Type Channels using FLIPR
Grant Number: NS050771-01

T-type Ca2+ channels are also called low voltage-activated channels because they open at voltages near the resting membrane potential of most cells. In many types of neurons, Ca2+ influx through T-type channels triggers low-threshold spikes, which in turn trigger a burst of action potentials mediated by Na+ channels (1). Burst firing is thought to play an important role in the synchronized activity of the thalamus observed in absence epilepsy, and also in a wider range of neurological disorders characterized by thalamocortical dysrhythmia (2). Prominent T-currents are also observed in dorsal root ganglion neurons, with subsets of nociceptors expressing more T-current than high voltage-activated Ca2+ currents (3). Considerable evidence supports the notion that a T-channel antagonist would be a useful drug for the treatment of pain and epilepsy (4).
Protocol
The purpose of this assay was to test the small molecule library provided by the Molecular Libraries Small Molecule Repository (MLSMR, distributed by DPI-Biofocus) for the ability to modulate calcium fluoresence in a Cav3.2 expressing cell line. All compounds were tested in single at 10uM final concentration.

High-Throughput Screening
Cells were plated in poly-D-lysine coated, clear-bottomed, black-walled 384-well plates at 20,000/well and cultured overnight at 37 degrees C in the presence of 5% CO2. Cells were washed and loaded with Fluo-4 dye in assay buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM glucose, 10 mM HEPES pH 7.3). The plates were loaded into the Hamamatsu FDSS 6000 and test compound was added to give 10uM final concentration. After 20 minute, the cells were stimulated with 5X stimulation buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 50 mM CaCl2, 5 mM glucose, 10 mM HEPES pH 7.3) and fluoresence measurements recorded.

Data Analysis and Statistics
The kinetic fluorescence values (F) from each well were divided by the initial frame of the read (F0) to give the static ratio (F/F0) which corrects for variability in cell number and dye loading. The maximum of the static ratio from 12 to 40 seconds was calculated and the value (Value) compared with the average distribution of the remaining compound wells assumed to Gaussian. Hits were picked using a combination of Z score and B score (5) with 99.7% confidence intervals in an automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). Compounds with Z or B score outside of the confidence interval were denoted "Active" and assigned a score of "100". Compounds with Z or B score within the confidence interval were assigned as "Inactive" and given a score of "0". Compound KK-3-118 and vehicle control (assay buffer + 0.1% DMSO) wells were included on every plate and used to calculate the Z' (6).

1. Perez-Reyes, E: Molecular physiology of low-voltage-activated T-type calcium channels. Physiol. Rev. 2003; 83: 117-161.
2. Llinas, R R, Ribary, U, Jeanmonod, D, Kronberg, E, and Mitra, P P: Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoencephalography. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 15222-15227.
3. Nelson, M T, Joksovic, P M, Perez-Reyes, E, and Todorovic, S M: The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors. J. Neurosci. 2005; 25: 8766-8775.
4. Nelson, M, Todorovic, S, and Perez-Reyes, E: The role of T-type calcium channels in epilepsy and pain. Curr Pharm Des 2006; 12: 2189-2197.
5. Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R.Statistical practice in high-throughput screening data analysis.Nat Biotechnol. 2006 Feb;24(2):167-75.
6. Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.J Biomol Screen. 1999;4(2):67-73.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1VUIDVanderbilt internal identifierString
2ValueThe raw fluorescence intensities were divided by the initial fluorescence, and the maximum from 12 to 40 seconds after stimulus addition was calculated and labeled 'Value.'Float
3BscoreBscore was calculated using 'Value' normalized for row and column effects on a per-plate basis.Float
4Value_meanMean 'Value' on a per-plate basisFloat
5Value_stddevStandard deviation of 'Value' on a per-plate basisFloat
6BScore_meanMean of 'Bscore' on a per-plate basisFloat
7BScore_stddevStandard deviation of 'Bscore' on a per-plate basisFloat
8KK3118_meanMean of the KK3118 control on a per-plate basisFloat
9KK3118_stddevStandard deviation of the KK3118 control on a per-plate basisFloat
10AB_meanMean of the vehicle control (Assay Buffer + 0.1% DMSO) on a per-plate basisFloat
11AB_stddevStandard deviation of the vehicle control (Assay Buffer + 0.1% DMSO) on a per-plate basisFloat
12zprimeZ factor calculation of vehicle and KK3118 control populations. (http://en.wikipedia.org/wiki/Z-factor)Float
Additional Information
Grant Number: NS050771-01

Data Table (Concise)
Classification
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