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BioAssay: AID 489005

Inhibitors of T-Type Calcium Channel

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(895)
 
 
Active(703)
 
 
Inactive(192)
 
 
 Tested Substances
 Tested Substances
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Active(703)
 
 
Inactive(192)
 
 
AID: 489005
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (T-Type Calcium Channel: Dose Response Testing)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-11-15
Modify Date: 2011-03-09

Data Table ( Complete ):           Active    All
Target
Sequence: voltage-dependent T-type calcium channel subunit alpha-1H isoform a [Homo sapiens]
Description ..   

Gene:CACNA1H     Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 703
Depositor Specified Assays
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AIDNameTypeComment
449739Inhibitors of Cav3 T-type Calcium Channels: Primary ScreenscreeningPrimary screen at 10uM compound concentration
463087Inhibitors of Cav3 T-type Calcium ChannelssummaryProject summary
504426Mode of action assay-Dose response assay for compounds that inhibit T-type calcium channel subunit Cav3.2 on automated patch clampconfirmatory
493041Inhibitors of T-Type Calcium Channels (SynthLib3)confirmatory
504584Inhibitors of T-Type Calcium Channels (Ancillary Pharm)other
504619Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell CRC)confirmatory
493023Inhibitors of T-Type Calcium Channels (SynthLib2)confirmatory
1053190Confirmed inhibitors of the Cav3 T-type Calcium Channelother
493021Inhibitors of T-Type Calcium Channelsconfirmatory
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
504628Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell)other
504579Inhibitors of T-Type Calcium Channels (rat DRG neuron currents)other
493022Inhibitors of T-Type Calcium Channels (SynthLib1)confirmatory
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).

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.
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 fluorescence in a Cav3.2 expressing cell line. All compounds were tested in single with a 10 point dose-response curve.

High-Throughput Screening
Cells were plated in poly-D-lysine 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. The plates were loaded into the Hamamatsu FDSS 6000 and test compound added to give 10uM final concentration. After 20 minutes, 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 fluorescence 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) versus compound concentration fit to a 4 parameter logistic fit using automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). Compound KK-3-118 and vehicle control wells were included on every plate and used to calculate the Z'.
Comment
A score of 100 was assigned to all active compounds and 0 was assigned for all inactive compounds.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Value1_30uM (30μM**)The 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_ConcUnit.'Float
2Value1_10uM (10μM**)The 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_ConcUnit.'Float
3Value1_3.44uM (3.34μM**)The 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_ConcUnit.'Float
4Value1_1.25uM (1.25μM**)The 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_ConcUnit.'Float
5Value1_313nM (0.313μM**)The 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_ConcUnit.'Float
6Value1_122nM (0.122μM**)The 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_ConcUnit.'Float
7Value1_38.1nM (0.0381μM**)The 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_ConcUnit.'Float
8Value1_15.3nM (0.0153μM**)The 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_ConcUnit.'Float
9Value1_3.81nM (0.0381μM**)The 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_ConcUnit.'Float
10AB_mean1Mean of the vehicle control (Assay Buffer + 0.1% DMSO) on a per-plate basisFloat
11AB_stddev1Standard deviation of the vehicle control (Assay Buffer + 0.1% DMSO) on a per-plate basisFloat
12KK3118_mean1Mean of the KK3118 control on a per-plate basisFloat
13KK3118_stddev1Standard deviation of the KK3118 control on a per-plate basisFloat
14zprime_AB_KK3118_1Z factor calculation of vehicle and KK3118 control populations. (http://en.wikipedia.org/wiki/Z-factor)Float
15FitConverged_1R statistics reports TRUE when algorithm converges on an equation with a fit value; Otherwise R statistics reports FALSEBoolean
16FitCategory_1Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit"String
17bottom_1If the fit converged, then this is the calculated value for the bottom parameterFloat
18bottom_stderr_1Standard error for the calculated bottom valueFloat
19bottom_pr_1p value for the calculated bottom valueFloat
20top_1If the fit converged, then this is the calculated value for the top parameterFloat
21top_stderr_1Standard error for the calculated top valueFloat
22top_pr_1p value for the calculated top valueFloat
23logEC50_1If the fit converged, then this is the calculated value for theLogEC50 parameterFloat
24logEC50_stderr_1Standard error for the calculated EC50 valueFloat
25logEC50_pr_1p value for the calculated logEC50Float
26slope_1If the fit converged, then this is the calculated value for the slope parameterFloat
27slope_stderr_1Standard error for the calculated slope valueFloat
28slope_pr_1p value for the calculated slopeFloat
29EC50_1*EC50 value in micromolarFloatμM
30EC50_stderr_1Standard error for the calculated EC50 valueFloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NS050771-01

Data Table (Concise)
Classification
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