Bookmark and Share
BioAssay: AID 493023

Inhibitors of T-Type Calcium Channels (SynthLib2)

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 ..
_
   
 Tested Compounds
 Tested Compounds
All(29)
 
 
Active(25)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Active(25)
 
 
Inactive(4)
 
 
AID: 493023
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (Cav3 Inhibitor SynthLib2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2011-01-11
Modify Date: 2011-03-09

Data Table ( Complete ):           View Active Data    View All Data
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: 25
Related Experiments
Show more
AIDNameTypeComment
449739Inhibitors of Cav3 T-type Calcium Channels: Primary ScreenScreeningdepositor-specified cross reference
463087Inhibitors of Cav3 T-type Calcium ChannelsSummarydepositor-specified cross reference
489005Inhibitors of T-Type Calcium ChannelConfirmatorydepositor-specified cross reference
493041Inhibitors of T-Type Calcium Channels (SynthLib3)Confirmatorydepositor-specified cross reference
504579Inhibitors of T-Type Calcium Channels (rat DRG neuron currents)Otherdepositor-specified cross reference
504584Inhibitors of T-Type Calcium Channels (Ancillary Pharm)Otherdepositor-specified cross reference
504619Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell CRC)Confirmatorydepositor-specified cross reference
504628Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell)Otherdepositor-specified cross reference
493021Inhibitors of T-Type Calcium ChannelsConfirmatorysame project related to Summary assay
493022Inhibitors of T-Type Calcium Channels (SynthLib1)Confirmatorysame project related to Summary assay
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 clampConfirmatorysame project related to Summary assay
504426Mode of action assay-Dose response assay for compounds that inhibit T-type calcium channel subunit Cav3.2 on automated patch clampConfirmatorysame project related to Summary assay
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.
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.
Protocol
The purpose of this assay was to follow-up on the confirmatory and dose-response testing of 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. This confirmatory assay tested 29 compounds synthesized at Vanderbilt and tested in 11-point 3-fold CRC experiments in triplicate with both the transfected (HH8) and untransfected cell lines (native HEK293). The native HEK cells contain a Ca2+ conductance that may be L-type and this was used as a first-tier selectivity screen.

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 log linear final concentrations ranging from 0.00316uM to 316uM. 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) 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). Compound KK-3-118 and vehicle control wells were included on every plate and used to calculate the Z (6).
Comment
The PUBCHEM_ACTIVITY_SCORE value was assigned a value of 0 for 'inactive', 100 for 'active' with an EC50 less than 10uM and 50 for 'active' with an EC50 equal to or greater than 10uM.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50*Average effective concentration 50 (EC50) value for modulation of Cav3 T-Type Calcium response (micromolar)FloatμM
2EC50_UPPER_CLIf the fit converged, then this is the calculated value for the EC50 upper confidence limit (micromolar)FloatμM
3EC50_LOWER_CLIf the fit converged, then this is the calculated value for the EC50 lower confidence limit (micromolar)FloatμM
4% EMAX TOPAverage percentage of maximum effect in kinetic time window from 12-40 seconds at highest concentrationFloat
5% EMAX TOP_UPPER_CLUpper confidence limit for the top kinetic time windowFloat
6% EMAX TOP_LOWER_CLLower confidence limit for the top kinetic time windowFloat
7% EMAX BOTTOMAverage percentage of maximum effect in kinetic time window from 12-40 seconds for the lowest concentrationFloat
8% EMAX BOTTOM_UPPER_CLUpper confidence limit for the bottom kinetic time windowFloat
9% EMAX BOTTOM_LOWER_CLLower confidence limit for the bottom kinetic time windowFloat
10SLOPEIf the fit converged, then this is the calculated value for the slope parameter for the effect in kinetic time window from 12-40 secondsFloat
11SLOPE_UPPER_CLUpper confidence limit for the calculated value of the slope parameterFloat
12SLOPE_LOWER_CLLower confidence limit for the calculated value of the slope parameterFloat

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
PageFrom: