Inhibitors of T-Type Calcium Channels (SynthLib3)
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 ..
BioActive Compounds: 24
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.
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 32 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.
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).
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.
* Activity Concentration.
Data Table (Concise)