ANG-2 screening poster presented at SLAS 2012
A group from Merck's Department of Exploratory Sciences and Screening presented a poster evaluating Asante Natrium Green-2 in a 1536 well voltage -gated sodium channel Assay.
Evaluation of the Sodium Sensing Dye Asante Natrium Green 2 in a Voltage-gated Sodium Channel Assay in 1536-well Format
Gregory T. O'Donnell, Kelli Solly, Carissa Quinn, Brian Squadroni, Eric Johnson, Jeffrey Hermes, and Michael Finley.
Department of Exploratory Sciences and Screening Merck Research Laboratories 140-154 Wissahickon Ave, North Wales, PA 19454, USA
High-throughput screening (HTS) of voltage-gated sodium channels has required the use of indirect measurements of channel activity partly due to the lack of a robust sodium sensitive dye. One popular screening method utilizes a dye that responds to changes in the membrane potential that results from sodium channel opening. Another approach, atomic absorption spectroscopy, makes use of surrogate ion transport through the voltage-gated ion channel of interest, substituting lithium for sodium. While these assays have been shown to be amenable to high-throughput screening, the direct measurement of sodium flux in an HTS-friendly read-out would be beneficial in lead identification efforts. Herein we describe a 1536-well FLIPR screening assay for antagonists to a voltage-gated sodium channel expressed in human embryonic kidney cells (HEK293) using a sodium sensing dye, Asante Natrium Green 2 (ANG-2, Teflabs). Addition of 60 μM of the site 2 agonist veratridine induced a signal-to-background ratio (S/B) of 1.3-1.6 fold (control wells versus wells treated with an IC100 of tetracaine). The assay was benchmarked against three known voltage-gated sodium channel blockers, tetracaine, flecainide, and mexilitene and exhibited acceptable sensitivity with IC50s of 2.7, 15.3, and 29 uM, respectively. The assay was then used to screen a library of 27,978 small molecules to assess the performance under screening conditions and compared to results using the same compound set screened with a membrane potential dye (Blue component A, Molecular Devices). The sodium dye assay gave robust statistics with Z' values averaging 0.71 and S/B averaging 1.58-fold over a 48 plate screening run. While the sodium dye showed good internal consistency (R2 = 0.80 when plotting duplicate data for each compound against each other), the membrane potential and sodium dye assays showed a weaker correlation with an R2 = 0.46. Using a 40% activity cut-off, 2237 compounds showed overlapping activity in both assays. However, each assay identified a large number of unique hits with 886 sodium dye only hits and 1186 membrane potential dye only hits. Data on the same compounds obtained from the IonWorks Quattro (Molecular Devices) system revealed that the sodium dye identified 39.3 % of all the electrophysiology positives, while the membrane potential dye identified 39.4 %. However, the sodium dye detected 129 electrophysiology actives that the membrane potential dye missed, while the membrane potential dye identified 130 compounds missed in the sodium dye assay. While neither assay appears capable of identifying all the potential inhibitors that may be uncovered using an electrophysiology read-out, both dyes provided robust read-outs in 1536-well format and would allow for the screening of large compound libraries. Additionally, the use of both membrane potential and sodium dyes in an HTS strategy may prove beneficial as both identify unique hits.
A total of 27,978 compounds were screened in the three different assays. The Venn diagram shows the hit distribution and overlap of those hits. Although the overall hit rates at 40% inhibition or greater for the sodium and membrane potential dye assays were similar (13.5% and 14.6%, respectively), each assay identified large numbers of assay specific hits. Combining the sodium and membrane potential assay hits, only 47% of all the electrophysiology hits were identified in the FLIPR screens.