High Throughput Screening Flux Assays

Aurora Biomed’s screening technology

High Throughput Screening (HTS), is very important in drug discovery. Aurora’s ICR could eliminate the bottlenecks in which HTS requires high sensitivity and small sample amount testing as well as quick results. Flux assays are functional, robust, and widely used by various pharmaceutical companies. ICR measurements of the ions are very sensitive with an extremely low percentage of CVs among the replicates. Assay volumes are less than 100 μL, minimizing the amount of compound used. The screens are carried out in the presence of 1% DMSO, minimizing compound solubility issues. Z’ values of the screens are always higher than 0.8 indicating high robustness and low variability. With a large window of detection presented by the screens, test compounds emerge as either clear blockers/non-blockers or activators/non-activators. Well-established compounds of known activity against specific ion channel targets are used as positive controls. Drug potencies determined by flux screens are highly correlated with those of electrophysiology. Calculated drug rank orders between the two methods are identical. Flux assays involve about1.38×105 cells per well and thus provide multicellular response compared to other techniques measuring only a few ion channels.

For ion channel screening, Aurora Biomed employs flux assay technology in which non-radioactive ions are used as tracers for specific ion channels. Tracer ions are loaded either into the cells or into the extracellular solution.The cells are then bathed in a solution containing the compound (s) of interest to determine its effect on channel activity. The amount of tracer ion present in the intra-and extra cellular portions of the cells is measured using Aurora Biomed’s sensitive, robust, and high-throughput proprietary atomic absorbance-based Ion Channel Reader (ICR) series. The following tracers are used for different ion channels and transporters:

Potassium ion (K+) channels: Rubidium(Rb+)

Sodiumion (Na+) channels: Lithium(Li+)

Chloride ion (Cl-) channels: A known concentration of silver (Ag+)is used to precipitate Cl- from the samples as AgCl, and then freeAg+ is measured

Calcium ion (Ca2+) channels: Ca2+ or Strontium (Sr2+)

Na+,K+-ATPase: Rb+ for K+ ion

K+,Cl-co-transporter: Ag+ precipitation for Cl- ion