Applications
Cell-Based Applications >> Ligand-Gated Ion Channels
BIND® technology enables cell-based detection of all GPCR subtype activation (Gi, Gq, Gs, G12/13-coupled) using a single assay. BIND is highly sensitive and can be used with both recombinant and endogenous GPCRs, requiring as few as 1000 cells per well in 1536-well format. BIND assays can identify agonist, antagonist, partial agonist and inverse agonists. The use of a single technology for GPCR applications streamlines assay development and screening, while the information-rich nature of BIND kinetic data increases the power and efficiency of compound profiling.

Ion channel regulation has implications in the treatment of a variety of disorders and thus has become a target of interest in many drug discovery efforts. Current ion channel assays are limited in their use in screening and profiling programs. BIND technology provides a novel label-free approach to studying ion channel activity with the higher throughput capacity and specificity required for efficient screening and profiling campaigns.

Application Features
  • Label-free Assay with Simplified Assay Development
  • Live Cell Assay with Kinetic Measurement
  • 96-, 384- and 1536-well Formats
  • Single Technology Unifies Assay Development, Screening and Profiling
Small Molecule Inhibitor Identification

hERG Ion Channel Inhibtors

hERG Ion Channel Inhibtors Agonists and antagonists of surface receptors including ligand-gated ion channels, can be identified, potencies assessed and even scrutinized for the mechanisms of action using BIND assays. Figure 1 depicts the results of a small molecule inhibitor identification campaign for the hERG receptor. Dose-dependent responses were detected upon hERG stimulation. These response were significantly decrease upon addition of a K+ channel inhibitor.
Figure 1 - Cells overexpressing hERG were incubated with various concentrations of a known activator in the presence or absence of a small molecule K+ channel inhibitor. A. Cell responses were measured over time with peak response observed 10 minutes post activation. B. Summary of responses at 10 minutes
Agonist/Antagonist Characterization

Capsaicin Dose-Dependent Channel Activation

TRPV1 is a nonselective cation channel (conducts Na+, Ca2+and K+) activated by capsaicin, low pH and high temperature. Modulation of TRPV1 function provides a novel approach to treatment of disorders such as chronic pain and inflammation.

The BIND TRPV1 assay is highly sensitive, allowing detection of the weak response induced by the putative natural activator anandamide and NADA (data not shown), in addition to the robust, sustained response stimulated by capsaicin(Figure 2A). Capsaicin responses were dependent on Ca2+ influx and involved protein kinases and microtubule rearrangement (Figure 2B).
Figure 2 - A. 2x104 CHO cells stably transfected with human TRPV1 per well into 384-well BIND plates pre-coated with collagen. For kinetic measurements, compounds were directly added to the cells and the PWV shift measured every second. B. TRPV1 expressing cells were plated in a variety of buffers and in the presence of different inhibitors. Capsaicin was added and responses measured. Responses 20 minutes following capsaicin activation are plotted. Data courtesy of AstraZeneca

TRPV1 Antagonist Characterization

The BIND assay was used to characterize a panel of known activators (data not shown) and inhibitors of TRPV1. The EC50, IC50 and efficacy values derived from the assay were in general agreement with literature report. With the new assay, we have uncovered different modes of action for different classes of TRPV1 inhibitors.

Figure 3 summarizes studies of TRPV1 ion channel antagonist. 9 antagonists were tested using capsaicin-stimulated cells (panel A). The potency and rank order of all compounds were in agreement with reported literature values. Compounds SB-366791 (panel B) and Ruthenium Red (panel C) were subsequently tested against a range of capsaicin concentrations. SB-366791 was identified as a competitive binder and Ruthenium Red as a non-competitive binder.
Figure 3 - To assess TRPV1 antagonist activity, the cells were incubated with the compounds for 15 min, and after a baseline reading, capsaicin was added to the cells to activate TRPV1 and delta PWV was measured 20-30 min after capsaicin addition. A. Dose response curves of known TRPV1 inhibitors stimulated with 0.1µM capsaicin. B. Capsaicin dose response curves of inhibitor SB-366791. C. Capsaicin dose response curves of Ruthenium.