Development and Validation of a Thallium Flux-Based Functional Assay for the Sodium Channel NaV1.7 and Its Utility for Lead Discovery and Compound Profiling

Abstract

Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.

Prediction of hERG Liability – Using SVM Classification, Bootstrapping and Jackknifing

“…[16] The thallium flux assay was comprehensively validated for its capability of identifying small molecules with potential to block hERG and induce LQTS. [17] Using U2OS cells, 4,323 small molecules were screened for hERG channel inhibition at concentrations ranging from 10nM to 46uM in a quantitative high throughput screening (qHTS) format. [18] Curve fitting is based on a grid method, and curve classes are in turn assigned according to the type of concentration–response curves observed.…”

“…One high‐throughput alternative is to detect inhibition of the hERG channels by measuring flow of a surrogate ion, thallium, in a homogenous assay format . The thallium flux assay was comprehensively validated for its capability of identifying small molecules with potential to block hERG and induce LQTS . Using U2OS cells, 4,323 small molecules were screened for hERG channel inhibition at concentrations ranging from 10nM to 46uM in a quantitative high throughput screening (qHTS) format .…”

Deep Mutational Scan of a cardiac sodium channel voltage sensor

“…Simultaneous treatment with both classes of drug was required for negative selection, and the highest selection was observed with two channel agonists (Figure 2), consistent with previous findings that veratridine and brevetoxin act in a synergistic manner. 25,40 Channel agonists are commonly used in other high-throughput screening approaches, 41,42 and therefore might be used to help develop analogous functional assays for other ion channels.…”

Emerging neurotechnology for antinoceptive mechanisms and therapeutics discovery

“…However, FLIPR blue dye assays do not enable sodium response times on the order of single action potentials (1-10 ms), instead offering changes in fluorescence on the order of tens of seconds. Surrogate measures of specific sodium channels have been proposed to overcome these challenges, such as thallium flux-based functional assays for the sodium channel Nav1.7 (Du et al, 2015). While the fluorescence yield and dynamics of these surrogate measures are more amenable to high-throughput screening, they are still an indirect measure of membrane excitability.…”