Search

Significance Subtype-selective modulation of ion channels is often important, but extremely difficult to achieve for drug development. Using Nav1.7 as an example, we show that this challenge could be attributed to poor design in ion channel assays, which fail to detect most potent and selective compounds and are biased toward nonselective mechanisms. By exploiting different drug binding sites and modes of channel gating, we successfully direct a membrane potential assay toward non–pore-blocking mechanisms and identify Nav1.7-selective compounds. Our mechanistic approach to assay design addresses a significant hurdle in Nav1.7 drug discovery and is applicable to many other ion channels., Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed.

The voltage-gated sodium channel Nav1.7 is one of the most validated pain targets, as gain-of-function mutations cause excessive pain, whereas loss-of-function mutations produce insensitivity to pain. Targeting Nav1.7 holds great promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 selective chemical scaffolds exist. So far millions of compounds have been screened by using fluorescence membrane potential (MP) assays, but very few selective hits have been identified. Here we show that the conventional MP approach, which uses Nav1.7 WT channel and the activator veratridine, is intrinsically flawed: it is biased toward non-selective pore blockers which compete with veratridine, and fails to detect highly potent, selective compounds such as the aryl sulfonamide PF-771. By using a mutant channel and an alternative activator, we developed a novel method that not only robustly detects known Nav1.7 blockers, but also decreases the number of non-selective pore blocker hits. We conducted a high-throughput screen using this methodology and identified novel Nav1.7-selective blockers.