Ion channels are increasingly common targets for drug discovery, but traditional plate-based screening methods, such as FRET and calcium flux assays, provide only indirect measurements of ion conductance and may not correlate closely with in vivo electrophysiology. While manual patch-clamp techniques remain the gold standard for screening ion channels, automated electrophysiology has not yet achieved sufficiently high throughput for use in large-scale screening and typically requires secondary assays to confirm hits and eliminate artifacts.
There remains a “gap between manual patch clamp and high throughput,” noted Juna Kammonen, senior scientist at Pfizer. A shift to plate-based screening will require assays that can be performed in smaller numbers of primary cells. Kammonen described his group’s adoption of two new strategies that have enabled higher-throughput whole cell electrophysiology assays.
They are using the FluxOR™ thallium flux assay from Invitrogen (a Life Technologies) to measure ion flow across an ion channel, together with Genedata’s Screener® Kinetics Analyzer software to enable multiparametric and time-series data analysis to support decision making on which compounds to take forward.
Pfizer is also working to achieve higher-throughput implementation of the IonWorks Quattro automated patch-clamp system (Molecular Devices) and improved correlation with PatchXpress® to yield greater confidence in the data generated. Furthermore, the company is using Port-a-Patch (Nanion Technologies) technology in automated plate-based applications for biophysical characterization of new ion-channel cell lines.
Experimentation with the Fluxion Biosciences IonFlux automated patch-clamp system is yielding promising results, and is an example of how technology is “starting to bridge the gap between plate-based assays and electrophysiology.” Kammonen reported that the instrument’s microfluidics technology provides fast, precise perfusion. A single instrument can perform a dose-response assay on 32 compounds in about 15 minutes, and multiple units can be stacked on a robotic platform to increase throughput.
He noted, in particular, the design of the system, with 12 interconnected wells per experimental zone: eight compound wells, two cell groups, and two cell suspension wells that provide built-in redundancy. This 12-well pattern is repeated in 32 experimental zones on a 384-well plate; screening of eight compounds with two recordings generated for each yields 512 data points/plate. The system’s fast liquid exchange rate allows for detection of rapid responses and screening of fast-gated channels such as GABA.
“Creating a holistic screening strategy for label-free technology in a plate based screening group” was the topic of a talk by Rachel Russell, associate director, Pfizer Global R&D, in which she detailed how label-free technology in assay development and screening is facilitating the use of primary cells in place of engineered cell lines for primary screening applications.
“Know what you are asking in the screen,” when you perform reagent validation and assay design using a label-free platform, Russell cautioned. She posed key questions, such as whether frozen primary cells are suitable for use in a label-free system, and whether a native cell grown in isolation on a plastic plate will behave as it would in the body, or whether soft substrates would promote more natural cell growth and differentiation and yield more relevant pharmacological and biological outcomes.
Russell mentioned several innovative technologies that are contributing to the successful development of label-free cell-based assays, including the CellKey® 96-well bioimpedance-based label-free detection system (Molecular Devices), the BIND® SCANNER plate-based instrument using photonic crystal technology (SRU Biosystems), the PathHunter® cell-based assay systems (DiscoveRx), and the Epic® optical biosensor-based screening platform (Corning).
“Label-free is part of the way forward,” she concluded, noting that it provides a method for measuring physiological responses across multiple pathways in parallel.