Ion Channel Screening
Metrion delivers accurate ion channel screening data, tailored to your target and delivered promptly to meet your specific needs.
Our ion channel and drug discovery screening capabilities enables us to provide reliable, high-quality data to accelerate and validate your drug discovery programmes.
An introduction to Metrion’s Ion Channel Screening services
At Metrion Biosciences, we specialise in delivering high-quality ion channel screening services using a variety of electrophysiology, label-free and fluorescence-based platforms.
Our assay technologies include Qube, QPatch and Patchliner automated electrophysiology, conventional manual patch clamp electrophysiology, plate-based impedance and microelectrode array techniques, and plate-based imaging on the FlexStation.
Table of Contents
Kv1.3 Ion Channel Screening Case Study
Ion channel assays used for selectivity and SAR screening don’t just need to be stable during the testing of individual compounds (minutes), but also stable over the entire duration of a drug discovery programme (months-years).
Primary screening assays
In the example below, we show the exceptional reliability of the optimised primary target Kv1.3 (Figure 1) and gene family selectivity Kv1.5 counter-screening assays (Figure 2) used as part of a long-term pharma collaboration. The positive control IC50 was plotted over the course of 2 years.
Metrion scientists also developed a complete gene family selectivity panel of human Kv1.x ion channel cell lines and assays to complement the primary Kv1.3 screening assay. These assays were critical in supporting this long-term drug discovery project and for successfully identifying selective small molecule modulators able to treat auto-immune disease.
Species selectivity assay
As no commercial rat Kv1.3 cell line was available, Metrion scientists created a stable CHO cell line to meet the species selectivity needs of our Kv1.3 drug discovery project. Several clones were assessed on the QPatch screening platform and the optimised assay was used successfully over a 2 year period, revealing minimal species shift in the small molecule scaffolds developed as part of this pharma collaboration.
Cell Line and Assay Optimisation
In some cases, ion channel cell reagents are not optimal for electrophysiology assays and need improved ‘patchability’ and expression of the protein of interest in a functional state at the cell surface.
Metrion’s scientists have over a decade of experience in developing, optimising and validating automated and manual patch clamp assays for ion channel targets. We can fully customise ion channel assays at different stages of the screening cascade, from hit finding and medium throughput structure activity studies, through to specialised biophysical and mechanism-of-action studies for hit series or lead compounds.
A neuronal Na+ ion channel used as the primary counter-screening target in a major pharma drug discovery collaboration is notoriously difficult to express in heterologous cells, and under standard conditions the original cell line yielded a low success rate assay (Figure 6a).
We tested a number of different cell culture, cell biology and experimental conditions (Figure 6c) to develop an optimised assay on the Patchliner (Nanion) automated patch clamp platform. Our efforts significantly improved the low expression seen in the original reagent without affecting ‘patchability’, yielding a highly efficient gene family selectivity assay (Figure 6b).
During our work with the FDA’s CiPA cardiac safety testing consortium, it became apparent that a ‘late’ Nav1.5 assay would be an important part of their selectivity panel to help predict the safety of new drug candidates. We therefore created stable CHO and HEK cell lines expressing the ΔKPQ LQT3 mutant protein (Figure 7) and assessed their utility as reagents for automated patch clamp assays that would be compliant with CiPA HTS sub-team requirements (Figure 8).
Assay-ready cells for APC
Biological reagents are inherently variable and it is typical for the performance of ion channel assays to fluctuate during extended passaging of stable cell lines.
Minimising biological reagents variation
To minimise this variation, during extended SAR screening campaigns, Metrion scientists adopted the approach pioneered in GPCR assays and created so-called ‘assay ready’ cells that are grown under conditions optimised for automated patch clamp assays and then frozen down to fix their state for later use. Assay-ready cells are thawed out on the day and used directly on APC platforms.
This guarantees optimum performance, provides consistent screening results, and increases workflow flexibility to run multiple ion channel assays at short notice.
Assay Development and Validation
Ion channels are large, complex transmembrane proteins that can be difficult to express with the correct folding and associated auxiliary subunit and scaffolding protein complexes, making it essential to validate cell line reagents and assays before they are used for drug discovery screening. Along with sequence verification, Metrion scientists determine the functional biophysical and pharmacological profile of each cell line reagent on our assay platforms prior to their use in drug discovery screening cascades.
ASIC1a QPatch assay
We recently validated a cell line expressing human ASIC1a, a ligand-gated receptor which is implicated in stroke and ischemia. Assay development efforts concentrated on creating a stable assay on the QPatch automated patch clamp platform (Figure 9) and confirming the correct agonist and antagonist pharmacology using a selection of reference and literature compounds (Figure 10).
Dynamic hERG assay
It is also possible to create new screening assays from existing cell lines.
As part of our efforts to create an expanded panel of CiPA-compliant cardiac safety assays, we developed and validated an optimised version of the very challenging ‘Milnes’ dynamic hERG voltage protocol suitable for automated patch clamp.
This protocol is designed to measure the potential for test compounds to become trapped inside the hERG channel pore, and Metrion’s dynamic hERG assay is the first to be validated on an automated patch clamp platform.
Ion channel cell line case studies
ND7-23 Neuroblastoma Assay
Rodent neuroblastoma cell lines provide a useful alternative to native peripheral neurons as they endogenously express many important membrane receptors and channels, thereby meeting 3Rs objectives and helping to translate the most relevant compounds towards preclinical efficacy models.
In this example Metrion scientists developed and validated a high throughput automated patch clamp assay using immortalised rodent ND7-23 neuroblastoma cells that express endogenous TTX-sensitive Na+ channels relevant to pain drug discovery.
Read publication Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening.
Neuronal Kv Channel mechanism-of-action studies
A highly desirable mechanism-of-action for small molecule modulators of ion channels is state-dependence, whereby a compound exhibits preferential binding for a particular conformational state or functional mode of the transmembrane protein.
In this way an ion channel drug discovery programme can achieve higher potency and improved selectivity, which promises superior efficacy and safety for lead candidates in vivo.
In this case study we show how Metrion scientists developed a state-dependent assay for the Kv1.1/(1.2)3 channel on the QPatch. In this way it was possible to acquire detailed mechanistic information alongside target potency for compounds at the top of the screening cascade, thereby enriching their SAR profile and accelerating the identification of optimum compounds to treat Multiple Sclerosis. This state-dependent assay format has been successfully employed for other voltage gated Kv1.x channels, including Kv1.3.
Ion Channel Screening Resources
Ion Channel Screening Resources
- Ion channel drug discovery and modern medicine.
- Marc Rogers (Metrion Director and CSO) outlines the benefits of targeting ion channels for pain and some of the hurdles in developing successful ion channel modulators.
- Ion Channel Discovery – Partnering to Access Specialized Expertise.
- Investigating the correlation between thallium flux and automated patch-clamp for ion channel activators. ELRIG Drug Discovery Annual Meeting, Liverpool, 2019
- Marc Rogers (Metrion Director and CSO) presents a talk at the Nanion Virtual User Meeting 2020 entitled “Designing multiple assay protocols for ligand gated ion channels using the stacked-tip feature on the Patchliner and SP384i platforms”. 14th October 2020.
- ASIC1a Ligand Gated Ion Channel Assay (App Note)
- Domainex collaboration TRPA1 case study (flyer)
- Identification of novel ion-channel binders: TRPA1 antagonist case study. (Collaboration with Domainex). World Preclinical Congress (WPC), Boston, 2019
- Identification of Novel Scorpion Venom Peptide Inhibitors of the Kv1.3 Ion Channel and their Potential as Drug Discovery Leads for Human T-Cell Mediated Disease. RSC Ion Channels Symposium, Cambridge, 2020
- The development of a set of novel small molecule inhibitors of the Kv1.3 ion channel. Biotech Outsourcing Strategy (BOS) Basel, 2019
- A drug discovery collaboration between Japanese pharma and a UK SME CRO successfully developed novel small molecule inhibitors of the Kv1.3 channel to treat autoimmune disease. The Best of Both Worlds: Innovation, Collaboration and Synergy between CROs and their Client Partners, Stevenage, 2018.
- Clathrodin, hymenidin and oroidin, and their synthetic analogues as inhibitors of the voltage-gated potassium channels.
- Novel K+ Channel Targets in Atrial Fibrillation Drug Development – Where Are We?
- Human Electrophysiological and Pharmacological Properties of XEN-D0101: A Novel Atrial-Selective Kv1.5/IKur Inhibitor.
- Recent advances in electrophysiology-based screening technology and the impact upon ion channel discovery research.
Cardiac Safety Screening Resources
- Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.
- A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm
- The Nav 1.5 Late Current in WT and Nav 1.5 ΔKPQ Mutant Channels: An Automated Patch Clamp LQT3 Electrophysiological Assay Comparison. Safety Pharmacology Society Virtual Meeting 2020.
- NaV1.5-ΔKPQ late INa current properties and pharmacology on the SyncroPatch 384i
- Recent advances in targeting ion channels to treat chronic pain.
- Marc Rogers (Metrion Director and CSO) takes part in a collaborative webinar with Nanion Technologies entitled “Validation and optimization of automated patch clamp voltage-gated Ca2+ channel assays”.
- Open access to the KCNQ channel: Retigabine and second generation M-current openers.
- Development of native and stem cell-derived electrophysiological assays for neurotoxicology screening and translational drug discovery
- Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening.
- Optimising a difficult Nav1.8 cell line assay for automated patch clamp screening. Ion Channel Retreat, Vancouver, 2015
- Synthesis and biological evaluation of piperazine derivatives as novel isoform selective voltage-gated sodium (Nav) 1.3 channel modulators
- Action of Clathrodin and Analogues on Voltage-Gated Sodium Channels
- Novel state-dependent voltage-gated sodium channel modulators, based on marine alkaloids from Agelas sponges
- Ligand- and structure-based virtual screening for clathrodin-derived human voltage-gated sodium channel modulators
Translational Assay Resources
- The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients
- Assessment of human induced pluripotent stem cell-derived cardiomyocytes for evaluating drug-induced arrhythmias with multi-electrode array
- Development of an impedance based screening assay for cardiac safety and cardiotoxicity detection in stem cell derived cardiomyocytes
- Validation of an impedance-based phenotypic screening assay able to detect multiple mechanisms of chronic cardiotoxicity in human stem cell-derived cardiomyocytes
- Electrophysiological characterisation of Cellular Dynamics International ventricular iCell2 iPSC-derived cardiomyocytes
- Functional characterisation of human iPSC-derived atrial cardiomyocytes
Let’s work together
What are your specific ion channel screening requirements?
If you have any questions, or would like to discuss your project, we will put you directly in touch with a member of our scientific team. Contact us today to discover more.