Combinatorial Chemistry & High Throughput Screening - Volume 12, Issue 1, 2009

Ion channel proteins are fascinating molecules that play key roles in many physiological processes. In fact, no mammalian cell type has been identified which does not express a complement of ion channel proteins. However, while all cell types express ion channels, the specific role these channels play can vary significantly. In excitable cells such as neurons, skeletal muscle and heart, ion channels act as molecular switches to either trigger excitation or reset the membrane potential to a baseline, resting level depending on which type of ion channel is activated. In non-excitable cells, channels can play other roles such as regulation of fluid movement, salt secretion or resorption, and triggering of signal transduction cascades that eventually lead to gene transcription. Over the past several years, there has been an explosion of data elucidating the role of specific channels in genetic disorders due to mutations which typically affect gating and/or expression. These ‘channelopathies’ further validate the role various ion channels play in maintaining normal physiological processes. The genetic data also suggest that modulation of ion channel function is a good means to intervene in pathophysiological processes and restore normal function. One good example of the genetics leading to a greater understanding of the role for a particular channel is a series of mutations in Kv7 channels that cause a neonatal seizure disorder [1]. The genetic data highlight the key role of these channels in regulating CNS excitability, and since decreases in channel expression lead to seizures activation of Kv7 channels may be a useful therapeutic approach to treating epilepsy [2]. Many other examples in a variety of diseases or disorders also exist including pain, myotonias and arrhythmia [3-5]. Channelopathies as a whole have been nicely reviewed by Ashcroft [6]. Given the key role ion channels play in health and disease and the wealth of new data validating various ion channel subtypes as possible therapeutic drug discovery targets, there has been something of a resurgence in ion channel drug discovery. Facilitating this resurgence have been advances in the technology available to academic and industrial scientists, which can be applied to investigate ion channel pharmacology [7]. In this volume, we have included reviews and original research by a number of investigators who are at the forefront in the development and/or application of new technologies for the characterization of ion channel function and pharmacology. The volume begins with an excellent review on the ion channel genome, which describes the landscape with respect to potential molecular targets for drug discovery, relevant comparisons across species and thoughts regarding the potential for overlapping pharmacology among gene families. The remainder of the volume describes several of the most widely applied technologies available to the research community including planar patch clamp technology, advanced semi-automated micropipette applications, use of voltage-sensitive dyes, and high-throughput Xenopus oocyte recordings. In addition, a number of the articles focus on real-world experiences from pharmaceutical/biotech investigators applying several of these technologies in an industrial setting. While all available methods and/or instrumentation can not be covered in a single volume, the articles cover many of the most widely used approaches and the investigators report their experiences and suggest how best to make progress using these technologies in a research setting and/or a drug screening environment. We hope the readers find the articles informative and helpful in advancing their own ion channel research and drug discovery programs.

Ion channels are intimately involved in virtually every physiological process of consequence in humans. Their importance is underscored by the identification of numerous “channelopathies”, human diseases caused by ion channel mutations. Ion Channels have consequently been viewed as fertile ground for drug discovery and, indeed, they represent one of the largest target classes for current medicines. The future prospects of ion channels as a target class are tied to the functional characterization of the human ion channel set on a genomic scale. The focus of this review is to describe the molecular diversity and conservation of human ion channels. The human genome contains at least 232 genes that encode the pore-forming subunits of plasma membrane ion channels. Comparative genome analysis shows that most human ion channel gene families have their origins in the earliest metazoans but the human genes are largely derived from duplications that took place in the vertebrate lineage. The mouse and human ion channel gene sets are virtually identical, but differ significantly from fish channel sets. Genome comparisons highlight a number of highly conserved channel families that do not yet have specifically defined functional roles in vivo. These channel families are likely to have non-redundant functions in metazoans and represent some of the best new opportunities for channel target prospecting. Furthermore, genome- wide patterns of sequence conservation can now be used to refine strategies for the identification of gene-specific channel probes.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually by a single operator, one cell at a time. However, methods of high throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications.

Planar chip technology has strongly facilitated the progress towards fully automated electrophysiological systems that, in contrast to the traditional patch clamp technology, have the capability of parallel compound testing. The throughput has been increased from testing below 10 compounds per day to a realized capacity approaching high throughput levels. Many pharmaceutical companies have implemented automated planar chip electrophysiology in their drug discovery process, particularly at the levels of lead optimization, secondary screening and safety testing, whereas primary screening is generally not performed. In this review, we briefly discuss the technology and give examples from selected NeuroSearch ion channel programs, where one of the systems, the QPatch, has been evaluated for use in lead optimization and primary screening campaigns, where high information content was a requirement.

Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of Ih (also known as ‘funny’ If and ‘queer’ Iq), a hyperpolarization- activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.

To facilitate automated patch clamp measurements of ion channels in cells, the development of an all-glass Chiptip pipette is reported that may be combined with the previously described Flip-the-Tip technology. A single measurement requires less than 50 cells, and the addition of drugs for screening can be limited to very low volumes down to 1 μL. This apparatus is suitable for the study small cells, subcellular organelles and bacteria.

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.

Voltage dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders such as epilepsy and pain as well as cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic and antiarrhythmic sodium channel blockers. While patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery in which there is often a need to evaluate many thousands to hundreds of thousands of compounds. Fortunately over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms like the PatchXpressTM and Ion- WorksTM, which are now widely used in drug discovery. This review highlights the strengths and weaknesses of these two high throughput devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpressTM and IonWorksTM electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state dependent modulation of sodium channels. IonWorksTM has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpressTM has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery.

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