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Ion Channel Technologies: Progress and Potential IPT 29 2009 10/6/09 15:32 Page 22 Discovery Technology Ion Channel Technologies: Progress and Potential By Frank H Yu, Barbara A New developments in screening platforms provide rapid and cost-effective Wible, Stephen S Smith approaches to interrogate a representative library of ion channel- and Arthur M Brown at ChanTest Corporation expressing cell lines using automated electrophysiological instruments. Ion channels are a structurally diverse group of membrane Drug development for ion channel targets has been difficult proteins highly specialised for fast, precise control of ion for several reasons. First, the large number of human ion transport in organisms from bacteria to mammals. In channel genes generates a greater number of mature ion complex multi-cellular organisms, ion channels provide channel types through assembly of various combinations of the functional basis for rapid, accurate transmission of pore-forming and modulatory subunits. Alternative splicing information among cells and tissues using electrical signals and post-translational modifications of the ion channel gene in the form of action potentials. Associated intracellular products further multiply their diversity. For maximal calcium signals control contraction of muscle, synaptic efficacy and minimal side effects, drugs are often required to transmission for processing information in the brain, target a specific channel within a subgroup of related secretion of hormones, transepithelial ion fluxes, gene channels. This necessitates screening drugs against many ion expression and cell division. Ion channel proteins span the channel targets to monitor selectivity. Panels of cell lines membrane lipid bilayer and fold to form an ion expressing a diverse array of ion channel targets have not conduction pathway or a pore through which ions flow at been available. Secondly, functional analysis of drug effects rates sufficiently rapid to change membrane potential and on ion channels has required manual patch clamp (MPC) resistance. The pore is a small but essential component of electrophysiology – a technique that is both expensive and ion channels, while the remainder of these large integral labour-intensive. This has largely prohibited the screening proteins regulate whether the pore is open or closed. of compounds in functional ion channel assays in the In the human genome, ion channels are the third largest early stages of drug discovery. The relatively recent group of signalling molecules after protein kinases and introduction of higher-throughput automated patch clamp G-protein coupled receptors (1,2). It is estimated that there instrumentation has removed this obstacle for some ion are 200-300 genes encoding the ion channel pore-forming channel types, and brought early-stage ion channel drug subunits, including at least 143 voltage-gated like ion discovery and safety screening within reach. channels, 21 chloride channels and 71 ligand-gated ion channels (2,3). As a group, ion channels comprise about 15 ION CHANNEL TECHNOLOGY per cent of drug targets for a variety of diseases including cardiac arrhythmias, hypertension, heart failure, epilepsy, Manual Patch Clamp asthma, diabetes, urinary retention and incontinence, Manual patch clamp (MPC) electrophysiology is a powerful irritable bowel syndrome, Parkinsonism, Alzheimer’s method and represents the ‘gold standard’ for measuring ion disease and cancer (4). Among the ~180 US FDA- channel function. It involves using fine glass pipettes to approved drugs that target ion channels are some with make high-resistance giga-ohm seals to the cell membrane. multi-billion dollar annual sales – such as amlodipine for Rupturing the patch of membrane underneath the pipette hypertension (L-type calcium channel blocker, Pfizer) and permits access to and voltage-clamp control of the gabapentin for epilepsy (targeting calcium channel α2δ membrane potential, to allow measurement of the electrical auxiliary subunit, Pfizer). Interestingly, the vast majority of currents generated by ion channels expressed in the cell. ion channel targeted drugs were identified serendipitously Although MPC produces high quality data and is able to prior to the early 1990s, and only six new molecular probe the biophysical intricacies of ion channel function – entities have been approved by the FDA from 2000 to such as open, closed and inactivated states of the channel, 2006 (5). Importantly, ion channels are also targets for and their respective interactions with drugs – it is very drug safety concerns via unwanted interactions. A well- labour-intensive, low-throughput and requires highly skilled known example is the cardiotoxicity (drug-induced long scientists. For these reasons, MPC is impractical for drug QT syndrome) associated with the block or trafficking discovery screening and has been utilised more effectively at inhibition of the human ether-à-go-go-related gene (hERG, later stages in the ion channel drug discovery pipeline, such KV11.1) potassium channel (6). as GLP safety pharmacology profiling. 22 Innovations in Pharmaceutical Technology IPT 29 2009 10/6/09 15:33 Page 24 Automated Patch Clamp Platform per day). Fluorescent dyes that monitor changes in the Great strides in the automation of patch clamp concentration of ions – such as calcium, chloride and electrophysiology in the last decade have increased the thallium (a potassium congener) – as well as changes in throughput from 15- to 45-fold over manual measurements membrane potential and pH are available. Limitations of with little loss of accuracy. Commercialisation of planar fluorescence-based assays for ion channel function chip-based patch clamp technology replaced MPC glass include compound autofluorescence, lack of voltage pipettes with multi-well arrays capable of well-specific control and the consequent inability to study state- voltage control in IonWorks HT (7) and its successor dependent effects of drugs, depolarisation triggered by IonWorks Quattro (IWQ) (MDS Analytical Technologies). changing extracellular potassium, and the lack of a With IWQ, recordings from 384 cells (one cell per well of millisecond resolution of response. Nevertheless, a FLIPR a 384-well plate) can be obtained in single hole mode, or assay is very effective for monitoring ion channels that currents from up to 64 cells per well can be averaged in induce changes in Ca2+ concentrations like voltage-gated population patch mode. In spite of medium resistance calcium channels and transient receptor potential (TRP) seals (hundreds of mega-ohms) and the necessity for using channels. For very large-scale ion channel screening the perforated patch configuration with ionophore campaigns, fluorescence assays – albeit an indirect amphotericin B, IWQ has proven to be an invaluable measure of channel activity – have the advantages of workhorse for ion channel cell-line screening/development lower cost and higher throughput compared with and drug-activity profiling for many different channels. electrophysiological approaches. An additional advantage In particular, IWQ is especially well-suited to assay in ion channel screening campaigns is that FLIPR assays voltage-gated ion channels. It provides a relatively higher measure response in a population of cells rather than throughput (more than 1,000 data points per day) individual cells. This is beneficial for mechano-sensitive compared to other automated planar array patch-clamp ion channels such as TRPC6 that are stimulated by cell platforms and allows both tonic and phasic drug block stretch and perform better in a population-based FLIPR (state-dependent activity) to be measured, something that is assay than in an electrophysiology assay. not possible with higher throughput assays of ion channel activity such as FLIPR or ion flux. IWQ is not suitable for A LIBRARY OF ION CHANNEL CELL LINES measuring drug block of rapidly desensitising channels or ligand-gated channels, however. Since IWQ is configured As discussed above, one barrier to ion channel drug with separate current recording and fluid delivery heads that discovery has been largely removed with the cannot be positioned in the well at the same time, the development of efficacious and economical automated systematic time delay between activation (fluid delivery) and assays of ion channel function. A second barrier has been recording makes resolution of ligand-gated events or fast the lack of available cell lines overexpressing ion blocking events impossible. channels. Recently, efforts undertaken by several The introduction of several instruments that produce companies have yielded a representative collection of high resistance (giga-ohm) seals of patched cell membrane stable mammalian cell lines heterologously for better voltage control and concurrent ion channel overexpressing individual ion channels. These cell lines recording and fluid delivery has allowed the higher permit compound library screening of specific ion throughput assay of ligand-gated channels and rapidly channel targets or off-target screens of promising lead desensitising channels. The PatchXpress 7000A (MDS- candidates in early phases of the drug discovery process Analytical Technologies), QPatch HT (Sophion to reduce the overall cost of pharmaceutical Biosciences) and Patchliner (Nanion Technologies GmbH) development. As an example, ChanTest Corporation has fall into this category (8,9,10). Although these automated established a catalogue of 82 diverse ion channel targets platforms provide higher fidelity recording capabilities
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