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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 - 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 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 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 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 , ion channels are the third largest early stages of drug discovery. The relatively recent group of signalling molecules after 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 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) (6). as GLP safety pharmacology profiling.

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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 expressed in human embryonic kidney (HEK293) comparable to MPC, they are lower throughput than IWQ, and/or Chinese hamster ovary (CHO) cells (Reference 4, only capable of generating hundreds of data points per day Table 1, page 26). The catalogue includes: representatives rather than thousands. of the voltage-gated subfamilies (sodium, potassium and calcium channels) important in neuronal signalling and Automated Fluorescence Assay Platform muscle contraction; ligand-gated channels opened by

Compared with automated patch-clamp platforms, ion GABAA, acetylcholine, NMDA or ATP (P2X receptors); channels that are amenable to automated fluorescence transient receptor potential (TRP) channels involved in measurements in a fluorescence plate reader – such as signalling; chloride channels and acid-sensing ion FLIPRTETRA (MDS-Analytical Technologies) – can be channels (ASIC) involved in ion homeostasis and/or assayed at even higher throughput (~10,000 datapoints pain and inflammation. The pairing of a library of ion

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Table 1: Composition of ChanTest Catalogue of Ion Channel Library channel cell lines with automated screening platforms Voltage-gated sodium Calcium-activated potassium Chloride opens up exciting new opportunities in ion channel drug Nav1.1 KCa1.1 (BK) CFTR discovery and safety pharmacology. Nav1.2 KCa2.1 (SK1) CLC-1 Nav1.3 KCa2.2 (SK2) CLC-2 Nav1.4 KCa2.3 (SK3) CLC-KA/barttin THE FUTURE Nav1.5 KCa3.1 (IK) CLC-KB/barttin Nav1.6 Nav1.7 Inward rectifier potassium Ligand-gated The future of ion channel drug discovery and safety Nav1.8 Kir1.1 GABA-A receptor (α2β2γ2) pharmacology is very bright. The marriage of higher Nav1.9 Kir2.1 GABA-A receptor (α5β2γ2) Kir3.1/Kir3.2 Nicotinic receptor (α3/β4) throughput automated patch clamp platforms with recent Voltage-gated potassium kir3.1/Kir3.4 Nicotinic receptor (α7) advances in human embryonic stem cell technology Kv1.1 Kir6.1/SUR2B NMDA receptor (NR1/NR2A) Kv1.2 Kir6.2/SUR1 NMDA receptor (NR1/NR2B) provides new opportunities in drug discovery/safety Kv1.3 Kir6.2/SUR2A P2X1 pharmacology of ion channels. Human stem cells should Kv1.4 Kir6.2/SUR2B P2X2 Kv1.5 P2X2/P2X3 provide a virtually unlimited supply of cells for cardiac risk Kv2.1/Kv9.3 Cyclic nucleotide-gated P2X3 assessment of off-target drug effects on human cardiac ion Kv3.4 CNGA1/CNGB1 P2X4 channels. Furthermore, in the era of post-human genome Kv4.1 CNGA3/CNGB3 P2X7 Kv4.2/KChiP2.2 sequencing, opportunities exist for customisation of ion Kv4.3 Hyperpolarization-gated Acid-sensing channel cell lines and embryonic stem-cell derived cells with Kv7.1(KvLQT1)/mink HCN1 ASIC1a Kv7.2/Kv7.4 (KCNQ2/KCNQ4 HCN2 ASIC2a disease-relevant mutations or patient-specific polymorphic Kv7.2/Kv7.3 (KCNQ2/KCNQ3) HCN3 ASIC3 variants. Modulation of channel expression levels through Kv7.3/Kv7.5 (KCNQ3/KCNQ5) HCN4 Kv7.4 (KCNQ4) Exchanger siRNA technology in native cells should also facilitate the Kv10.1 (EAG1) Transient receptor potential NCX1 discovery of novel pharmacotherapies. Kv11.1 (hERG) TRPA1 TRPC1 Voltage-gated calcium TRPC4 References Cav1.1 TRPC6 1. Harmar AJ, Hills RA, Rosser EM et al, IUPHAR-DB: The Cav1.2/β2/α2δ TRPM2 Cav2.1/β4/β2δ TRPM4 IUPHAR database of G protein-coupled receptors and ion Cav2.2/β3/2δ TRPM8 channels, Nucleic Acids Res 37, 2008 Cav3.2 TRPP1/TRPP2 TRPV1 2. Venter JC, Adams MD, Myers EW et al, The sequence TRPV4 of the human genome, Science 291, 2001 TRPV6 3. Yu FH and Catterall WA, The VGL-chanome: A protein Channel names in italics are under development and have not completed validation superfamily specialized for electrical signaling and ionic homeostasis, Sci STKE re15, 2004 Frank Yu, PhD, has been Study Director at ChanTest Corporation since 2007. He 4. Wible BA, Kuryshev YA, Smith SS et al, An ion channel holds a PhD in Physiology from McGill University (Montreal, Canada) and has library for drug discovery and safety screening on actively published numerous research articles during his 18 years of research on ion automated platforms, Assay Drug Dev Tech 6, 2008 channels and ion transporters. Before joining ChanTest, Frank was a faculty member 5. Lu Q and An WF, Impact of novel screening of the Department of Pharmacology, University of Washington (Seattle, WA). technologies on ion channel drug discovery, Comb Barbara Wible, PhD, has been with ChanTest Corporation since 2002 and currently Chem HTS 11, 2008 serves as Head, Cell and Molecular Biology. Barbara holds a PhD in Biology from 6. Brown AM and Rampe D, Drug-induced long QT Marquette University (Milwaukee, WI), and has previously held faculty positions at syndrome: Is HERG the root of all evil? Pharm Baylor College of Medicine (Houston, TX) and Case Western Reserve University News 7, 2000 (Cleveland, OH). 7. Schroeder K, Neagle B, Trezise DJ and Worley J,

Stephen Smith, PhD, has been at ChanTest Corporation since 2007. Steve Ionworks HT: A new high-throughput electrophysiology was a Senior Scientist in the Pharmaceutical Discovery Group at Lexicon measurement platform, J Biomol Screen 8, 2003 Pharmaceutical, and prior to this, he was with Axon Instruments working with 8. Dubin AE, Nasser N, Rohrbacher J et al, Identifying the group that developed and deployed the PatchXpress 7000A, an automated modulators of hERG channel activity using electrophysiology platform. Steve holds a PhD in Physiology from the University the PatchXpress planar patch clamp, J Biomol of Michigan (Ann Arbor, MI). Screen 10, 2005 Arthur ‘Buzz’ Brown, MD, PhD, is Chief Executive Officer of ChanTest Corporation. 9. Mathes C, QPatch: The past, present and future Before coming to ChanTest, Buzz was Vice President for Research at the of automated patch clamp, Expert Opin Ther Targets Rammelkamp Center for Education and Research at the MetroHealth Medical 10, 2006 Center (Cleveland, OH), and Professor and Chairman of the Department of 10. Farre C, Stoelzle S, Haarman C et al, Automated ion Molecular Physiology and Biophysics, Baylor College of Medicine (Houston, TX). channel screening: patch clamping made easy, Expert Email: [email protected] Opin Ther Targets 11, 2007

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