Vol. 2, No. 2 2005
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Screening technology
Ion channel screening technologies today Georg C. Terstappen Sienabiotech S.p.A., Discovery Research, Via Fiorentina 1, 53100 Siena, Italy
For every heartbeat, movement and thought, ion channels have to open and close, and thus, it is not surprising that malfunctioning of these membrane proteins leads to serious diseases. Today, only 7% of all marketed
Section Editors: Jeff Brockman – Psychiatric Genomics Inc., Gaithersburg, MD, USA Mark Divers – AstraZeneca, Mo¨lndal, Sweden
drugs act on ion channels but the systematic exploitation of this important target class has started mainly enabled by novel screening technologies. Thus, the
associated with transient changes in membrane potential (Fig. 1).
discovery of selective and state-dependent drugs is on the horizon, hopefully leading to effective novel medicines.
Introduction If no information is available about a target protein structure or ligands, drug discovery typically starts with high-throughput screening (HTS) of hundreds of thousands of chemical molecules. Although assays that are compatible with the requirements of HTS have been in use since the beginning of the 1990s for target classes such as G protein-coupled receptors and enzymes, such assays for ion channels have become available only more recently. Ion channels are poreforming membrane proteins which enable rapid membrane passage (flux) of ions along a concentration gradient. Their ion conductivity is often highly specific and has been used for general classification into sodium, potassium, calcium, chloride and nonselective cation channels (Box 1). Opening and closing (‘gating’) is regulated by various stimuli including transmembrane voltage and ligand binding (Box 1). Functional screening assays exploit the fact that channel activation leads to flux of charged molecular species which is E-mail address: G.C. Terstappen ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.05.011
Screening assay systems exploiting ion flux Radioactive flux assays The use of radioactive isotopes of ions that pass through the channel under study – and hence, can serve as tracers for these ions in cellular assay systems – has a long tradition. In addition to using radioactive isotopes of the naturally conducting ion species as tracers, such as 22Na+ [1], 45Ca2+ [2] and 36Cl [3], other radioactive ion species, which are conducted by the channel can be employed as well. For instance, [14C]-guanidinium has been used for the study of sodium channels [1] and 86Rb+ for analysis of potassium and nonselective cation channels [4]. Based on ion conductivity and transmembrane concentration gradient (Fig. 1) influx of radiotracer is usually measured for sodium, calcium and chloride channels whereas efflux is measured for potassium channels upon activation. In these experiments, cells are usually grown in microplates and voltage-gated channels (Box 1) are activated by adding a ‘depolarizing’ concentration of 50 mM KCl to the cell medium whereas other channels are activated by adding an appropriate concentration of ligand (Box 1). Counting of radioactivity using standard equipment is either carried out in the cell supernatant, the cell lysate or both matrices. Dual measurements allow calculation of the relative flux of radiotracer, thus eliminating potenwww.drugdiscoverytoday.com
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Glossary Atomic absorption spectrometry (AAS): AAS is a technique traditionally used for the detection of trace elements in environmental, biological and medical samples. It utilizes thermal energy to generate free ground-state atoms in a vapor phase that absorb light of a specific wavelength (in the case of rubidium this is 780 nm). This absorption is proportional to the concentration of the element measured. Channelopathies: the term ‘channelopathies’ to define inherited ion channel disorders was introduced in the early 1990s with the discovery that several diseases associated with alterations in muscle membrane excitability were caused by missense mutations in voltage- and ligandgated ion channels. Focused libraries: as compared to ‘random’ compound libraries which consist of 100,000s of chemical entities, focused (often also called targeted or biased) compound libraries usually contain 1000–10,000s of compounds which are selected based on knowledge of the target protein and/or ligands binding to it. Fluorescence resonance energy transfer (FRET): FRET is a technique based on the transfer of energy from the excited state of a donor moiety to an acceptor. Because the measurable transfer efficiency depends on the distance between donor and acceptor, FRET can be used to estimate intramolecular distances (200–1000 nm) and to investigate interactions between macromolecules. States of ion channels: ion channels are highly dynamic macromolecules, which exist in multiple states such as open, closed and inactivated. The interconversions between these states are currently not well understood. Xenopus oocytes: Oocytes taken from the frog Xenopus laevis are widely employed as an efficient transient expression system for the study of ion channels. These several millimeter-sized cells faithfully and efficiently translate injected mRNA and cDNA leading to functional surface expression of ion channels. For screening purposes the lipid-rich yolk sack is an issue because it might adsorb relatively hydrophobic compounds.
tial well-to-well differences in cell densities and tracer loading. Radioactive flux assays, typically performed in 96- or 384-well plates, were in routine use in major pharmaceutical companies by the mid 1990s, mainly for HTS of calcium, potassium and nonselective cation channels. Since these assays represent a direct measure of channel activity, they are robust and insensitive to disturbances. By contrast, their temporal resolution is relatively low (seconds–minutes) and the transmembrane potential cannot be controlled. The main disadvantage is the use of radioisotopes, which is associated with significant costs, safety and environmental (e.g. disposal) problems.
Flux assays based on atomic absorption spectrometry A nonradioactive Rb+ efflux assay for screening of potassium and nonselective cation channels was introduced more recently [5]. This assay employs ATOMIC ABSORPTION SPECTROMETRY (AAS) for the determination of rubidium (see Glossary). Cells are loaded with Rb+, simply by replacing KCl with RbCl in a cell-compatible buffer. After a series of wash steps to remove extracellular Rb+, channel activation leads to Rb+ efflux, which is determined by measuring rubidium in the cell supernatant and cell lysate by AAS. Since its first description, this nonradioactive assay has found widespread application in drug discovery and greatly replaced 86Rb+ assays in the 134
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pharmaceutical industry [6]. In conjunction with the recent development of an innovative AAS instrument (ICR 12000; Aurora Biomed; http://www.aurorabiomed.com/) measurements of 12 samples at a time utilizing 96- or 384-well plates
Box 1. Classification of ion channels and mechanisms of activation (‘gating’) Ion channels can generally be classified into sodium, potassium, calcium, chloride and nonselective cation channels. The schematic drawings represent the membrane topology of the first channel type mentioned for each category. The pore region is highlighted in purple color. Na+ channels:
voltage-gated Na+ channels (Nav); amiloride-sensitive epithelial Na+ channel (ENaC), modulated by a variety of hormones that control activity via phosphorylation, GTP binding proteins and lipid metabolites; acid-sensing ion channels (ASIC), activated by pH 6.9 or higher H+ concentrations. K+ channels:
voltage-gated K+ channels (Kv); calcium-activated K+ channels (KCa); ATP-sensitive K+ channels (KATP); two-pore K+ channels (e.g. TWIK, TASK and TREK), activated by various physical and chemical stimuli including membrane stretching, temperature, acidosis, lipids and inhaled anaesthetics.
Ca2+ channels:
voltage-gated Ca2+ channels (Cav); ligand-gated Ca2+ channels comprise the ryanodine (RYR) and the IP3 (IP3R) receptors.
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Cl channels:
Drug Discovery Today: Technologies | Screening technology
allow a throughput of up to 60,000 samples per day (as stated in http://www.aurorabiomed.com/ICR12000.htm) making the nonradioactive Rb+ efflux assay compatible with the throughput requirements of HTS. Because of the high sensitivity of AAS for the determination of Li+ ions, influx experiments for screening of sodium channels, which display a high conductivity for Li+ [7], should also be possible in principle. Although no results have been published yet in scientific journals, preliminary results at a conference in the year 2003 were presented which looked promising (http://www.aurorabiomed.com/main-1.htm). A more indirect application for chloride channels was noted as well [8]. The major advantage of flux assays based on atomic absorption spectrometry relies in the fact that no radioactive isotopes are necessary.
Flux assays based on ion-specific fluorescence dyes
ligand-gated Cl channels comprise the glycine and GABAA receptors; voltage-gated Cl channels (CLC); cystic fibrosis conductance regulator (CFTR), activated by cAMP. Nonselective cation channels:
purinergic receptors (P2X), activated by ATP; cyclic nucleotide-gated ion channels, activated by cAMP or cGMP; are weakly voltage dependent (CNG channels) or require hyperpolarization (HCN channels); nicotinic acetylcholine receptors (nAChR), activated by the neurotransmitter acetylcholine; ionotropic glutamate receptors are classified based on their ligands as AMPA, NMDA and kainate receptors; 5-hydroxytryptamine 3 (5-HT3) receptors, activated by serotonin; transient receptor potential (TRP) channels, activated by stimuli such as temperature, touch, pain, osmolarity, pheromones and taste; polycystin kidney disease proteins (PKD) and the vanilloid receptors (TRPV) belong to this superfamily of ion channels.
At the beginning of the 1990s, scientists in major pharmaceutical companies with an interest in ion channels investigated the potential of ion-specific fluorescence indicator dyes (Molecular Probes, http://www.probes.com/) for screening assay development which exist for many ions including K+, Na+, Cl and Ca2+ [9–12] Upon ion binding, emission characteristics of such dyes change, often associated with an increased fluorescence intensity which can be measured. Apart from fluorescence probes for Ca2+, results were rather disappointing mainly owing to an often low ion binding affinity/selectivity and fluorescence quantum yield of indicator dyes in combination with small electrochemical concentration gradients for K+, Na+ and Cl ions (Fig. 1). Today, sensitive cell-permeable Ca2+ indicators with high binding affinities (Kd 300–400 nM) such as Fluo-3 and Fluo-4 [13] are routinely employed in Ca2+ flux assays for screening of calcium channels and nonselective cation channels with conductivity for Ca2+. Assay development is greatly facilitated by a steep electrochemical concentration gradient of about four orders of magnitude (Fig. 1). Typically, cells which are cultivated in microplates are loaded with cell-permeable acetoxymethyl (AM) ester derivatives of Fluo-3/4 which are trapped inside the cell upon enzymatic cleavage by cellular esterases. After removal of excessive extracellular dye by a wash step (which can be omitted if a fluorescence quencher such as Brilliant Black is added), channel activation leads to Ca2+ influx, an increased Ca2+ binding to indicator dye molecules and a concomitant increase in fluorescence emission which is typically measured at 525 nm (lEx 488 nm). The development of highly specialized fluorescence plate reader instrumentation such as the fluorescence imaging plate reader (FLIPR; Molecular Devices; http://www.moleculardevices.com/) systems, which measure through the bottom of microplates (intracellular fluorescence) and allow temporal resolution of transient Ca2+ peaks, was essential for the widespread routine application of this Ca2+ flux assay for HTS [14]. Today, these FLIPR systems exist already in third and fourth generations and www.drugdiscoverytoday.com
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Figure 1. Ion flux (current) along a concentration gradient leads to changes in transmembrane potential which can be quantified by the Nernst equation. Both properties are exploited for the development of functional ion channel screening assays. Whereas ion flux assays represent a direct measure of channel activity, assays measuring changes in membrane potential are indirect read-out sytems. Intra- and extracellular ion concentrations have been taken from Ref. [7].
allow measurements of 10,000s of data points per day in 384well plate format. ‘False positive’ hits in this assay might be obtained as a consequence of compound effects on cellular processes that change intracellular Ca2+ concentrations independent from the channel under study (e.g. calcium transporters and calcium release from intracellular organelles). Further disadvantages are the lack of transmembrane potential control and the limited temporal resolution (seconds).
Flux assays based on electrophysiology Patch clamp electrophysiology [15] is the ‘gold standard’ for functional analysis of ion channels. This method measures ion flux (current) within milliseconds with an electrode (glass micropipette), which is attached to the cell membrane while keeping the transmembrane potential constant (‘voltageclamped’) employing electronic negative feedback circuits (Fig. 2a). The basis of this method is the formation of a highresistance seal (>1 GV) between the cell membrane and the glass electrode which minimizes ‘leak current’ and allows even measurements of single channels. Only this method allows bona fide identification of state-dependent (see Glossary) ion channel modulators but its throughput is very limited (10 s of compounds per day) and thus not applicable for HTS. Hence, much research has been carried out intensifying at the end of the 1990s to develop electrophysiological methods with higher throughput (Table 1) [16]. In principle, these novel electrophysiological methods can be divided into automated robotic systems for patch clamping of Xenopus oocytes (see Glossary) or mammalian cells and systems in which traditional glass electrodes are replaced by planar arrays (‘chips’) as recording interfaces (Fig. 2b). Whereas automated patch clamping systems can increase throughput by a factor of about ten as compared to 136
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Figure 2. Electrophysiology-based assays allow precise control of the transmembrane potential by employing electronic negative feedback circuits. (a) The classical patch clamp technique is illustrated in which access to a single cell is achieved with a glass capillary electrode. (b) One individual well of a planar array (‘chip’)-based patch clamp system is depicted in which two compartments are connected by a micrometer-sized aperture. Access to the cell is achieved by ionophores such as amphotericine (as in the case of IonWorks) or mechanical rupture (as in the case of PatchXpress) to establish a whole-cell recording configuration.
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Table 1. Commercially available ‘higher throughput’ electrophysiology systems Technology
Main product
Company
URL
Automated voltage clamp of Xenopus oocytes
OpusXpress 6000 A Roboocyte
Axon Instr./Molecular Devices IonGate
http://www.axon.com/ http://www.iongate.de/
Automated voltage clamp of mammalian cells
Flyscreen Apatchi-1 AutoPatch
Flyion Sophion Bioscience Xention Discovery
http://www.flyion.com/ http://www.sophion.dk/ http://www.xention.com/
Planar array-based recording interfaces
PatchXpress 7000 A CytoPatch Automat IonWorks1 HT system; IonWorks1 Quattro Port-a-Patch QPatch
Axon Instr./Molecular Devices Cytocentrics CCS Molecular Devices Nanion Technologies Sophion
http://www.axon.com/ http://www.cytocentrics.com/ http://www.moleculardevices.com/ http://www.nanion.de/ http://www.sophion.dk/
standard patch clamp technology, array-based technologies were reported to increase throughput 100–1000-fold [17]. The first commercially available instrument (IonWorks HT system, Molecular Devices), which was launched in 2002, uses special 384-well plastic microplates (‘PatchPlate’) of which each single well contains a micrometer-seized aperture. Cells are added to the wells and pulled into the apertures by applying negative pressure, which results in seal formations (success rates between 60 and 90%) with electrical resistances of 50–600 mV. Electrical access to the interior of the cells is obtained by applying chemicals such as amphotericine (which perforate the cell membranes) to the bottom reservoir of the microplate thus establishing a whole cell-recording configuration. It was reported that between 2000 and 3000 cells per day could be ‘patch-clamped’ with this system [17]. Compared to traditional patch clamping, the lower electrical resistance is an issue, which limits application to cell lines with high surface expression of functional ion channels. More recently, a second array-based system was made available commercially (PatchXpress 7000A; Axon Instr./Molecular Devices) which uses 16well glass ‘chips’. Although the throughput is lower (240 compounds per day), GV resistance seals can be formed with success rates of 20–70%. A second-generation version of IonWorks with fourfold increased throughput (IonWorks Quattro) was launched in February 2005. This system allows measurements of averaged currents of cell populations because each well contains multiple apertures as recording sites. Even with the latest advances in innovative electrophysiology, all available systems (Table 1) are currently not completely compatible with the requirements of HTS and most useful as secondary assays in screening cascades to guide structure–activity relationship (SAR) studies with quantitative concentration– response data.
Screening assay systems exploiting changes of membrane potential Fluorescence assays employing membrane potential-sensitive (‘voltage-sensing’) dyes Activation of ion channels and the concomitant ion flux lead to changes in membrane potential (Fig. 1). In cellular sys-
tems, fluorescence probes such as the anionic oxonol DiBAC4(3) partition across the cell membrane according to the membrane potential independent from the ion selectivity of the channel [18]. Cellular hyperpolarization which occurs upon potassium channel activation, leads to a net extrusion of the dye from the cells whereas depolarization results in an increase in cytoplasmic DiBAC4(3). Determining changes in intracellular DiBAC4(3) fluorescence (lEx 480 nm; lEm 520 nm) is thus a measure of ion channel activation. Such ‘voltage-sensing’ fluorescence dyes (‘Nernstian dyes’), which were known since the 1960s were first employed for the development of ion channel screening assays at the beginning of the 1990s. In fact, the development of a ‘screen’ for the ATP-sensitive K+ channels (KATP) was probably one of the first examples which also triggered the development of appropriate instrumentation which allows to measure cellular fluorescence through the bottom of microplates (FLIPR systems, see above), an essential feature in this case. Because the distribution equilibrium of these partitioning dyes is a function of the transmembrane potential, cell supernatant containing the fluorescence dye cannot be removed (this would immediately lead to dye redistribution and thus unstable signals). Because of the small fraction of fluorescence dye inside the tiny cell volume, as compared to supernatant volumes (typically 10–100s of microliters), measurements have to be made ‘inside the cells’ and not through the supernatant. Based on their ‘optical geometry’ laser-based FLIPR systems measure largely cellular fluorescence and eliminate most of the bulk fluorescence of the supernatant. Simpler fluorescence plate readers can also be used if a non-cell-permeable quenching dye (see above) is added to the cell supernatant. Many assays employing DiBAC4(3) have been configured in major pharmaceutical companies and are widely used for ion channel HTS [19,20]. The slow response time of this dye (minutes) is a significant disadvantage, in particular for fast inactivating channels. Moreover, high numbers of ‘false positive’ compounds are obtained in HTS owing to the indirect nature of the read-out system, compound autofluorescence and compound-dye interactions. www.drugdiscoverytoday.com
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[18,21–23] [18–20] [5,6,8]
[9–14]
[16]
[16,17] [1-4] Refs
Indirect measurement of channel activity; low sensitivity (1% fluorescence change/mV) Indirect measurement of channel activity; slow response time of dye (minutes); low sensitivity (1% fluorescence change/mV) Low success rates of seal formation (depending on the technology between 20 and 90%); seal formation often with sub-GOhm resistances; expensive ‘chip’ consumables Throughput only tenfold increased over traditional patch clamping Mainly limited to calcium and nonselective cation channels; low temporal resolution (seconds); no transmembrane voltage control Mainly limited to potassium and nonselective cation channels; low temporal resolution (seconds–minutes); no transmembrane voltage control Use of radioactive isotopes; low temporal resolution (seconds–minutes); no transmembrane voltage control Cons
HTS compatible; fast response time of dye system (seconds) Transmembrane voltage control; high temporal resolution (milliseconds); 100–1000-fold increased throughput over traditional patch clamping Transmembrane voltage control; high temporal resolution (milliseconds); seal formation not different from traditional patch clamping No use of radioactive isotopes; HTS compatible Direct measure of channel activity; no use of radioactive isotopes; HTS compatible Direct measure of channel activity; HTS compatible Pros
Molecular Probes http://www.probes.com/ Molecular Devices http://www.moleculardevices.com/ Axon Instr./Molecular Devices http://www.axon.com/ Cytocentrics CCS http://www.cytocentrics.com/ Molecular Devices http://www.moleculardevices.com/ Nanion Technologies http://www.nanion.de/ Sophion http://www.sophion.dk/ Axon Instr./Molecular Devices http://www.axon.com/ IonGate http://www.iongate.de/ Flyion http://www.flyion.com/ Sophion Bioscience http://www.sophion.dk/ Xention Discovery http://www.xention.com/ Molecular Devices http://www.moleculardevices.com/ Molecular Probes http://www.probes.com/
Fluorescence assays employing membrane potential-sensitive dyes Planar array (‘chip’)-based recording interfaces Automated robotic systems for patch clamping Nonradioactive flux assays based on fluorescence dyes Nonradioactive flux assays based on AAS Radioactive flux assays Ion channel screening technologies
As true for most other methods and technologies, also in the case of ion channel screening for drug discovery no ‘universal’ method exists that fits all needs (Table 2). In fact, the application of a particular method largely depends on the nature of the ion channel, the number of compounds that need to be screened and the associated costs. The arsenal of functional screening assays has increased significantly over the past 10 years and greatly displaced radioligand binding assays which were frequently used in the 1980/1990s but do not provide information about the effect of compounds on channel function. Currently, primary HTS of large sample collections (hundreds of thousands of compounds) is mainly carried out with functional fluorescence-based assay systems – employing recombinant ion channels stably expressed in suitable eukaryotic cell lines – which exist for sodium, potassium, calcium and nonselective cation channels (Box 1). The high throughput that these assays allow usually compensates for the relatively high number of initial ‘false positive’ hits which often can easily be eliminated by testing hits on the non-recombinant cell line which does not express the target ion channel. The more robust nonradioactive Rb+ efflux assay can be considered a reasonable alternative for HTS of potas-
Table 2. Comparison summary table
Conclusions
Aurora BioMed http://www.aurorabiomed.com/
Based on membrane potential-sensitive dyes, a FLUORESCENCE assay system (see Glossary) was developed employing the modified oxonol DiSBAC2(3) as FRET acceptor and the coumarin-linked phospholipid CC2DMPE as FRET donor [21]. The phospholipid derivative remains stationary owing to its attachment to the outer leaflet of the cell membrane [22]. After loading cells with these dyes, hyperpolarization leads to an increase in FRET within seconds whereas depolarization reduces it. Because fluorescence intensities are simultaneously measured at the emission maximum of the donor (460 nm) and acceptor (570 nm) and the calculated ratio is used as measure of channel activity, some common factors of variability (e.g. differences in cell numbers and dye loading) can be eliminated. For HTS purposes, specialized instrumentation is usually necessary such as the voltage ion probe reader systems (VIPR, Aurora Discovery; http://www.auroradiscovery.com/) which were specifically developed for this type of FRET assay [23] or a more recently introduced novel FLIPR system (FLIPRTetra, Molecular Devices). The much faster response time of these FRET assays, which is in the range of seconds, and the ‘ratiometric’ nature of measurements are the major advantages over other fluorescence assays employing ‘voltage-sensing’ dyes. Also in this case, transmembrane potential is not controlled and measurements represent an indirect signal of channel activity thus suffering from comparatively high numbers of ‘false positives’ in HTS. RESONANCE ENERGY TRANSFER (FRET)
Companies and their websites
FRET assays employing membrane potential-sensitive dyes
Fluorescence resonance energy transfer (FRET) assays employing membrane potential-sensitive (‘voltage-sensing’) dyes
HTS compatible
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Molecular Probes http://www.probes.com/ Aurora Discovery http://www.auroradiscovery.com/
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Links Ion channels, transmitters, receptors and disease: http:// www.neuro.wustl.edu/neuromuscular/mother/chan.html Ligand-gated ion channel database: http://www.pasteur.fr/ recherche/banques/LGIC/LGIC.html Channelopathies: http://www.channelopathies.org CFTR review page: http://opal.msu.montana.edu/cftr/defaultbu.htm Ion channels: http://ifcsun1.ifisiol.unam.mx/Brain/ionchan.htm Ion channel links: http://www.hmbg.utah.edu/sanguinetti/Links/ links.htm> Ion channel web links: http://www.geocities.com/ionchannels/ Voltage-gated potassium channel database: http:// vkcdb.biology.ualberta.ca/
sium and nonselective cation channels but its use is not so widespread yet. Innovative transmembrane voltage-controlled electrophysiological technologies are not yet completely compatible with the requirements of primary HTS and thus most widely employed as secondary assays in screening cascades to provide quantitative concentration–response data during SAR studies. However, with the latest advancements in ‘higher throughput’ electrophysiology, primary screening of focused compound libraries (1000–10,000s of compounds; see Glossary) becomes feasible as well, thus allowing bona fide identification of state-dependent (see Glossary) modulators. This will enable the discovery of drugs that target only the ‘disease-state’ of an ion channel, thereby largely reducing potential side effects. However, despite the fact that currently about 50 human CHANNELOPATHIES (see Glossary) are known, detailed knowledge of ion channels causing disease phenotypes is very limited, even more which particular ion channel ‘states’ might be involved in a disease. Additional complexity is generated because functional ion channels are often homo- or heteromeric protein complexes, which can coassemble with accessory (b- and further) subunits, thus creating a vast number of physiological ion channel complexes with different functions and pharmacology.
Related articles Bennett, P.B. and Guthrie, H.R.E. (2003) Trends in ion channel drug discovery: advances in screening technologies. Trends Biotechnol. 21, 563–569 Editorial (2004) The state of ion channel research in 2004. Nat. Rev. Drug Discov. 3, 237–278 Mattheakis, L.C. and Savchenko, A. (2001) Assay technologies for screening ion channel targets. Curr. Opin. Drug Discov. Dev. 4, 124–134 Worley, J.F. and Main, M.J. (2002) An industrial perspective on utilizing functional ion channel assays for high throughput screening. Receptors Channels. 8, 269–282 Xu, J. et al. (2001) Ion channel assay technologies: quo vadis? Drug Discov. Today 6, 1278–1287
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Thus, advances in ion channel screening technologies have reached a point where knowledge about the biology and patho/physiology of ion channels might become the ratelimiting factor for meaningful drug discovery.
Outstanding issues What are the disease-relevant ion channel species? How can screening assays be configured that faithfully express the disease relevant ion channel species that are multi-subunit complexes comprising auxiliary proteins? Can automated electrophysiology be developed further to become fully compatible with the requirements of HTS? How can subtype selectivity of drugs be obtained?
References 1 Willow, M. et al. (1984) Inhibition of voltage-sensitive sodium channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs diphenylhydantoin and carbamazepine. Mol. Pharm. 25, 228–234 2 Rasmussen, C.A. et al. (1987) Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells. Circ. Res. 60, 495–504 3 Thampy, K.G. and Barnes, E.M., Jr (1984) Gamma-aminobutyric acidgated chloride channels in cultured cerebral neurons. J. Biol. Chem. 259, 1753–1757 4 Weir, S.W. and Weston, A.H. (1986) The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on 86Rb efflux in rat blood vessels. Br. J. Pharmacol. 88, 121–128 5 Terstappen, G.C. (1999) Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay. Anal. Biochem. 272, 149–155 6 Terstappen, G.C. (2004) Nonradioactive Rb+ efflux assay and its applications in drug discovery and development. Assay Drug Dev. Technol. 2, 553–559 7 Hille, B. (1992) Ionic channels of excitable membranes. Sinauer Associates 8 Stankovich, L. et al. (2004) Atomic absorption spectroscopy in ion channel screening. Assay Drug Dev. Technol. 2, 569–574 9 Kasner, S.E. and Ganz, M.B. (1992) Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye PBFI. Am. J. Physiol. 262, 462–467 10 Minta, A. and Tsien, R.Y. (1989) Fluorescent indicators for cytosolic sodium. J. Biol. Chem. 264, 19449–19457 11 Verkman, A.S. et al. (1989) Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications. Anal. Biochem. 178, 355–361 12 Grynkiewicz, G. et al. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 13 Minta, A. et al. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178 14 Schroeder, K.S. and Neagle, B.D. (1996) FLIPR: A new instrument for accurate high-throughput optical screening. J. Biomol. Screen. 1, 75–80 15 Hamill, O.P. et al. (1981) Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches. Pflugers Arch. Eur. J. Physiol. 391, 85–100 16 Special issue (2003) Receptors Channels, 9, 1–66 17 Schroeder, K. et al. (2003) Ionworks HT: a new high-throughput electrophysiology measurement platform. J. Biomol. Screen. 8, 50–64 18 Epps, D.E. et al. (1994) Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) in model systems and cells. Chem. Phys. Lipids 69, 137–150 www.drugdiscoverytoday.com
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Terstappen, G.C. et al. (2001) Pharmacological characterization of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines. Neuropharmacology 40, 772–783 Whiteaker, K.L. et al. (2001) Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators. J. Biomol. Screen. 6, 305–312
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Gonzalez, J.E. and Tsien, R.Y. (1995) Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys. J. 69, 1272–1280 Gonzalez, J.E. and Tsien, R.Y. (1997) Improved indicators of cell membrane potential that use fluorescence resonance energy. Chem. Biol. 4, 269–277 Falconer, M. et al. (2002) High-throughput screening for ion channel modulators. J. Biomol. Screen. 7, 460–465
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Screening technology
Ion channel screening technologies today Georg C. Terstappen Sienabiotech S.p.A., Discovery Research, Via Fiorentina 1, 53100 Siena, Italy
For every heartbeat, movement and thought, ion channels have to open and close, and thus, it is not surprising that malfunctioning of these membrane proteins leads to serious diseases. Today, only 7% of all marketed
Section Editors: Jeff Brockman – Psychiatric Genomics Inc., Gaithersburg, MD, USA Mark Divers – AstraZeneca, Mo¨lndal, Sweden
drugs act on ion channels but the systematic exploitation of this important target class has started mainly enabled by novel screening technologies. Thus, the
associated with transient changes in membrane potential (Fig. 1).
discovery of selective and state-dependent drugs is on the horizon, hopefully leading to effective novel medicines.
Introduction If no information is available about a target protein structure or ligands, drug discovery typically starts with high-throughput screening (HTS) of hundreds of thousands of chemical molecules. Although assays that are compatible with the requirements of HTS have been in use since the beginning of the 1990s for target classes such as G protein-coupled receptors and enzymes, such assays for ion channels have become available only more recently. Ion channels are poreforming membrane proteins which enable rapid membrane passage (flux) of ions along a concentration gradient. Their ion conductivity is often highly specific and has been used for general classification into sodium, potassium, calcium, chloride and nonselective cation channels (Box 1). Opening and closing (‘gating’) is regulated by various stimuli including transmembrane voltage and ligand binding (Box 1). Functional screening assays exploit the fact that channel activation leads to flux of charged molecular species which is E-mail address: G.C. Terstappen ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.05.011
Screening assay systems exploiting ion flux Radioactive flux assays The use of radioactive isotopes of ions that pass through the channel under study – and hence, can serve as tracers for these ions in cellular assay systems – has a long tradition. In addition to using radioactive isotopes of the naturally conducting ion species as tracers, such as 22Na+ [1], 45Ca2+ [2] and 36Cl [3], other radioactive ion species, which are conducted by the channel can be employed as well. For instance, [14C]-guanidinium has been used for the study of sodium channels [1] and 86Rb+ for analysis of potassium and nonselective cation channels [4]. Based on ion conductivity and transmembrane concentration gradient (Fig. 1) influx of radiotracer is usually measured for sodium, calcium and chloride channels whereas efflux is measured for potassium channels upon activation. In these experiments, cells are usually grown in microplates and voltage-gated channels (Box 1) are activated by adding a ‘depolarizing’ concentration of 50 mM KCl to the cell medium whereas other channels are activated by adding an appropriate concentration of ligand (Box 1). Counting of radioactivity using standard equipment is either carried out in the cell supernatant, the cell lysate or both matrices. Dual measurements allow calculation of the relative flux of radiotracer, thus eliminating potenwww.drugdiscoverytoday.com
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Glossary Atomic absorption spectrometry (AAS): AAS is a technique traditionally used for the detection of trace elements in environmental, biological and medical samples. It utilizes thermal energy to generate free ground-state atoms in a vapor phase that absorb light of a specific wavelength (in the case of rubidium this is 780 nm). This absorption is proportional to the concentration of the element measured. Channelopathies: the term ‘channelopathies’ to define inherited ion channel disorders was introduced in the early 1990s with the discovery that several diseases associated with alterations in muscle membrane excitability were caused by missense mutations in voltage- and ligandgated ion channels. Focused libraries: as compared to ‘random’ compound libraries which consist of 100,000s of chemical entities, focused (often also called targeted or biased) compound libraries usually contain 1000–10,000s of compounds which are selected based on knowledge of the target protein and/or ligands binding to it. Fluorescence resonance energy transfer (FRET): FRET is a technique based on the transfer of energy from the excited state of a donor moiety to an acceptor. Because the measurable transfer efficiency depends on the distance between donor and acceptor, FRET can be used to estimate intramolecular distances (200–1000 nm) and to investigate interactions between macromolecules. States of ion channels: ion channels are highly dynamic macromolecules, which exist in multiple states such as open, closed and inactivated. The interconversions between these states are currently not well understood. Xenopus oocytes: Oocytes taken from the frog Xenopus laevis are widely employed as an efficient transient expression system for the study of ion channels. These several millimeter-sized cells faithfully and efficiently translate injected mRNA and cDNA leading to functional surface expression of ion channels. For screening purposes the lipid-rich yolk sack is an issue because it might adsorb relatively hydrophobic compounds.
tial well-to-well differences in cell densities and tracer loading. Radioactive flux assays, typically performed in 96- or 384-well plates, were in routine use in major pharmaceutical companies by the mid 1990s, mainly for HTS of calcium, potassium and nonselective cation channels. Since these assays represent a direct measure of channel activity, they are robust and insensitive to disturbances. By contrast, their temporal resolution is relatively low (seconds–minutes) and the transmembrane potential cannot be controlled. The main disadvantage is the use of radioisotopes, which is associated with significant costs, safety and environmental (e.g. disposal) problems.
Flux assays based on atomic absorption spectrometry A nonradioactive Rb+ efflux assay for screening of potassium and nonselective cation channels was introduced more recently [5]. This assay employs ATOMIC ABSORPTION SPECTROMETRY (AAS) for the determination of rubidium (see Glossary). Cells are loaded with Rb+, simply by replacing KCl with RbCl in a cell-compatible buffer. After a series of wash steps to remove extracellular Rb+, channel activation leads to Rb+ efflux, which is determined by measuring rubidium in the cell supernatant and cell lysate by AAS. Since its first description, this nonradioactive assay has found widespread application in drug discovery and greatly replaced 86Rb+ assays in the 134
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pharmaceutical industry [6]. In conjunction with the recent development of an innovative AAS instrument (ICR 12000; Aurora Biomed; http://www.aurorabiomed.com/) measurements of 12 samples at a time utilizing 96- or 384-well plates
Box 1. Classification of ion channels and mechanisms of activation (‘gating’) Ion channels can generally be classified into sodium, potassium, calcium, chloride and nonselective cation channels. The schematic drawings represent the membrane topology of the first channel type mentioned for each category. The pore region is highlighted in purple color. Na+ channels:
voltage-gated Na+ channels (Nav); amiloride-sensitive epithelial Na+ channel (ENaC), modulated by a variety of hormones that control activity via phosphorylation, GTP binding proteins and lipid metabolites; acid-sensing ion channels (ASIC), activated by pH 6.9 or higher H+ concentrations. K+ channels:
voltage-gated K+ channels (Kv); calcium-activated K+ channels (KCa); ATP-sensitive K+ channels (KATP); two-pore K+ channels (e.g. TWIK, TASK and TREK), activated by various physical and chemical stimuli including membrane stretching, temperature, acidosis, lipids and inhaled anaesthetics.
Ca2+ channels:
voltage-gated Ca2+ channels (Cav); ligand-gated Ca2+ channels comprise the ryanodine (RYR) and the IP3 (IP3R) receptors.
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Cl channels:
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allow a throughput of up to 60,000 samples per day (as stated in http://www.aurorabiomed.com/ICR12000.htm) making the nonradioactive Rb+ efflux assay compatible with the throughput requirements of HTS. Because of the high sensitivity of AAS for the determination of Li+ ions, influx experiments for screening of sodium channels, which display a high conductivity for Li+ [7], should also be possible in principle. Although no results have been published yet in scientific journals, preliminary results at a conference in the year 2003 were presented which looked promising (http://www.aurorabiomed.com/main-1.htm). A more indirect application for chloride channels was noted as well [8]. The major advantage of flux assays based on atomic absorption spectrometry relies in the fact that no radioactive isotopes are necessary.
Flux assays based on ion-specific fluorescence dyes
ligand-gated Cl channels comprise the glycine and GABAA receptors; voltage-gated Cl channels (CLC); cystic fibrosis conductance regulator (CFTR), activated by cAMP. Nonselective cation channels:
purinergic receptors (P2X), activated by ATP; cyclic nucleotide-gated ion channels, activated by cAMP or cGMP; are weakly voltage dependent (CNG channels) or require hyperpolarization (HCN channels); nicotinic acetylcholine receptors (nAChR), activated by the neurotransmitter acetylcholine; ionotropic glutamate receptors are classified based on their ligands as AMPA, NMDA and kainate receptors; 5-hydroxytryptamine 3 (5-HT3) receptors, activated by serotonin; transient receptor potential (TRP) channels, activated by stimuli such as temperature, touch, pain, osmolarity, pheromones and taste; polycystin kidney disease proteins (PKD) and the vanilloid receptors (TRPV) belong to this superfamily of ion channels.
At the beginning of the 1990s, scientists in major pharmaceutical companies with an interest in ion channels investigated the potential of ion-specific fluorescence indicator dyes (Molecular Probes, http://www.probes.com/) for screening assay development which exist for many ions including K+, Na+, Cl and Ca2+ [9–12] Upon ion binding, emission characteristics of such dyes change, often associated with an increased fluorescence intensity which can be measured. Apart from fluorescence probes for Ca2+, results were rather disappointing mainly owing to an often low ion binding affinity/selectivity and fluorescence quantum yield of indicator dyes in combination with small electrochemical concentration gradients for K+, Na+ and Cl ions (Fig. 1). Today, sensitive cell-permeable Ca2+ indicators with high binding affinities (Kd 300–400 nM) such as Fluo-3 and Fluo-4 [13] are routinely employed in Ca2+ flux assays for screening of calcium channels and nonselective cation channels with conductivity for Ca2+. Assay development is greatly facilitated by a steep electrochemical concentration gradient of about four orders of magnitude (Fig. 1). Typically, cells which are cultivated in microplates are loaded with cell-permeable acetoxymethyl (AM) ester derivatives of Fluo-3/4 which are trapped inside the cell upon enzymatic cleavage by cellular esterases. After removal of excessive extracellular dye by a wash step (which can be omitted if a fluorescence quencher such as Brilliant Black is added), channel activation leads to Ca2+ influx, an increased Ca2+ binding to indicator dye molecules and a concomitant increase in fluorescence emission which is typically measured at 525 nm (lEx 488 nm). The development of highly specialized fluorescence plate reader instrumentation such as the fluorescence imaging plate reader (FLIPR; Molecular Devices; http://www.moleculardevices.com/) systems, which measure through the bottom of microplates (intracellular fluorescence) and allow temporal resolution of transient Ca2+ peaks, was essential for the widespread routine application of this Ca2+ flux assay for HTS [14]. Today, these FLIPR systems exist already in third and fourth generations and www.drugdiscoverytoday.com
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Figure 1. Ion flux (current) along a concentration gradient leads to changes in transmembrane potential which can be quantified by the Nernst equation. Both properties are exploited for the development of functional ion channel screening assays. Whereas ion flux assays represent a direct measure of channel activity, assays measuring changes in membrane potential are indirect read-out sytems. Intra- and extracellular ion concentrations have been taken from Ref. [7].
allow measurements of 10,000s of data points per day in 384well plate format. ‘False positive’ hits in this assay might be obtained as a consequence of compound effects on cellular processes that change intracellular Ca2+ concentrations independent from the channel under study (e.g. calcium transporters and calcium release from intracellular organelles). Further disadvantages are the lack of transmembrane potential control and the limited temporal resolution (seconds).
Flux assays based on electrophysiology Patch clamp electrophysiology [15] is the ‘gold standard’ for functional analysis of ion channels. This method measures ion flux (current) within milliseconds with an electrode (glass micropipette), which is attached to the cell membrane while keeping the transmembrane potential constant (‘voltageclamped’) employing electronic negative feedback circuits (Fig. 2a). The basis of this method is the formation of a highresistance seal (>1 GV) between the cell membrane and the glass electrode which minimizes ‘leak current’ and allows even measurements of single channels. Only this method allows bona fide identification of state-dependent (see Glossary) ion channel modulators but its throughput is very limited (10 s of compounds per day) and thus not applicable for HTS. Hence, much research has been carried out intensifying at the end of the 1990s to develop electrophysiological methods with higher throughput (Table 1) [16]. In principle, these novel electrophysiological methods can be divided into automated robotic systems for patch clamping of Xenopus oocytes (see Glossary) or mammalian cells and systems in which traditional glass electrodes are replaced by planar arrays (‘chips’) as recording interfaces (Fig. 2b). Whereas automated patch clamping systems can increase throughput by a factor of about ten as compared to 136
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Figure 2. Electrophysiology-based assays allow precise control of the transmembrane potential by employing electronic negative feedback circuits. (a) The classical patch clamp technique is illustrated in which access to a single cell is achieved with a glass capillary electrode. (b) One individual well of a planar array (‘chip’)-based patch clamp system is depicted in which two compartments are connected by a micrometer-sized aperture. Access to the cell is achieved by ionophores such as amphotericine (as in the case of IonWorks) or mechanical rupture (as in the case of PatchXpress) to establish a whole-cell recording configuration.
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Table 1. Commercially available ‘higher throughput’ electrophysiology systems Technology
Main product
Company
URL
Automated voltage clamp of Xenopus oocytes
OpusXpress 6000 A Roboocyte
Axon Instr./Molecular Devices IonGate
http://www.axon.com/ http://www.iongate.de/
Automated voltage clamp of mammalian cells
Flyscreen Apatchi-1 AutoPatch
Flyion Sophion Bioscience Xention Discovery
http://www.flyion.com/ http://www.sophion.dk/ http://www.xention.com/
Planar array-based recording interfaces
PatchXpress 7000 A CytoPatch Automat IonWorks1 HT system; IonWorks1 Quattro Port-a-Patch QPatch
Axon Instr./Molecular Devices Cytocentrics CCS Molecular Devices Nanion Technologies Sophion
http://www.axon.com/ http://www.cytocentrics.com/ http://www.moleculardevices.com/ http://www.nanion.de/ http://www.sophion.dk/
standard patch clamp technology, array-based technologies were reported to increase throughput 100–1000-fold [17]. The first commercially available instrument (IonWorks HT system, Molecular Devices), which was launched in 2002, uses special 384-well plastic microplates (‘PatchPlate’) of which each single well contains a micrometer-seized aperture. Cells are added to the wells and pulled into the apertures by applying negative pressure, which results in seal formations (success rates between 60 and 90%) with electrical resistances of 50–600 mV. Electrical access to the interior of the cells is obtained by applying chemicals such as amphotericine (which perforate the cell membranes) to the bottom reservoir of the microplate thus establishing a whole cell-recording configuration. It was reported that between 2000 and 3000 cells per day could be ‘patch-clamped’ with this system [17]. Compared to traditional patch clamping, the lower electrical resistance is an issue, which limits application to cell lines with high surface expression of functional ion channels. More recently, a second array-based system was made available commercially (PatchXpress 7000A; Axon Instr./Molecular Devices) which uses 16well glass ‘chips’. Although the throughput is lower (240 compounds per day), GV resistance seals can be formed with success rates of 20–70%. A second-generation version of IonWorks with fourfold increased throughput (IonWorks Quattro) was launched in February 2005. This system allows measurements of averaged currents of cell populations because each well contains multiple apertures as recording sites. Even with the latest advances in innovative electrophysiology, all available systems (Table 1) are currently not completely compatible with the requirements of HTS and most useful as secondary assays in screening cascades to guide structure–activity relationship (SAR) studies with quantitative concentration– response data.
Screening assay systems exploiting changes of membrane potential Fluorescence assays employing membrane potential-sensitive (‘voltage-sensing’) dyes Activation of ion channels and the concomitant ion flux lead to changes in membrane potential (Fig. 1). In cellular sys-
tems, fluorescence probes such as the anionic oxonol DiBAC4(3) partition across the cell membrane according to the membrane potential independent from the ion selectivity of the channel [18]. Cellular hyperpolarization which occurs upon potassium channel activation, leads to a net extrusion of the dye from the cells whereas depolarization results in an increase in cytoplasmic DiBAC4(3). Determining changes in intracellular DiBAC4(3) fluorescence (lEx 480 nm; lEm 520 nm) is thus a measure of ion channel activation. Such ‘voltage-sensing’ fluorescence dyes (‘Nernstian dyes’), which were known since the 1960s were first employed for the development of ion channel screening assays at the beginning of the 1990s. In fact, the development of a ‘screen’ for the ATP-sensitive K+ channels (KATP) was probably one of the first examples which also triggered the development of appropriate instrumentation which allows to measure cellular fluorescence through the bottom of microplates (FLIPR systems, see above), an essential feature in this case. Because the distribution equilibrium of these partitioning dyes is a function of the transmembrane potential, cell supernatant containing the fluorescence dye cannot be removed (this would immediately lead to dye redistribution and thus unstable signals). Because of the small fraction of fluorescence dye inside the tiny cell volume, as compared to supernatant volumes (typically 10–100s of microliters), measurements have to be made ‘inside the cells’ and not through the supernatant. Based on their ‘optical geometry’ laser-based FLIPR systems measure largely cellular fluorescence and eliminate most of the bulk fluorescence of the supernatant. Simpler fluorescence plate readers can also be used if a non-cell-permeable quenching dye (see above) is added to the cell supernatant. Many assays employing DiBAC4(3) have been configured in major pharmaceutical companies and are widely used for ion channel HTS [19,20]. The slow response time of this dye (minutes) is a significant disadvantage, in particular for fast inactivating channels. Moreover, high numbers of ‘false positive’ compounds are obtained in HTS owing to the indirect nature of the read-out system, compound autofluorescence and compound-dye interactions. www.drugdiscoverytoday.com
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[18,21–23] [18–20] [5,6,8]
[9–14]
[16]
[16,17] [1-4] Refs
Indirect measurement of channel activity; low sensitivity (1% fluorescence change/mV) Indirect measurement of channel activity; slow response time of dye (minutes); low sensitivity (1% fluorescence change/mV) Low success rates of seal formation (depending on the technology between 20 and 90%); seal formation often with sub-GOhm resistances; expensive ‘chip’ consumables Throughput only tenfold increased over traditional patch clamping Mainly limited to calcium and nonselective cation channels; low temporal resolution (seconds); no transmembrane voltage control Mainly limited to potassium and nonselective cation channels; low temporal resolution (seconds–minutes); no transmembrane voltage control Use of radioactive isotopes; low temporal resolution (seconds–minutes); no transmembrane voltage control Cons
HTS compatible; fast response time of dye system (seconds) Transmembrane voltage control; high temporal resolution (milliseconds); 100–1000-fold increased throughput over traditional patch clamping Transmembrane voltage control; high temporal resolution (milliseconds); seal formation not different from traditional patch clamping No use of radioactive isotopes; HTS compatible Direct measure of channel activity; no use of radioactive isotopes; HTS compatible Direct measure of channel activity; HTS compatible Pros
Molecular Probes http://www.probes.com/ Molecular Devices http://www.moleculardevices.com/ Axon Instr./Molecular Devices http://www.axon.com/ Cytocentrics CCS http://www.cytocentrics.com/ Molecular Devices http://www.moleculardevices.com/ Nanion Technologies http://www.nanion.de/ Sophion http://www.sophion.dk/ Axon Instr./Molecular Devices http://www.axon.com/ IonGate http://www.iongate.de/ Flyion http://www.flyion.com/ Sophion Bioscience http://www.sophion.dk/ Xention Discovery http://www.xention.com/ Molecular Devices http://www.moleculardevices.com/ Molecular Probes http://www.probes.com/
Fluorescence assays employing membrane potential-sensitive dyes Planar array (‘chip’)-based recording interfaces Automated robotic systems for patch clamping Nonradioactive flux assays based on fluorescence dyes Nonradioactive flux assays based on AAS Radioactive flux assays Ion channel screening technologies
As true for most other methods and technologies, also in the case of ion channel screening for drug discovery no ‘universal’ method exists that fits all needs (Table 2). In fact, the application of a particular method largely depends on the nature of the ion channel, the number of compounds that need to be screened and the associated costs. The arsenal of functional screening assays has increased significantly over the past 10 years and greatly displaced radioligand binding assays which were frequently used in the 1980/1990s but do not provide information about the effect of compounds on channel function. Currently, primary HTS of large sample collections (hundreds of thousands of compounds) is mainly carried out with functional fluorescence-based assay systems – employing recombinant ion channels stably expressed in suitable eukaryotic cell lines – which exist for sodium, potassium, calcium and nonselective cation channels (Box 1). The high throughput that these assays allow usually compensates for the relatively high number of initial ‘false positive’ hits which often can easily be eliminated by testing hits on the non-recombinant cell line which does not express the target ion channel. The more robust nonradioactive Rb+ efflux assay can be considered a reasonable alternative for HTS of potas-
Table 2. Comparison summary table
Conclusions
Aurora BioMed http://www.aurorabiomed.com/
Based on membrane potential-sensitive dyes, a FLUORESCENCE assay system (see Glossary) was developed employing the modified oxonol DiSBAC2(3) as FRET acceptor and the coumarin-linked phospholipid CC2DMPE as FRET donor [21]. The phospholipid derivative remains stationary owing to its attachment to the outer leaflet of the cell membrane [22]. After loading cells with these dyes, hyperpolarization leads to an increase in FRET within seconds whereas depolarization reduces it. Because fluorescence intensities are simultaneously measured at the emission maximum of the donor (460 nm) and acceptor (570 nm) and the calculated ratio is used as measure of channel activity, some common factors of variability (e.g. differences in cell numbers and dye loading) can be eliminated. For HTS purposes, specialized instrumentation is usually necessary such as the voltage ion probe reader systems (VIPR, Aurora Discovery; http://www.auroradiscovery.com/) which were specifically developed for this type of FRET assay [23] or a more recently introduced novel FLIPR system (FLIPRTetra, Molecular Devices). The much faster response time of these FRET assays, which is in the range of seconds, and the ‘ratiometric’ nature of measurements are the major advantages over other fluorescence assays employing ‘voltage-sensing’ dyes. Also in this case, transmembrane potential is not controlled and measurements represent an indirect signal of channel activity thus suffering from comparatively high numbers of ‘false positives’ in HTS. RESONANCE ENERGY TRANSFER (FRET)
Companies and their websites
FRET assays employing membrane potential-sensitive dyes
Fluorescence resonance energy transfer (FRET) assays employing membrane potential-sensitive (‘voltage-sensing’) dyes
HTS compatible
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Molecular Probes http://www.probes.com/ Aurora Discovery http://www.auroradiscovery.com/
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Links Ion channels, transmitters, receptors and disease: http:// www.neuro.wustl.edu/neuromuscular/mother/chan.html Ligand-gated ion channel database: http://www.pasteur.fr/ recherche/banques/LGIC/LGIC.html Channelopathies: http://www.channelopathies.org CFTR review page: http://opal.msu.montana.edu/cftr/defaultbu.htm Ion channels: http://ifcsun1.ifisiol.unam.mx/Brain/ionchan.htm Ion channel links: http://www.hmbg.utah.edu/sanguinetti/Links/ links.htm> Ion channel web links: http://www.geocities.com/ionchannels/ Voltage-gated potassium channel database: http:// vkcdb.biology.ualberta.ca/
sium and nonselective cation channels but its use is not so widespread yet. Innovative transmembrane voltage-controlled electrophysiological technologies are not yet completely compatible with the requirements of primary HTS and thus most widely employed as secondary assays in screening cascades to provide quantitative concentration–response data during SAR studies. However, with the latest advancements in ‘higher throughput’ electrophysiology, primary screening of focused compound libraries (1000–10,000s of compounds; see Glossary) becomes feasible as well, thus allowing bona fide identification of state-dependent (see Glossary) modulators. This will enable the discovery of drugs that target only the ‘disease-state’ of an ion channel, thereby largely reducing potential side effects. However, despite the fact that currently about 50 human CHANNELOPATHIES (see Glossary) are known, detailed knowledge of ion channels causing disease phenotypes is very limited, even more which particular ion channel ‘states’ might be involved in a disease. Additional complexity is generated because functional ion channels are often homo- or heteromeric protein complexes, which can coassemble with accessory (b- and further) subunits, thus creating a vast number of physiological ion channel complexes with different functions and pharmacology.
Related articles Bennett, P.B. and Guthrie, H.R.E. (2003) Trends in ion channel drug discovery: advances in screening technologies. Trends Biotechnol. 21, 563–569 Editorial (2004) The state of ion channel research in 2004. Nat. Rev. Drug Discov. 3, 237–278 Mattheakis, L.C. and Savchenko, A. (2001) Assay technologies for screening ion channel targets. Curr. Opin. Drug Discov. Dev. 4, 124–134 Worley, J.F. and Main, M.J. (2002) An industrial perspective on utilizing functional ion channel assays for high throughput screening. Receptors Channels. 8, 269–282 Xu, J. et al. (2001) Ion channel assay technologies: quo vadis? Drug Discov. Today 6, 1278–1287
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Thus, advances in ion channel screening technologies have reached a point where knowledge about the biology and patho/physiology of ion channels might become the ratelimiting factor for meaningful drug discovery.
Outstanding issues What are the disease-relevant ion channel species? How can screening assays be configured that faithfully express the disease relevant ion channel species that are multi-subunit complexes comprising auxiliary proteins? Can automated electrophysiology be developed further to become fully compatible with the requirements of HTS? How can subtype selectivity of drugs be obtained?
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