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Technology Review High Throughput Assay Technologies for Ion 5315_16_p543-552 11/5/04 12:35 PM Page 543 ASSAY and Drug Development Technologies Volume 2, Number 5, 2004 © Mary Ann Liebert, Inc. • Technology Review • High Throughput Assay Technologies for Ion Channel Drug Discovery Wei Zheng,1 Robert H. Spencer,2 and Laszlo Kiss2 Abstract: Ion channels represent a class of membrane spanning protein pores that mediate the flux of ions in a variety of cell types. To date, Ͼ400 ion channels have been cloned and characterized, and some of these channels have emerged as attractive drug targets. Several existing medications elicit their therapeutic effect through the modulation of ion channels, underscoring the importance of ion channels as a target class for modern drug discovery. To meet the increasing demand for high- throughput screening of ion channels, assay technologies have evolved rapidly over the past 5–10 years. In this article, the authors review the technologies that are currently used for the screening of ion channels. The technologies discussed are binding assays, ion flux assays, fluorescence-based assays, and automated patch-clamp instrumentation. Introduction in several cases, produce a lethal ventricular arrhythmia known as torsade de pointes.6–8 The mechanism under- ON CHANNELS ARE membrane-spanning proteins that lying this toxic effect involves inhibition of one or more Iform pores through which inorganic ions, such as Naϩ, of the cardiac ion channels: (1) the hERG potassium ϩ 2ϩ Ϫ K , Ca , and Cl , can rapidly traverse the cell mem- channel (Ikr); (2) the KCNQ1/KCNE1 potassium chan- 9,10 brane down their electrochemical gradient (Table 1). It nel (Iks); and (3) the SCN5A sodium channel. is well established that ion channels play a vital role in Ion channels can be broadly grouped into two major neuronal signal transduction, neurotransmitter release, classes11,12: ligand-gated and voltage-gated ion channels muscle contraction, cell secretion, enzyme activation, and (Table 2). The primary distinguishing feature is that volt- gene transcription. To date, upwards of 400 different hu- age-gated ion channels do not have endogenous li- man ion channel genes have been identified. Mutations gands, but are activated by changes in the membrane that disrupt or alter channel function have been associ- potential. Ion channels can exist in multiple states such ated with many diseases (“channelopathies”), including as the closed, open, and inactivated states. Voltage-gated hypertension, cardiac arrhythmia, diabetes, cystic fibro- ion channels transition (gate) between these states in re- sis, and a variety of neuronal disorders.1–5 Thus, ion chan- sponse to changes in membrane potential. In contrast, li- nels represent an important class of molecular targets for gand-gated channels transition between these states in drug development. response to the binding and unbinding of a ligand. In the The role of ion channels in drug safety has also open state, ions can flow through a single ion channel emerged as an important issue in the last several years. pore at prodigious rates of over 107 ions/s. Cell-based Since 1985, five drugs have been withdrawn from the functional assays are an essential requirement for the market because they prolong the cardiac QT interval and, screening of ion channels at both the primary and sec- Departments of 1Automated Biotechnology and 2Molecular Neurology, Merck Research Laboratories, West Point, PA. ABBREVIATIONS: AAS, atomic absorbance spectrometry; CCD, charge-coupled device; FLIPR, fluorescent-imaging plate reader; FRET, fluorescence resonance energy transfer; hERG, human ether-go-go-related gene; HTS, high throughput screening; IC50, 50% inhibitory concen- tration; SPA, scintillation proximity assay. 543 5315_16_p543-552 11/5/04 12:35 PM Page 544 544 Zheng et al. TABLE 1. ION CONCENTRATIONS INSIDE Demands on Ion Channel Screening Assays AND OUTSIDEOFACELL Concentration The current demands for ion channel screening in the drug discovery process can be grouped into three main Ion Extracellular Intracellular areas: primary screening assays, secondary screening as- says, and ion channel safety assessment. Naϩ 145 mM 12 mM Kϩ 4.5 mM 140 mM Ca2ϩ 1.8 mM 0.1–0.2 ␮M (cytosol, resting cell) 100 ␮M (cytosol, stimulated cell) Primary screening assays (HTS) Mg2ϩ 1.5 mM 0.8 mM ClϪ 116 mM 4 mM HTS has seen a tremendous advance during the last pH 7.4 7.1 10 years and remains the first critical step in the dis- covery of lead chemical structures for novel drug tar- gets. To increase the probability of success for finding new leads from HTS, many companies have invested ondary levels. Traditional methods developed for HTS heavily in expanding both the diversity and quality of of ion channels, such as binding, ion flux, and fluores- their compound libraries. For most mid- and large-sized cent probes, measure ion channel activity indirectly. companies, the library collection has grown to 400,000– Patch-clamp electrophysiology is regarded as the “gold 1,000,000 (or more) compounds. The standard para- standard” for measuring ion channel activity and phar- digms used to screen these libraries have evolved to au- macology. Patch-clamp allows for the direct, real-time tomated 384-well or higher-density single-compound measurement of ion channel activity, but in its traditional test formats. Minimal throughput of 30,000 (ideally format is low-throughput and requires a high degree of Ͼ100,000) compounds/day has become the requisite. operator skill. Hence, drug screening assays for ion chan- An important consideration in screening compound li- nels, in comparison to those for enzyme and receptor tar- braries of this size is the materials used in assay devel- gets, have compromised data quality for throughput. Re- opment (including dead volume during robotic screen- cently, a number of new screening technologies have been ing, positive and negative controls, etc.), which can developed and improved for ion channel assays and are typically account for 30–60% of the total reagent and poised to change this.13–15 A summary of the currently consumable requirements for completion of a single tar- available screening technologies is listed in Table 3. get screen. TABLE 2. CLASSIFICATION OF ION CHANNELS Channel type Activator Ion permeability TM domains a Ligand-gated ion channels 2ϩ IP3RIP3 Ca 6-TM CNG cAMP Naϩ, Kϩ, Ca2ϩ 6-TM nAChR ACh, nicotine Naϩ, Kϩ, Ca2ϩ 4-TM ϩ ϩ 2ϩ 5-HT3 5-HT Na , K , Ca 4-TM Ϫ GABAA,C GABA Cl 4-TM Glycine GABA ClϪ 4-TM NMDA Glutamate, NMDA Naϩ, Kϩ, Ca2ϩ 3-TM AMPA Glutamate, AMPA Naϩ, Kϩ, Ca2ϩ 3-TM Kainate Glutamate Naϩ, Kϩ, Ca2ϩ 3-TM P2X, P2Z ATP Naϩ 2-TM Voltage-gated ion channels Kϩ channels membrane potential Kϩ 6-TM Naϩ channels membrane potential Naϩ 24-TM Ca2ϩ channels membrane potential Ca2ϩ 24-TM ClϪ channels membrane potential ClϪ 12-TM TM, transmembrane; IP3, inositol trisphophate; IP3R, IP3 receptor; CNG, cyclic nucle- otide-gated; cAMP, cyclic adenosine monophosphate; ACh, acetylcholine; nAChR, nico- tinic ACh receptor; 5-HT, 5-hydroxytryptamine (serotonin); GABA, ␥-aminobutyric acid; NMDA, N-methyl-D-aspartate; AMPA, ␣-amino-3-hydroxy-5-methylisoxazole-4-propi- onic acid. aThe pore-forming subunit of these channels. 5315_16_p543-552 11/5/04 12:35 PM Page 545 Assay Technologies for Ion Channels 545 ) Comments ⍀ permeable channels ϩ 2 highest plate density available. channels ϩ shifted to the right in certain types of channels seal but low throughput 50 ⍀ IC Classic and gold standard ARGETS T a HANNEL C ON I Median-high Limited by ligand availability and structure. Not functional HighVery highMedianLow G Low–median Low seal resistance (G Median–high Low signal-to-noise ratio, not for HTS Low–median Limited for Ca For K b b b b b ECHNOLOGIES FOR b T b b b 384-well (SPA) Median CREENING 3. S ABLE T ) Low 96-well ϩ Rb 86 , ϩ Na 22 , ϩ 2 Ca 45 dye High 96/384-well ϩ 2 Membrane potential kit High 96/384-well FRET-based High 96/384-well Heterogeneous assay (cell wash is required). Cost per well is calculated only for the special consumable reagent (e.g., SPA beads, dye, or plate/chip) based on Ca Membrane potential Radioisotopes ( Voltage-clamp/patch-clampRb flux assay (atomic absorbance) Very low Mediuma Single cell b 96/384-well PatchXpress 7000 Low 16-well IonWorks HT Low-medium 384-well Membrane binding Medium/high 96-well Fluorescence dye Ion flux assay Assay typeElectrophysiology Throughput Format Cost per well 5315_16_p543-552 11/5/04 12:35 PM Page 546 546 Zheng et al. Secondary screening assays (HTS hit confirmation fer of energy from labeled ligands to SPA beads in prox- and lead optimization) imity. This SPA binding assay can be miniaturized into 384- and 1,536-well formats with a throughput of 50,000– The throughput requirement for these types of assays 100,000 compounds per day at moderate cost. is much lower than HTS, but the demands on data qual- Binding assays provide no information about the effect ity are higher. Unlike primary screening, compound titra- of novel agents on ion channel function. For example, an tion with eight to 10 different concentrations in duplicate agonist cannot be distinguished from an antagonist in a is usually needed to determine the IC value for each 50 binding assay. Additionally, if a compound interacts with compound tested in a secondary screen. Often one or the channel protein at a site distinct from the labeled li- more different types of assays are performed to confirm gand, it will not be detected in a binding assay. An ex- the activity of a compound. The required screening ample of this is the hERG 3H-MK-499 binding assay. It throughput for secondary assays is on the order of tens has been reported that the potency of certain compounds to hundreds of compounds per day.
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