High-throughput Ion Channel Screening: A “Patch”-work Solution
2010; Future Science Ltd; Volume: 48; Issue: 1 Linguagem: Inglês
10.2144/000113339
ISSN1940-9818
Autores Tópico(s)Mass Spectrometry Techniques and Applications
ResumoBioTechniquesVol. 48, No. 1 Tech NewsOpen AccessHigh-throughput Ion Channel Screening: A "Patch"-work SolutionJeffrey M. PerkelJeffrey M. PerkelPublished Online:3 Apr 2018https://doi.org/10.2144/000113339AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail When the Nobel Assembly at the Karolinska Institute awarded the 1991 Nobel Prize in Physiology or Medicine to Erwin Neher and Bert Sakmann, the committee announced that their work on ion channels "opened a way to develop new and more specific drugs." But nearly two decades later, that promise remains largely unfulfilled.The problem is that while ion channels might make for fantastic drug targets—accounting for an estimated $12 billion in pharmaceutical company revenue in 2002 (1)—they also make for lousy screens. Those drugs currently on the market are the fruits of serendipity, not high-throughput screening (HTS), says Michael Dabrowski, head of AstraZeneca's three-year Global Ion Channel Initiative (GICI). GICI is a technology development program launched in 2006 whose goal "is to enable … and to raise increased awareness about ion channel lead generation internally at AstraZeneca as well as externally.""Ion channels were always underserved because the throughput—the rate at which you could study how drugs affect them—was very low," says Arthur "Buzz" Brown, president and CEO of ChanTest, an ion channel screening service provider located in Cleveland, Ohio. Thanks to technologic developments over the past decade, however, that trend is beginning to change.The "Art" of the ScreenIn a business where speed and cost are everything, ion channels don't fit neatly into the traditional process of drug discovery. Pharmaceutical companies cannot easily apply their million-compound libraries to ion channels, because these channels don't behave like other proteins. Ion channels are membrane-spanning macromolecules that regulate the flow of charged molecules such as Na+, K+, Cl−, and Ca2+ across an otherwise impermeable barrier. They don't catalyze enzymatic reactions, and induce no secondary, amplified signal. Instead, upon activation—whether by changes in voltage, ligand binding, or mechanical force—these channels open, creating a pore in the membrane through which charged molecules can flow.In traditional patch clamping, a skilled operator must manually manipulate cells and a pipet to form a seal, perhaps recording from just 10 cells in a day "if [he] were lucky," says Arthur "Buzz" Brown.In contrast, planar patch clamping uses an aperture bored into the bottom of a glass or plastic consumable, opening the process up to automation and higher-throughput analysis. Illustration by Lauren E. Wool.The resulting currents are small and fast—on the order of picoamperes and often occurring on a millisecond timescale. Yet they are substantial enough to kick-start a chain reaction that, depending on the context and location, results in effects as varied as pain, muscle contraction, or even fertilization.The bottom line is that it's not enough to know whether a potential drug compound binds a particular ion channel; scientists need to actually measure its impact on that flow of ions across the cell membrane. Researchers can measure these electrophysiologic changes indirectly, by using voltage-or ion-sensitive fluorescent dyes or atomic absorption spectroscopy for instance. But to do it right—that is, to directly measure the current flow across individual ion channels—requires the technique that won Neher and Sakmann their Nobel prize: patch clamping.In traditional patch clamping, a glass microelectrode (basically a drawn-out micrometer-wide glass pipet with buffer and an electrode inside) is coupled to a cell membrane under a microscope. Suction is then applied to form a seal between the pipet tip and the piece of membrane to which it is attached (the "patch"). In such a system, ion flow across the membrane (mediated by ion channels) produces current that can be recorded by the electrode. The goal is to hold the transmembrane voltage difference steady—that is, "clamp" it—and measure the current. That's not to say the voltage remains constant throughout the experiment: oftentimes, current is recorded while modulating the voltage to mimic, for instance, a cardiac action potential.Cellectricon's Dynaflow HT System, developed in conjunction with AstraZeneca, patches up to six cells in parallel per microfluidic channel.Total throughput: about 10,000 data-points per shift. Courtesy of Cellectricon.On MDS Analytical Technologies' IonWorks Quattro, the signals from up to 64 patched cells are averaged per well of a 384-well plate in a strategy called population patch clamping (PPC).PPC enables the system to overcome one technical hurdle characteristic of patch clamping: cells that fail either to seal efficiently or to produce measurable recordings. Courtesy of MDS Analytical Technologies.Data-rich, the resulting technique is nonetheless a bottleneck. The low-throughput, manual process requires a skilled operator, is done one cell at a time, and is, says David Yamane, MDS Analytical Technologies' senior director for drug discovery marketing, "more art than science." In contrast, planar patch clamp systems may be run by technicians, and are amenable to scale-up and robotic integration.Planar systems employ a flat substrate through which one or several apertures, functioning as microelectrodes, have been bored. This configuration opens planar systems to automation, because those holes can be bored in microtiter plate–like consumables, and cells can be patched via vacuum. The IonWorks Quattro from MDS Analytical Technologies has 64 holes per well of a 384-well PatchPlate, and records the average signal from 64 cells in a strategy the company calls "population patch clamping." Similarly, the PatchXpress (also from MDS) can measure 16 individual cells at once, and the QPatch HT (from Sophion) can record from 48. Two other systems set to launch in 2010—Nanion's SynchroPatch and Cellectricon's Dynaflow HT—will record 96 cells in parallel.Channeling Drug Discovery PossibilityAt the moment, electrophysiology, or "ephys," is a field in transition. Using automated patch clamp platforms, companies like ChanTest can record from thousands of cells per day, introducing ephys far earlier into the drug development pipeline than ever before. AstraZeneca routinely subjects libraries of thousands of chemical compounds to similar analyses, says Dabrowski.Yet that's still not enough to make electrophysiology a first-tier screening application, says Dabrowski. "It's about one digit off; a factor of 10 [is] still needed to do high-throughput screening on ephys platforms."According to Chris Mathes, vice president and general manager of Sophion, the highest quality data come from gigaohm or "gigaseal" connections, because only the current through the channel can flow to the electrode. Weaker "megaohm" seals are "leaky," he says, meaning potential loss of signal, baseline fluctuation, and noise."There are tricks you can use to get around that," says Mathes of megaohm's setbacks, "but most people agree that, if you can do gigaseal recording, it's better."Sophion's QPatch HT, one of a growing family of automated patch clamp systems that is bringing electrophysiology deeper into the drug development process.Courtesy of Sophion.Sophion's QPatch systems all produce gigaohm seals, as does MDS's Patch X-press and Nanion's SynchroPatch. MDS's IonWorks and Cellectricon's Dynaflow systems generate megaohm seals. The difference is in the planar substrate: silicon and glass can produce a gigaseal, and plastics usually cannot. But plastics are cheaper to manufacture. "If you change the substrate, you lose the gigaseal, but you can gain in throughput," Mathes observes.Another distinction among the various high-throughput systems lies in the fluidics. While some systems are well-based, the QPatch and Dynaflow use microfluidics for rapid fluid exchange across the cell. This feature is particularly useful in studies of ligand-gated channels because they often desensitize (that is, stop responding) to a compound, sometimes within milliseconds. As MDS's Yamane puts it, desensitization is like a baseball cap. "When you first put your cap on, you can feel it. Later, you forget it's there."In the case of the Dynaflow HT, micro-fluidics enable millisecond-scale solution exchange, which enables data collection before a receptor can desensitize. "We have essentially perfect control of the solution environment around the cell," says Mattias Karlsson, chief technical officer at Cellectricon.Developed in conjunction with Astra-Zeneca, the Dynaflow HT is not, per se, planar. Instead, the system integrates electrical and microfluidic circuitry in a microtiter plate format to probe up to six cells in parallel in each of 16 experimental modules. "It is more like a lateral patch clamp system," says Karlsson.The Dynaflow HT system can collect up to 10,000 datapoints in 8 to 12 hours, notes Karlsson, compared to 2,000 per 6-hour shift on the IonWorks Quattro. But for Dabrowski, the calculus for determining the best platform is more complicated—a balance of throughput, cost, and reliability. Automated ephys platforms are simply too expensive for standard HTS at a company like AstraZeneca, he says. "Where I see the best fit for Dynaflow is in concentration-response screening."At the National Institutes of Health (NIH)–funded Johns Hopkins Ion Channel Center, director Min Li's compound-discovery workflow combines an IonWorks Quattro and Hamamatsu FLIPR (fluorometric imaging plate reading) system with Corning's label-free Epic instrument, which detects morphologic changes resulting from drug treatment. In one case, Li's team screened a 300,000-compound library for modulators of hERG proteins— voltage-gated potassium channels that help maintain cardiac rhythm.Starting with an indirect fluorescent FLIPR assay on engineered cells expressing only the channel of interest, the team whittled their library down to only a few thousand possibilities. They then profiled those compounds on the IonWorks to understand each compound's pharmacology. Using the Epic device to assess the drugs' activities in native, non-engineered cells, the team ended with a final tally of 30 leads.If Johns Hopkins can do that, so too can pharmaceutical companies. Indeed, at least one such compound is in phase 2 trials: AstraZeneca's AZD1386, a capsaicin receptor antagonist. From this perspective, then, ion channel drug development is right on schedule: it takes 10 to 15 years to develop a drug, and automated ephys systems only entered the market in 2002. "It's likely that in the next five years or so, we will see new drugs developed based on automated patch clamp," says Mathes. "For the first time, drug companies are, in a more serious way, looking at ion channel targets as real targets of drug development."References1. Xie, M., M.H. Holmqvist, and A.Y. Hsia. 2004. Ion channel drug discovery expands into new disease areas. Current Drug Discovery 4:31–33.Google ScholarFiguresReferencesRelatedDetailsCited ByAdvancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms1 January 2022Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia18 July 2017 | British Journal of Pharmacology, Vol. 175, No. 12Screening methods for influenza antiviral drug discovery22 March 2012 | Expert Opinion on Drug Discovery, Vol. 7, No. 5 Vol. 48, No. 1 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print January 2010 Information© 2010 Author(s)PDF download
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