June 1, 2006 (Vol. 26, No. 11)

Discovery Strategies and Channelopathies Interest a Wide Group of Bioresearchers

More is not necessarily better, as was previously examined in an article about the pitfalls of high-throughput screening (GEN, March 15, 2006, pg. 24), not the least of which is the terabytes of information it creates and the time it takes to cull through the resulting information.

At the Strategic Research Institute’s 6th Annual Ion Channels in Drug Discovery and Development conference, held recently, expert speakers from academia and industry presented leading approaches and concepts pertinent to ion channel drug discovery and development. Major themes this year included new targets, discovery strategies and technologies, channelopathies, and ion-channel approaches for CNS and neurodegeneration indications.

GEN spoke with some of the presenters about this expanding arm of drug discovery, its progress in the last several years, and some of the challenges that lay ahead.

Molecular Devices (www.moldev.com) leads the way in creating the capability for ion-channel exploration. Ion channels are a viable and under-represented target class in the pharmaceutical industry, noted James Costantin, application scientist at Molecular Devices.

Population Patch Clamp Technology

The era of ion-channel screening using a direct electrophysiological assay has commenced, and several instruments are commercially available, including the IonWorks HT and PatchXpress 7000A systems from Molecular Devices.

These systems offer considerably higher throughput than conventional patch clamping, yet there is a need to match the throughput of surrogate assays, as well as to reduce the cost per compound assay. In addition, conventional patch clamping of a single cell at a time, the gold standard for studying ion channels, is labor intensive and not amenable to screening large numbers of compounds in early-stage drug discovery, says Costantin. Hence, Molecular Devices developed the IonWorks Quattro system with Population Patch Clamp (PPC) technology.

We introduced PPC a year ago, Costantin said. It is a second-generation technology that records the ensemble current from up to 64 cells in each recording chamber, resulting in nearly perfect success rates and highly consistent responses from one well to the next.

Costantin discussed the emergence of innovative assays that are only possible using the PPC technique, including mutational analysis, multiplexing, and a highly parallel screening assay for ligand-gated ion-channel targets.

Since biological variability, such as cell health and size and channel-expression levels, all contribute to reduced success rates in planar patch clamp systems, Costantin explained that IonWorks HT system had to find a way to compensate and increase the likelihood of obtaining at least one successful recording for every compound. Hence, IonWorks HT system pipettes compounds in quadruplicates. Thus, the probability of obtaining at least one recording for every compound using a cell line with 70% success rate is over 99%, Constantin stated, but at the expense of lower throughput.

To eliminate the quadruplicates, PPC records averaged ionic currents from a population of cells expressing a recombinant voltage-gated ion channel. Cells are plated into a 384-well PatchPlate PPC substrate, in which each well contains multiple recording sites.

The increased throughput of the IonWorks Quattro system is ideal for generating comprehensive pharmacological determinations for dozens of compounds in a single experiment, Constantin commented. Using 96- or 384-well compound plates, a wide variety of dose-response experiments can be designed, including any number of titrations from 3󈝸 concentrations, with or without replicates.

High-Throughput Electrophysiology

Ion-channel discovery research requires extensively validated stable cell lines, information-rich screening data, and high-quality selectivity profiling information. BioFocus has invested in the IonWorks Quattro platform to address all of these requirements and complement our existing ion flux and fluorescence-based high-throughput screening platforms, said Andrew Southan, head of ion-channel pharmacology at BioFocus (www.biofocus.com).

By adopting high-throughput electrophysiology, we have significantly reduced the time taken to generate and characterize new cell lines stably expressing ion-channels. We can now rapidly obtain high-quality data on expression level, stability, and pharmacology and deliver well-validated screening tools to our clients.

Furthermore, by offering both selectivity and safety profiling in an electrophysiology format, BioFocus can now provide its collaborators with the detailed information necessary to progress hit molecules directed toward ion-channel targets. And as a service company, it is crucial that we have this kind of capability, Southan said.

In certain circumstances high-throughput electrophysiology can now be used for the primary screen, Southan noted. The IonWorks Quattro Population Patch Clamp recording mode enables focused library screening (1󈝶,000 compounds) to be used as the first approach to voltage-gated ion-channel targets.We believe that the use of ion-channel directed compound collections, such as the BioFocus SoftFocus ion-channel targeted libraries, will reduce the need for extensive diverse screening campaigns, looking at 100,000 or more compounds. At the end of the day, it is all about getting the highest quality data as economically as possible, Southan said.

Future of the Patch Clamp

Will true high-throughput screeninggreater than 10,000 compounds per dayever really be possible economically with automated patch clamp? This is the question that Sophion Bioscience’s (www.sophion.com) Chris Mathes, Ph.D., vp and general manager, North America, posed in his panel. Patch clamp is a low-throughput technique. It is time-consuming and demands the presence of a skilled operator. Sophion Bioscience has an automated parallel ion-channel screening technology system, the QPatch, for throughput in the range of hundreds of data-points per day. Upscaling to such figures moves patch clamp toward the HTS regime and revolutionizes future ion-channel drug discovery.

Dr. Mathes noted that HTS has not always been the most efficient process. Doing true HTS allows screening around 30,000 compounds a day, which is not possible with patch clamp. But examining ion channels with the QPatch allows you to target much smaller libraries that may be much more relevant to the target you are trying to find. There hasn’t been a whole lot in the way of blockbuster drug discovery via HTS, and I think that high-information screening, afforded by automated patch clamp, is a huge opportunity for us.

Also, lower throughput allows screeners to factor safety pharmacology much earlier in the drug discovery process. Cost, however, still remains a factor. Lowering the chip price is going to be a key issue, particularly in getting academia on board, Dr. Mathes commented.

Leak Potassium Channels

In this way, Sophion has gotten buy-in from pharma giant Merck (www.merck.com). Automated patch clamp technology has come a long way in the last five years, said Andrew Swensen, senior research biologist, Merck. And the advances made have resulted in a means to get high-quality, medium-throughput electrophysiological data from a range of ion channels.

Despite the fact that automated instruments for voltage-dependent and ligand-gated ion channels have been proven to work, less data is available on how well leak potassium and inward rectifier channels can be screened on these platforms. These channels produce currents that by conventional patch methods can be challenging to study, Swensen explained. For many of these channels, the lack of selective pharmacological probes and their voltage-independent nature makes it challenging to differentiate true channel current from nonspecific leak current.

One of the chief questions we are looking to answer is how feasible is it to look at these particular ion channels on automated patch platforms, Swensen stated. It is possible to get high-quality data from these channels that allow you to confirm hits and eliminate the false positives that often come from higher-throughput screens. Of course, if you get false negatives in the higher-throughput screens, you are never going to know the difference.

As Dr. Mathes noted earlier, the biggest limitation, Swensen agrees, is the price of consumables. That is part of the reason for taking drug discovery through the electrophysiology route. You get better numbersbetter control over voltage states with the benefits of HTS.

Applications of Planar Patch Clamp

GlaxoSmithKline (www.gsk.com), meanwhile, looks to planar patch clamp on the IonWorks platform to ease its drug discovery bottleneck. Four years ago, we used patch clamp electrophysiology for all phases of ion-channel drug discovery, said Claire Townsend, investigator. Patch clamp is the gold-standard technology for the characterization of ion-channel biophysics and pharmacology. However, the use of this technology in today’s drug discovery paradigm is quite limited due to its very low throughput and high cost.

The introduction of higher-throughput automated electrophysiology, such as planar patch clamp, has revolutionized ion-channel drug discovery. The IonWorks technology allows us to do parallel testing of multiple reagents, drugs, or targets, Townsend said.

Townsend reviewed the current positioning and applications of planar patch clamp in the ion-channel drug discovery pipeline, from reagent generation to liability profiling. In the ion-channel field, it used to be only possible to work with one cell at a time, which was time-consuming and expensive. Automating with PPC revolutionized the process and removed a lot of the bottlenecks, such as reagent characterization and validation or hit validation.

The IonWorks Quattro platform has opened the door to electrophysiology-based focused screening on small libraries of compounds (

This technology has some limitations though. It cannot be applied to fast ligand-gated channels and covers approximately 30% of the ion-channel genome. Also, as noted earlier, it is still costly compared to the cost of other assays. It is pretty expensive, but high-quality data pays for itself, Townsend said.

Voltage-Gated Channels

And just when automated patch clamp technology establishes itself as the de facto standard in ion-channel drug discovery, along comes Vertex Pharmaceuticals (www.vrtx.com) to shake up the industry with a new entrant to the field. Vertex has introduced an electro-optical technology and automated instrument, E-VIPR, which employs extracellular electrical field stimulation and cellular fluorescent probes to measure the activity of voltage-gated sodium (NaV) and calcium channels, said Tito Gonzalez, senior director of biology.

This technology is a real game changer. E-VIPR has high sensitivity and correlation compared with voltage clamp recording for several known drugs in cells expressing a neuronal NaV. Furthermore, we screened approximately 400 drugs and observed that a large percentage have NaV blocking activity, which is particularly enriched in certain drug classes. The convenience, sensitivity, and robustness of this technology has potential to provide unprecedented access to voltage-gated targets.

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