November 1, 2005 (Vol. 25, No. 19)

Tansmembrane Proteins Offer Significant Therapeutic Opportunities

Fifteen of the top-100 best-selling drugs target ion channels, and ion channel function is implicated in a large number of diseases as disparate as Alzheimer’s, epilepsy, heart disease, neuromuscular disease, and even cancer. Ion channels also represent a significant class of emerging therapeutic opportunities.

Being transmembrane proteins, ion channels have structures that are both some of the most challenging and most impressive in all of biology. Illustrated variously as long-stemmed flowers, multilobed mushrooms, or gigantic, science-fictional faceted crystalline tunnels, ion channels seem to be not only gateways for potassium, sodium, calcium, and other small ions, but gateways to understanding the physiology of the cell systems they protect. Reflecting rapidly increasing interest in the subject, Select Conferences’ ( inaugural meeting and exhibition, “Ion Channel Targets,” was held recently.

Structure studies of ion channels have long been complicated by the hydrophobic nature of the molecules. In order to crystallize them, it is necessary to solubilize them with detergent, a procedure drastic enough to cast the shadow of doubt upon the ultimate structure.

Andreas Namslauer, Ph.D., a researcher at Sidec Technologies (Kista, Sweden), presented the results of a collaboration with AstraZeneca in which they studied the subunit composition of an unidentified, proprietary ion channel target.

The technology, protein tomography, is based on the same principles as tomography methods used in medical imagery. A number of two-dimensional images from different angles are captured and combined into a three-dimensional model. Sidec uses a method wherein frozen, gold-labeled protein samples are visualized with a powerful electron microscope.

The resulting resolution is around 20 angstroms and is limited not primarily by the capabilities of the microscope, but the nature of the sample. Sidec’s intellectual property is based on getting optimal results within this limitation.

“When working with biological sampes, you can’t use too high an electron dose, you’ll destroy the sample. It’s too soft a material. When you use the low electron dose, you get a large noise level. What our intellectual property algorithms do is reduce the noise and enhance the signal, which enables users to get a high resolution from biological material,” said Dr. Namslauer.

Sidec’s tomography method can reveal a number of aspects of gross ion channel structure, including subunit composition, protein conformation, and complexes with other molecules which can help in understanding the role of ion channels in disease.

Early and Accurate Screening

Whatever the specific disease or therapeutic area, there is great demand for accurate screens for drug compound activity against ion channel targets, and the trend is for earlier screening and elimination of unsuitable compounds. Patch-clamp assays are the favored method for measuring ion channel activity, but they are generally slow and cumbersome.

As a quick and convenient alternative, most early ion channel screens rely on the use of a fluorescent dye. However, some of the compounds themselves have fluorescent properties, resulting in a large number of false positives and negatives.

Hydra Biosciences (Cambridge, MA) has developed an assay that uses two different fluorescent dyes, and rescues compounds that would otherwise have been discarded from an ion channel screen due to confounding effects of compound fluorescence.

Christopher Fanger, Ph.D., the director of lead discovery at Hydra, presented results of a sample screen in which two structural groups were rescued from a screen using a dual-assay system that would have been discarded using a standard, single, fluorescent dye.

“By doing this we were able to focus in on just a small number of compounds that are dual hits, the ones that hit in both assays. And since the target is an ion channel, what we could do is follow-up these compounds in manual patch clamp assays. We found that around eight percent of those compounds turned out to block our target channel, which is a really fantastic rate of hit confirmation,” said Dr. Fanger.

“The conclusion from all of this is that by doing a dual screen, we were able to increase our percentage of hits that really were hits. We were able to get a much better rate of true positives, and not waste much time at all on the false positives.”

Primary areas of emphasis at Hydra include cardiovascular therapeutic areas and pain. As it turns out, much of the interest in ion channels centers on cardiovascular issues, and not just the search for cures for heart disease. Adverse cardiovascular effects from noncardiovascular drugs are also an area of interest.

Many promising drugs have been removed from the market after reports of increased adverse cardiac events such as arrhythmia and heart attack. The most famous of these recently has been Vioxx, but drugs such as seldane and propulsid have also been pulled for this reason.

Thus far, all adverse cardiac side effects have been found to affect an ion channel called hERG. The fatal physiological effect is called Tosade de Pointes (TdP). Cardiac safety assays now focus on hERG and drug-induced long QT syndrome, a cardiac arrhythmia known to precede TdP (although TdP does not always follow long QT syndrome).

ChanTest (Cleveland, OH) specializes in cardiac safety testing, particularly GLP safety assays for hERG activity. “What we’re looking at primarily is how the compounds affect electrical behavior of the ion channels. What we’ve found is, for example, the target for noncardiac drugs that produce lethal arrhythmias 99 percent of the time is the hERG channel,” said ChanTest’s CEO, Arthur Brown, M.D., Ph.D.

“We’ve also identified that there are drugs that work not directly on the hERG channel, but with its trafficking to the membrane. These particular drugs do their thing not by direct action but by trafficking to the cell surfacefor example, arsenic trioxide.”

The whimsically named human etheragogo related gene, or hERG, is responsible for repolarizing a current in the cardiac action potential. The gene earned its name when it was noted that fruit flies who had the gene would shake when anaesthetized with ether.

In addition to hERG assays, ChanTest has a number of cell lines designed for purposes such as testing compounds against “pacemaker” channels, contractile function assays for calcium channels, or prevention of arrhythmia by blocking sodium channels. ChanTest has tested over 6,000 drugs.

“We’ve also shown that there are drugs on the market that block the hERG channel and do not have lethal side effects,” said Dr. Brown.

Not Just hERG

Cytomyx (Cambridge, U.K.) has invested in a panel of eight ion channels as a combined cardiac safety screen. Cytomyx maintains 180,000 highly characterized human tissue samples. Twenty of them are ion channel cell lines that customers can buy off-the-shelf, with more than 50 in development.

“We’re promoting the notion that hERG is not the only channel that people can screen their compounds against, there are other channels that people can screen against as well, and make an informed decision as to whether that compound can be taken through clinical trials. Not all compounds that block hERG give you long QT syndrome, you can’t just look at hERG, even though the FDA recommends looking at hERG at the moment,” said Umesh Patel, Ph.D., Cytomyx’ director of biological operations.

Cytomyx presented its cardiac safety panel at the “Ion Channel Conference,” emphasizing eight ion channels including: Nav 1.5, Kv 4.3, KCNQ1/Mink, HCN4, L-type calcium channel, and Kir 2.1. According to Dr. Patel, the main advantage of running the cardiac safety panel is to eliminate inappropriate compounds early, before a lot of resources have been wasted on them.

“We believe this is important because it will save a lot of time, a lot of effort, and a lot of money for drug companies to make clear and informed choices early in the drug discovery process about whether a drug will proceed to clinical trials or whether it will be rejected. That is the reason why we say not just hERG. You can still take compounds that block hERG, but do not give you the long QT syndrome or TdP.”

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