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.