Overton's rule is over!

It is the 1980s. Ernst Overton at the University of Zurich (Switzerland) declares that those substances that dissolve in lipids pass more easily into a cell than those that dissolve in water. He then diligently applies himself to the development of an equation that would allow the calculation of the time required by the substance to permeate through the cell barrier. Based on the lipophilicity (ability of a compound to dissolve in fats, oils and nonpolar solvents) of the substance he also goes as far as defining a parameter K, whose value dictates the speed of diffusion; that is, the higher the value of K, the faster the permeation rate. So far, so good. For over a century, scientists of a variety of disciplines have adhered to Overton's rule and used his equation to shape their studies.

Unfortunately, Overton was wrong, very wrong. It is 2008 and a team of researchers at Warwick University (UK) have now proved that exactly the opposite phenomenon occurs: studying the diffusion of a variety of acids through the cell membrane it is the most lipophilic compounds that are transported slowest. In fact, their research suggests that the real transport rates could be up to a hundred times slower that predicted by Overton's rule.

So how is it possible that Overton got it so wrong and that the rule has remained unchallenged for more than 100 years? It is worse than that. "The important point is really how other researchers have confirmed Overton's rule many times in the past two to three decades," Professor Patrick Unwin, lead researcher of the scientific team, says. He blames the technology: "Techniques in recent years have employed rather inefficient and fairly ill-defined mass transport to move molecules up to the membrane, which makes it difficult to accurately measure fast membrane transport with confidence." Additionally, measuring the rate of transport of compounds across a membrane is very challenging "because, at the simplest level, there are two processes in series — diffusion to and from the membrane and transport across it — and the slowest step is what is measured."

The technology developed by the scientific team at Warwick University allowed them to observe every step of the diffusion process and investigate up close what really happens at the cell boundary. "We use piezoelectric positioners, similar to those used in our other recent work with ultramicroelectrodes, except that by coupling electrochemistry and confocal microscopy, we can also visualise the electrode position with high accuracy. The electrode can thus be placed at a precise, quantifiable distance from the membrane without touching it."

The research team are excited with their discovery and are looking to use this technique to examine the diffusion of other compounds into the membrane cells. "We can tackle any process," says Unwin "where we can detect an ion or molecule by fluorescence, which opens up a wide range of possibilities." Other techniques will also be explored: "in addition to the solid electrodes used in this study, we can also use micro- and nanopipettes to deliver reagents locally in a controlled manner via an applied current or potential."

For the scientific community in general the implications of this finding are huge: "Text books will have to be rewritten," says Unwin. For drug developers in particular, this discovery is of paramount importance for the development and testing of drugs. "Drug companies may want to review whether an initial screen of candidate drug efficacy based the measurement of partition coefficient is necessarily a good indicator of permeation. There may be candidates that make it through initial screening, despite actually having poor membrane transport properties and, conversely, candidates with good transport dynamics that are rejected on the basis of partitioning alone. Our methodology could also readily be developed to look at protein and ion channel activity in model membranes and living cells."