Breaking the bonds of pain

Slightest touch can evoke pain in patients suffering from nerve injuries. Researchers of the MDC now have suppressed this type of neuropathic pain in mice by applying a chemical substance to the skin. The method could work in humans.

It is a vision that motivates work at the MDC and translational research everywhere: that a detailed understanding of basic processes in cells and organisms will yield new approaches to the treatment of disease. For a decade, Gary Lewin's lab has been assembling a picture of the mechanisms behind a severe form of pain. The pieces have now come together in a therapy that works in mice and may one day be tried in human patients. The story appears in the Dec. 12 issue of Nature Neuroscience.

Imagine experiencing alarming, excruciating pain with the slightest brush of something against your skin. Many people are burdened by this condition, which can arise through an injury that damages nerves or as a result of diabetes. It may also underlie the irritation felt by many autistic children as they get dressed: their skin is so sensitive that they can barely tolerate the sensations of cloth brushing against it.

For years Gary Lewin's lab has been trying to understand what transforms the perception of a gentle touch into severe pain. Both sensations involve nerve cells called mechanoreceptors, whose branch-like extensions lie in the skin and sense when it moves. This opens pore-like channels in the nerves' membranes, opening passages for charged particles called ions. By entering the cell they alter its charge, turning it into a sort of biological battery. It generates an electrical signal that races along the cell to its other end in the spine, where it stimulates the next nerve cell and evokes a similar signal that is transmitted to the brain.

This basic account describes the activity of many sensory nerves, but it doesn't explain how a soft touch can trigger such intense pain. Without that knowledge, treatments are non-specific. "Most current therapies rely on anesthetics that shut down the nerves entirely," Gary says. "While this reduces pain, it also prevents some important signals from getting through."

An unexplored landscape on the surface of nerves

Ideally a way could be found to block pain without interfering with other activities of nerves in the skin – but that would require identifying the mechanisms that caused it. Ten years ago Kate Poole and other members of Gary's lab aimed their sights on the problem. The current work represents how far they have come: at the time almost nothing was known about the way mechanoreceptors sensed very light touch.

Human cells can make at least 300 ion channels. There was no easy, direct way to screen them to find the one responsible, but there might be another route to getting a handle on the problem. Most ion channels require help from other membrane proteins that undergo physical rearrangements upon stimulation. In some cases they are directly attached to the channel and open it mechanically; in others this process is mediated by other proteins. Even without knowing the channel's identity, the scientists might be able to gain control of its behavior through such a protein.

Gary's lab had already identified proteins called stomatins that seemed to facilitate the  opening of channels in mechanoreceptors. Evolution had produced five types of stomatins in animals such as mice and humans. One of them controls the ion channel for fine touch sensation. The lab started developing strains of mice in which specific stomatins could be deactivated.

In 2007, the analysis of one of these mouse strains led to an important paper in the journal Nature. Christiane Wetzel, another member of the lab, had taken the lead on analyzing mice with a non-functioning version of the molecule Stomatin-like protein 3, or Stoml3. Disabling it, she found, stopped the generation of the fine touch signal by about a third of the mechanoreceptors in the skin. If the mice suffer a nerve injury, this sense normally becomes painful in a condition called neuropathic pain, which is also common to many human patients. But in the absence of Stoml3, the animals didn't develop this typical type of touch-evoked pain. The findings meant that it might be dependent on the way stoml3 in regulated the ion channels that detect light touch.

"Potentially this had important implications for the development of a therapy," Gary says. "It meant that blocking Stoml3 might be enough to stop neuropathic pain."

"Unfriending" stomatins

That idea looked even more promising in 2014 after Ardem Patapoutian's group at the Scripps Research Institute in La Jolla, California, finally identified the elusive ion channel: a protein called Piezo2. The finding was quickly followed by other studies revealing that the channel played a wider role. As well as sensing other types of touch, it was crucial to proprioception: an animal's sense of the relative positions of its limbs and other parts of the body.

"This told us that any therapy that focused on the channel itself might interrupt pain, but would probably also dangerously disrupt this other important system," Gary says. What about Stoml3? "As far as we could tell, its functions were restricted to light touch and pain after an injury. That made it a far more promising therapeutic target."

This put the focus on finding something that could interrupt the channel-related functions of the molecule. Kate Poole had already started setting up experiments to be carried out in the Small Molecule Screening Platform, which was jointly operated by the MDC and its campus neighbor, the Leibniz Institute for Molecular Pharmacology (FMP). The facility has a large library of compounds acquired through collaborations with other institutes and pharmaceutical companies. Jens Peter von Kries and his staff at the facility began helping the scientists design a test for Stoml3 inhibitors.

One aspect of the molecule's behavior suggested a way to find such a molecule. "Copies of stomatin proteins typically bind to each other in pairs or more to carry out their functions," Gary says. "There was no way to predict in advance what type of substance would succeed, so in the end 35,000 different small molecules were applied."

Carrying out that many experiments meant that the scientists had to design an experiment whose results could be evaluated automatically by computer. That meant finding a simple way to distinguish cells in which stomatins could bind from those with single copies that couldn't, due to the obstructive influence of an inhibitor. Kate solved the problem by creating versions of the molecule attached to a fluorescent tag. Pairs of Stoml3 would yield a much brighter signal than single copies, and the binding of more copies would be even brighter – something that could be measured by automated analysis.

The initial screen yielded 21 compounds that left the protein stranded as single copies. Two of those compounds passed another round of tests with more stringent controls. One of the substances, which the scientists called Oligomerization Blocker 1 (OB-1), potently blocked not only Stoml3 but also other forms of stomatins. When these results had been verified using mouse versions of the proteins, the experiment was repeated in cells outfitted with their human counterparts. The substance had equally potent effects on the human version of the protein.

Of mice and men

Further work by Gary's team confirmed that when Stoml3 couldn't cluster, the ion channel remained closed. They carried out precise measurements of the electrochemical signal generated by mechanoreceptors treated with OB-1. The substance performed as they had hoped: it blocked signals triggered by mechanical stimuli but left signals from other types of stimuli intact.

Finally it was time to test the substance on mice. "James Poulet's lab helped design a set of elegant behavioral experiments in which the animals could 'talk' to us," Gary says. "Small amounts of OB-1 were administered to the mouse paw. The paw was then gently tapped. The mice had been trained to reach for a reward when they felt it."

Here, too, treatments with OB-1 blocked a mouse's perception of the tap without affecting other types of sensation. Later, after the effects of the drug had worn off, the animals' sensitivity returned to normal levels. This meant that OB-1 had fulfilled the main criteria for a potential treatment.

The results were encouraging for many reasons, Gary says. "What this represents is a new strategy that arose from understanding the mechanisms that turn sensations of touch into pain," he says. "From what we can tell so far, OB-1 only affects a very specific type of mechanoreceptor that has both Stoml3 proteins and Piezo channels. By blocking Stoml3, we can dampen touch sensation in healthy animals. More interestingly, when animals experience touch-evoked pain after a nerve injury, for example as a consequence of diabetes, the Stoml3 blocking drug can alleviate the pain.

"We would now like to develop a Stoml3-blocking drug that is suitable for human trials. This is a long road, but if such substances work in a similar way in humans, we think we'll have accomplished a major step toward treating a neuropathology that has a devastating effect on the lives of many people."

Christiane Wetzel1,10, Simone Pifferi1,9, Cristina Picci1,2, Caglar Gök1, Diana Hoffmann1,3,
Kiran K Bali4, André Lampe5, Liudmila Lapatsina1, Raluca Fleischer1, Ewan St John Smith1,6,
Valérie Bégay1, Mirko Moroni1, Luc Estebanez1,3, Johannes Kühnemund1, Jan Walcher1, Edgar Specker5, Martin Neuenschwander5, Jens Peter von Kries5, Volker Haucke5, Rohini Kuner4, James F A Poulet1,3, Jan Schmoranzer7, Kate Poole1,8,10 & Gary R Lewin1,3 (2016): Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity. Nature Neurosciencedoi:10.1038/nn.4454

1Department of Neuroscience, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. 2Department of Biomedical Sciences, Section of Cytomorphology, University of Cagliari, Monserrato (California), Italy. 3Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité – Universitätsmedizin, Berlin, Germany. 4Institute of Pharmacology, Heidelberg University, Heidelberg, Germany. 5Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany. 6Department of Pharmacology, University of Cambridge, Cambridge, UK. 7Freie Universität Berlin, Berlin, Germany. 8Department of Physiology and EMBL Australia Node for Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia. 9Present address: Neurobiology Group, SISSA, International School for Advanced Studies, Trieste, Italy. 10These authors contributed equally to this work. Corresponding authors: Gary R. Lewin and Kate Poole.

Featured image: A microfabricated cell substrate with tiny elastic pillars (red) with an adherent cell (green). Image: Kate Poole, MDC.