New possibilities for the medical use of botulinum toxin A1

PSI researchers have discovered a surprising trick that could expand the possibilities for the medical use of botulinum toxin A1, better known as Botox, as an active agent. They developed antibody-like proteins that accelerate the enzyme’s effect on the transmission of nerve signals. This suggests that Botox might, for example, be able to relieve pain more quickly than before.

The article is published in the journal Natural communications.

Botulinum neurotoxin A1, better known by the brand name Botox, is actually a nerve toxin produced by bacteria. It has gained notoriety among the public through its use as a cosmetic aid. Many people inject it into wrinkles to rejuvenate them.

The substance blocks signal transmission from nerves to muscles, thereby relaxing them and leaving facial features smooth. What is less known: Botox is also very often used in therapeutic medicine to treat conditions that can be attributed to muscle cramps or faulty nerve signals, including pain, spasms, urinary leakage, grinding teeth and misalignments, for example of the eyes. Botox is even used in the treatment of stomach cancer, to block the vagus nerve and thus slow tumor growth.

In any therapy it is crucial to use this highly effective medication in a very targeted manner and with careful dosage, as Botox is the most powerful natural nerve toxin of all, which can lead to dangerous paralysis in a clinical picture called botulism. A hundred nanograms administered intravenously can be enough to kill a person, as the toxin paralyzes respiratory muscles, as well as others.

Different types of botox

Botulinum neurotoxins are classified into seven so-called serotype groups designated by the letters A to G. Botox used in cosmetics comes from the first group. To be precise, it is designated subtype A1. It is known that three other serotypes – B, E and F – can also lead to botulism in humans, with E and F acting much more quickly but not as long as A and B.

The effect becomes evident after just a few hours and lasts for a few weeks, which opens up important perspectives in the field of pain treatment and orthopedics, for example. Types C and D are effective in certain animal species such as birds; To date, no cases of type G botulism have been observed.

The serotypes are mainly produced by different strains of the bacterium Clostridium botulinum. These microbes grow anaerobically, that is, in the absence of oxygen, and are mainly found in soil as well as marine and river sediments. If they get into food and are stored in airtight containers, as can be the case with canned products, there is a risk of toxin contamination. Eating it can cause botulism. However, the disease occurs very rarely; Over the past ten years, there have been only one or two cases per year in Switzerland.

Surprising results

As part of a research project, a team led by Richard Kammerer from the PSI Biomolecular Research Laboratory wanted to investigate whether it was possible to influence the action of the toxin.

“For this, we produced, in collaboration with biochemist Andreas Plückthun from the University of Zurich, 25 DARPins,” explains Kammerer. DARPins are small, artificially produced proteins that work similarly to antibodies. They are used in therapy and diagnosis as well as in basic medical research.

The idea was to find DARPins that selectively bind to the so-called catalytic domain of Botox serotype A1, the part of the enzyme responsible for its effect on nerves, by cutting up certain proteins. DARPins were expected to inhibit this function.

“In vitro, that is, on individual samples in a test tube, we identified a suitable candidate that limits the function of botulinum toxin,” reports Kammerer.

Through studies carried out at the Swiss Light Source SLS at PSI, researchers were able to precisely observe the DARPin complex and the catalytic domain down to the molecular level and discover how DARPin prevents cleavage.

But when the researchers also tested this DARPin on cell cultures, in collaboration with a team from the Institute of Biomedicine at the University of Padua in Italy, a completely different effect, in fact the opposite, suddenly appeared: the action toxic from Botox. The cleavage of proteins important for nerve signal transmission took effect even more quickly than usual.

“At first we thought we had done something wrong,” says Oneda Leka, postdoctoral researcher at the PSI Biomolecular Research Laboratory and first author of the study. But other experiments confirmed this contradictory result: instead of decreasing, the toxic effect of the Botox enzyme accelerated.

The researchers have now repeated the experiments with real muscles, the diaphragms of mice. These remain intact for a long time in a nutrient solution and constitute a preferred model for testing the effects of nerve toxins. Here too, the results indicated that with DARPin, the paralyzing effect of the toxin took hold more than twice as quickly.

New options for Botox therapy

Now the big question was: why is this so? The possible explanation is biochemically very complex. Simply put, DARPins destabilize the toxin in such a way that it is transported inside nerve cells more quickly. As a result, the toxin acts more quickly.

“For this reason, we believe that DARPin could expand the spectrum of possible uses of botulinum neurotoxin,” explains Oneda Leka.

Although the researchers did not perform any comparative tests as part of this study, it appears that botulinum neurotoxin A1 with DARPin acts much more quickly than A1 without the antibodies. At the same time, the duration of the effect remains significantly longer than that of E and F.

Thus, the addition of this DARPin provides an intermediate variant between serotype A and serotypes E and F. The result, however unexpected, opens up new possibilities for treating various diseases. According to Richard Kammerer, “in pain medicine, an additive that accelerates the onset of the effect of an extremely effective and long-term drug could be of interest.”