Research Team Finds New Approach For Sleeping Sickness Remedies

X-ray laser provides structure of a key enzyme of the Trypanosoma brucei pathogen

Using ultra-bright X-ray flashes, a team of researchers has identified a possible target for new drugs to treat sleeping sickness: The scientists have decoded the detailed spatial structure of a vital enzyme of the pathogen. The result provides evidence of a possible blueprint for an active ingredient that specifically blocks this enzyme and thus causes the pathogen to die, as the team led by Christian Betzel from the University of Hamburg, Lars Redecke from the University of Lübeck and DESY, and Henry Chapman from DESY im Specialist journal "Nature Communications" reports.

The sleeping sickness is triggered by the parasite Trypanosoma brucei , which is transmitted with the bite of the Tsetse fly native to southern Africa. The parasite first multiplies under the skin, in the blood and in the lymphatic system and then migrates into the central nervous system. If left untreated, the disease is considered fatal. Intensive control measures have drastically reduced the number of cases registered in recent years. Nevertheless, tropical doctors still count the infection as one of the most important tropical diseases. According to the Hamburg Bernhard Nocht Institute for Tropical Medicine, around 65 million people in 36 sub-Saharan Africa live in the risk area. War, displacement and migration could keep the disease flare up again and again.

In search of a possible starting point for drugs against the pathogen, the researchers had their sights set on a central enzyme of the unicellular organism, the so-called inosine 5'-monophosphate dehydrogenase (IMPDH). "This enzyme is part of the central inventory of every organism and is an interesting target for medication because it regulates the balance of two vital nucleotides in the cell: guanosine diphosphate and guanosine triphosphate," says Redecke. “The cell needs these nucleotides to supply energy and to build larger structures such as the genome. If you interrupt this cycle, the cell dies. ”

The enzyme has a type of on and off switch that is activated by docking its own molecules. A promising way is to block this switch with a tailor-made molecule. In order to construct such a blocking molecule, the exact spatial structure of the switch must be known. Structural biologists can use X-rays to determine the structure of biomolecules. To do this, they first grow small crystals of the biomolecules, which then generate characteristic scatter patterns in X-ray light, from which the atomic structure of the crystal and its building blocks, the biomolecules, can be calculated.

This path is often complicated because most biomolecules are difficult to crystallize. If such crystals succeed, they are usually very sensitive to high-energy X-rays and are quickly destroyed. "Although the structures of numerous IMP dehydrogenases are already known, crystal growth has so far not been successful with that of the parasite Trypanosoma brucei ," reports Betzel, who also researches in the CUI: Advanced Imaging of Matter Cluster of Excellence at the University of Hamburg and DESY.

The team therefore chose an alternative route: The group of co-author Michael Duszenko at the University of Tübingen has caused certain insect cells to crystallize biomolecules inside them. With the help of this so-called in-cellulo crystallization, the same team had already decoded another enzyme of the sleeping sickness pathogen, cathepsin B, which is also a potential target for medication. In fact, the modified insect cells also produced crystals of the dehydrogenase now under investigation. These tiny, needle-shaped crystals became around 5 thousandths of a millimeter (micrometer) thick and up to 70 micrometers long, so that they stood out from the cells that produced them.

The in-cellulo crystals are so small that very bright X-rays are necessary to analyze them. Because the larger a crystal is, the more atoms it has that scatter X-rays, the better the scatter pattern. The researchers therefore used the X-ray laser LCLS at the US research center SLAC in California for analysis. "X-ray lasers produce extremely intense flashes," explains Chapman, who is the lead scientist at DESY at the Center for Free-Electron Laser Science CFEL and one of the spokesmen for the CUI cluster of excellence: Advanced Imaging of Matter. "Although the sensitive crystals explode immediately, they first create a scatter pattern from which the structure can be obtained." This method is called serial femtosecond crystallography (SFX),

The team recorded the scatter images of more than 22,000 microcrystals and was able to use them to calculate the spatial structure of the enzyme with an accuracy of 0.28 millionths of a millimeter (nanometer) - roughly equivalent to the diameter of an aluminum atom. "The result shows not only the exact structure of the enzyme switch, the Bateman region, but also which molecules the cell uses to switch the enzyme and how these so-called co-factors dock on the enzyme switch," reports Karol Nass from DESY, who worked on this study as part of his doctoral thesis. Nass is currently researching at the Paul Scherrer Institute in Switzerland and, together with Redecke, is the main author of the publication.

According to the results, the switch is operated by the molecules adenosine triphosphate (ATP) and guanosine monophosphate (GMP). “The advantage of our method is not only that we can examine the enzyme at room temperature, ie at the temperature for which the enzyme is made, but also that the natural co-factors are bound to the enzyme during in-cellulo crystallization become, ”says Betzel. According to the researcher, the data could now provide an approach for blocking parasite dehydrogenase. "For example, it would be conceivable to construct a kind of clasp that overlaps the docking points of both co-factors."

However, there is still a challenge in designing the dehydrogenase blocker so specifically that it blocks the parasite enzyme, but not the human one. If this succeeds, the method could possibly be extended to other pathogens, explains Betzel. “Other parasites have a very similar structure, and possibly they could also be attacked via the respective IMP dehydrogenase. The enzyme is a very interesting target for medication, for example against the fox tapeworm or the causative agent of elephantiasis. "

The universities of Hamburg, Lübeck and Tübingen, the Russian Academy of Sciences, Arizona State University, the Lawrence Livermore National Laboratory in the USA, the Max Planck Institute for Medical Research, the US Research Center SLAC, and the university were involved in the study Gothenburg and DESY involved.