New design strategy for sleeping sickness drugs

Using ultra-bright x-ray flashes, a team of researchers have located a potential target for new sleeping sickness drugs: Scientists have decoded the detailed spatial structure of a vital enzyme in the pathogen , the parasite Trypanosoma brucei. The result provides a possible model for a drug that specifically blocks this enzyme and thus kills the parasite, 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 reports in the newspaper Nature Communication.

Sleep sickness (African trypanosomiasis) is a tropical disease caused by the parasite Trypanosoma brucei, which is transmitted by the bite of tsetse flies, which inhabit much of tropical Africa. In the body, the parasite first multiplies under the skin, in the blood and in the lymphatic system and then migrates to the central nervous system. If left untreated, the disease is almost always fatal. Thanks to intensive control measures, the number of registered cases has fallen considerably in recent years. Nevertheless, sleeping sickness is still considered one of the most important tropical diseases. According to the World Health Organization, more than 60 million people in rural areas of sub-Saharan Africa are at risk. War, displacement and migration can lead to an outbreak of the disease.

In the search for a possible starting point for drugs against the pathogen, the researchers had targeted a central enzyme of the unicellular organism, inosine-5′-monophosphate dehydrogenase (IMPDH). “This enzyme belongs to the central inventory of every organism and constitutes an interesting target for drugs because it regulates the concentration of two vital nucleotides in the cell: guanosine diphosphate and guanosine triphosphate”, explains Redecke. “The cell needs these nucleotides to provide energy and to build larger structures such as the genome. If you interrupt this cycle, the cell dies.

The enzyme has a kind of on / off switch that is activated by docking the cell’s own molecules. One promising approach is to block this switch with a precisely matched molecule. In order to construct such an inhibitor, the exact spatial structure of the switch must be known. Structural biologists can determine the structure of biomolecules using x-rays. They do this by first growing small crystals from the biomolecules, which then generate characteristic diffraction patterns when illuminated by rays. X-rays. From these models, the atomic structure of the crystal and its building blocks, the biomolecules, can be calculated.

This approach is often complicated by the intractability of most biomolecules against crystal formation. And while such crystals can be cultivated, they are usually extremely sensitive to high energy x-rays and are quickly destroyed. “Although the structures of many IMP dehydrogenases are already known, there has been no success in growing crystals of the Trypanosoma brucei version of the enzyme ”, reports Betzel, also a researcher in the Pole of Excellence CUI: Advanced Matter Imaging at the University of Hamburg and DESY.

The team therefore chose an alternative path: the group of co-author Michael Duszenko from the University of Tübingen induced certain insect cells to crystallize biomolecules within them. Using this so-called in cellulosic crystallization, the same team had previously deciphered another key enzyme in the sleeping sickness pathogen, cathepsin B, which is also a potential drug target. It has been found that the modified insect cells also produce crystals of the dehydrogenase currently under investigation. These crystals form tiny needles about 5 thousandths of a millimeter (5 microns) thick and up to 70 microns long, so that they protrude from producer cells.

In-cellulo crystals are so small that very bright X-rays are needed to analyze them. The larger a crystal, the more atoms inside that can scatter x-rays, causing a better diffraction pattern. The researchers therefore used the LCLS x-ray laser from the SLAC National Accelerator Laboratory in the United States for the analysis. “X-ray lasers generate extremely intense flashes,” explains Chapman, senior researcher at DESY at the Center for Free-Electron Laser Science CFEL and one of the spokespersons for the cluster of excellence. CUI: Advanced Matter Imaging. “Although the sensitive crystals evaporate immediately, they first generate a diffraction pattern from which structure can be obtained.” The method used here to harness these properties, called serial femtosecond crystallography, was developed earlier by many of the researchers involved in this study and was named one of the top ten breakthroughs of the year by Science revised in 2013.

The team recorded the diffraction patterns of more than 22,000 microcrystals and were able to calculate the spatial structure of the enzyme with an accuracy of 0.28 millionth of a millimeter (nanometer), which roughly corresponds to the diameter of an aluminum atom. “The result not only shows the exact structure of the enzyme switch, the Bateman region, but also which molecules in the cell activate the switch and how these so-called cofactors bind to the enzyme switch,” reports Karol Nass who carried out this study. work during his doctoral studies at DESY. He now works at the Paul Scherrer Institute in Switzerland and is, along with Redecke, the main author of the publication.

The switch is operated by the molecules of adenosine triphosphate (ATP) and guanosine monophosphate (GMP). “The advantage of our method is not only that we are able to study the enzyme at room temperature, at which the enzyme works naturally, but also that during in cellulosic crystallization the natural cofactors bind to the enzyme, ”Betzel said. According to the researcher, the data could now provide an approach to inhibit the parasite’s IMP dehydrogenase. “You could think of building some kind of clasp that would cover the binding sites of the two cofactors, for example.”

However, a remaining challenge is to design the IMP dehydrogenase inhibitor in such a specific way that it blocks the parasite enzyme, but not the human enzyme. If this is successful, the method could potentially be extended to other pathogens, says Betzel. “Other parasites have a very similar structure, and it might also be possible to attack them via the respective IMP dehydrogenase. The enzyme is a very interesting target for drugs, for example against fox tapeworm or the pathogen of elephantiasis.

The universities of Hamburg, Lübeck and Tübingen, the Russian Academy of Sciences, Arizona State University, the Lawrence Livermore National Laboratory in the United States, the Max Planck Institute for Medical Research, the US National Accelerator Laboratory SLAC, the University of Göteborg and DESY were involved in this research.

Reference Nass, et al. (2020) The in cellulo crystallization of Trypanosoma brucei IMP dehydrogenase allows the identification of true cofactors. Nature Communication, DOI: 10.1038 / s41467-020-14484-w

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