X-ray laser reveals structure of a key enzyme of the parasite Trypanosoma brucei
Biochemists of the University of Lübeck involved in a potential novel approach for sleeping sickness drugs
Using ultra-bright X-ray flashes, a team of researchers led by Lars Redecke from the University of Lübeck and DESY, Christian Betzel from the University of Hamburg, and Henry Chapman from DESY has identified a potential target for new drugs against sleeping sickness: The scientists have decoded the detailed spatial structure of a vital enzyme of the pathogen, the parasite Trypanosoma brucei. The result provides a possible blueprint for a drug that specifically blocks this enzyme and thus kills the parasite, as the team reports in the journal Nature Communications.
Sleeping sickness (African trypanosomiasis) is a tropical disease caused by the parasite Trypanosoma brucei, which is transmitted by the bite of tsetse flies, that 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 dramatically decreased in recent years. Nevertheless, sleeping sickness is still considered one of the most important tropical diseases. Around 65 million people in 36 sub-Saharan countries live in the risk area. War, displacement and migration can cause the disease to flare up.
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 is an interesting target for drugs because it regulates the concentration 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 kind of on/off switch that is activated by binding of the cell’s own molecules. A promising approach is to block this switch with a precisely tailored 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. To do this, they first grow small crystals from the biomolecules, which then generate characteristic diffraction patterns when illuminated with X-rays. From these patterns the structure of the crystal and its building blocks, the biomolecules, can be calculated.
This route is often complicated by the intractability of most biomolecules against forming crystals, a state that usually contradicts their natural function. And if such crystals can be grown, they are usually extremely sensitive to the high-energy X-rays and are quickly destroyed. Although the structures of numerous IMP dehydrogenases from other organisms are already known, there had been no success in growing crystals of the Trypanosoma brucei version of the enzyme so far.
The team therefore chose an alternative route, the crystallization of proteins in living insect cells, denoted as in cellulo crystallization. Using this approach the same team had already deciphered another key enzyme of the sleeping sickness pathogen, cathepsin B, which is also a potential drug target. It turned out that the modified insect cells also produce crystals of the dehydrogenase now investigated. These crystals form tiny needles around 5 micrometres in thickness and up to 70 micrometres in length, which protruded from the producing cells without affecting the cell viability.
The in-cellulo crystals are so small that very bright X-rays are required to analyse them. The larger a crystal is, the more X-rays it scatters, the better the diffraction pattern. The researchers therefore used the LCLS X-ray laser at the SLAC National Accelerator Laboratory in the US for the analysis, which generates extremely intense X-ray flashes. Although the sensitive crystals evaporate immediately, they first generate a diffraction pattern from which the structure can be obtained. The method used here to exploit these properties, called serial femtosecond crystallography, was developed earlier by many of the researchers involved in this study and named one of the top ten breakthroughs of the year by Science magazine in 2013.
The team recorded the diffraction patterns of more than 22,000 microcrystals and was able to calculate the spatial structure of the enzyme with an accuracy of 0.28 nanometre. The result does not only show the exact structure of the enzyme switch, the Bateman region, but also which molecules of the cell activate the switch and how these so-called co-factors dock to the enzyme. The switch is operated by the molecules adenosine triphosphate (ATP) and guanosine monophosphate (GMP). The advantage of the applied apporach is not only that the enzyme was investigate at room temperature, at which the enzyme naturally operates, but also that during in cellulo crystallisation the natural co-factors bind to the enzyme.
The data might now provide an approach for blocking the parasite’s IMP dehydrogenase. However, a remaining challenge is to design the IMP dehydrogenase iinhibitor in such a specific way that it blocks the parasite’s enzyme, but not the human enzyme. If this is successful, the method could potentially be extended to other pathogens, since other parasites have an IMPDH of highly similar structure.
The universities of Lübeck, Hamburg, 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 in Heidelberg, the US National Accelerator Laboratory SLAC, the University of Gothenburg and DESY were involved in this research.
In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors; Karol Nass, Lars Redecke et al.; Nature Communications, 2020; DOI: 10.1038/s41467-020-14484-w