Anders Hofer Lab

Nucleotides are needed as building blocks to manufacture DNA and RNA and can be categorized into purines and pyrimidines. Many pathogens lack important enzymes for making nucleotides, especially the ability to make purines is often lacking. These pathogens are therefore dependent on nucleotide precursors (ribonucleosides, deoxyribonucleosides and nucleobases), which can be taken up from the host organism. In the case of ribonucleosides and deoxyribonucleosides, they need to be phosphorylated in three steps before being usable as building blocks for RNA and DNA.

In one part of the project, we study the enzymes responsible for the first phosphorylating step in the pathogens and in a second part we study the regulation of the enzyme ribonucleotide reductase, which has a key role in making DNA building blocks.

Nucleotide metabolism in pathogens

Trypanosoma brucei is a unicellular parasite, which is spread by tsetse flies and causes the fatal disease African sleeping sickness. We exploit that the parasite cannot perform de novo synthesis of purines, and therefore is dependent on taking up and phosphorylating already existing purines, which are salvaged from the surrounding environment in the body. One of these enzymes is adenosine kinase, which is needed for the phosphorylation of adenosine that is a purine source in the blood. We exploit this weakness of the pathogen by developing substances (adenosine analogues), which resemble adenosine but kill the parasites after being activated by the enzyme.

Trypanosoma brucei is a protozoan parasite that is spread by tsetse flies and causes African sleeping sickness.

ImageAnders Hofer

The project has subsequently been expanded to more pathogens, including Giardia intestinalis, Trichomonas vaginalis and Borrelia burgdorferi. Giardia intestinalis causes giardiasis, a severe diarrhea which can sometimes become fatal whereas Trichomonas vaginalis causes trichomoniasis, a venereal disease coupled to cervical cancer. The treatments of giardiasis, trichomonas and African sleeping sickness give side effects and in many cases the diseases are not cured by current treatments. Borrelia burgdorferi causes lyme disease (borreliosis). The treatment options against early stage lyme disease are better than against the other three diseases but with an increasing antibotic resistance there is also in this case a need to develop new drugs. In contrast to existing medicines, our substances are targeting unique features of the pathogens, and there is thereby no risk that the drug resistance can spread between different species.

Giardia intestinalis and Borrelia burgdorferi lack many of the enzymes in nucleotide metabolism and one of the enzymes missing is ribonucleotide reductase. The pathogens can therefore not make the building blocks needed for de novo DNA biosynthesis. The DNA building blocks (deoxyribonucleosides) needs therefore to be taken up from the surroundings and be phosphorylated and the project is focused on the nucleoside kinases involved and how the process is regulated. We have also initiated studies of the protozoan parasite Trichomonas vaginalis, which is another organism lacking many of the enzymes needed to synthesize the nucleic acid building blocks. The dependence on salvage pathways in the pathogens can be exploited by nucleoside analogues, which upon activation by the nucleoside kinases harm the pathogens.

In a second project, we study how the production of the four DNA building blocks dCTP, dTTP, dGTP and dATP is regulated in the cell. A key enzyme in the process is ribonucleotide reductase, which can oligomerize and form different structures with higher and lower activity levels. The oligomers formed varies depending on the species and one part of the project is to study how the protein complexes differ from the mammalian enzyme in pathogens such as Pseudomonas aeruginosa, Clostridium botulinum and Helicobacter pylori. The project is technically driven in order to be able to measure which protein complexes can be formed at physiological concentrations of the enzyme and we use methods such as GEMMA (gas-phase electrophoretic macromolecular mobility analysis) and mass photometry for the analyses. We also develop new methods for the measurement of different nucleotides in various organisms and cell types to be able to increase the reliability and sensitivity of the analyses.

Farahnaz Ranjbarian makes an HPLC analysis of nucleotides.

ImageMattias Pettersson