Plasmodium falciparum infections pose a tremendous burden on global health, which is becoming increasingly aggravated by the worrying rise in P. falciparum drug resistance making the discovery of novel intervention strategies imperative. In line with this, the objective of this project is to explore the inhibition of a recently identified parasite vitamin biosynthesis pathway as a therapeutic strategy and to assess its potential as drug target (1). The absence of vitamin biosynthesis in humans suggests that specific targeting of the respective parasite pathways is feasible, as it has already been successfully proven for folate metabolism. VITBIOMAL focuses on vitamin B6 biosynthesis, as this important nutrient is required as a co-factor for a wide variety of essential metabolic functions in protein and amino acid metabolism and also has been implicated in the defence against oxidative stress in other eukaryotes.
With 300-500 million clinical cases and 1-3 million deaths a year, malaria is one of the most fatal tropical diseases. As current therapeutics become increasingly ineffective and a clinically available protective vaccine is not in sight, there is an urgent need to develop and pursue new therapeutic strategies.[+] Read More
The aim of VITBIOMAL was to specifically assess vitamin B6 de novo biosynthesis of Plasmodium as target for antimalarial drug development. To do so, the precise role of de novo biosynthesis versus uptake for the overall regulation of vitamin B6 homeostasis in the parasites will be established.
The expected results of this project are:
We furthermore anticipate that we will generate novel insight into the biochemical, biophysical and structural properties, as well as in putative interaction partners of the parasite vitamin B6 synthesising enzyme.
De novo vitamin B6 biosynthesis in Plasmodium occurs through the recently discovered desoxyxylulose 5-phosphate (DXP)-independent pathway (2). This pathway is distinguished by the presence of two genes, pdx1 and pdx2, encoding the synthase and the glutaminase subunit of a class I glutamine amido transferase. The understanding of the molecular interactions between Pdx1 and Pdx2 as well as their interplay with putative interaction partners is instrumental to the development of new effective drugs and will be addressed. In line with this, the expected results will be:
The results, which have been achieved so far, are:
Referring to 3: (i) Knockout parasites of the pdx1 (vitamin B6 biosynthesis) and of the pdxK gene (vitamin B6 uptake/salvage) have been generated in the mouse malaria model system Plasmodium berghei. PdxK, pyridoxal kinase, converts the B6 vitamers pyridoxine, pyridoxamine and pyridoxal into their phosphorylated forms, a prerequisite for the formation of pyridoxal 5`-phosphate - the biologically active form of vitamin B6. PdxK function is required upon vitamin B6 uptake and salvage. As knockouts of both genes were obtained neither vitamin B6 de novo biosynthesis nor vitamin B6 uptake/salvage are essential for the survival of blood stage forms of P. berghei.
(ii) Up to now, knockouts of the pdx1 and pdxK genes in P. falciparum were not obtained, implying that vitamin B6 homeostasis may differ among plasmodial parasites. Accordingly, vitamin B6 biosynthesis has neither been validated not ruled out as a target for the development of novel antimalarial drugs.
Referring to 1: (i) In constrast to the P. berghei pdxK knockout, which had no effect on parasite blood stages, the pdx1 knock out resulted in a growth delay of the erythrocytic forms, suggesting that vitamin B6 de novo biosynthesis is superior to vitamin B6 uptake/salvage in this phase of parasite development.
(ii) Both knockouts resulted in massive reduction of sporozoite numbers ranging from 90 (pdx1 knockout) to 99% (pdxk knockout), suggesting that both vitamin B6 de novo biosynthesis and vitamin B6 uptake/salvage are required for parasite development within the mosquito and accordingly for the progression through this part of the life cycle.
(iii) The PLP synthase and the pdxK are expressed in blood stages of P. falciparum, suggesting that vitamin B6 biosynthesis and B6 uptake/salvage are operating in this phase of parasite development (parasite growth within human erythrocytes results in a massive amplification of parasite numbers being responsible for the symptoms of the disease. Consequently, these stages are the target of most antimalarials).
(iv) Depletion of B6 vitamers from the growth medium had no effect on the development of P. falciparum blood stage forms indicating that vitamin B6 biosynthesis is sufficient to cover the need of pyridoxal 5-phosphate.
Referring to 4: (i) The plasmodial Pdx1 and Pdx2 proteins encode a functional pyridoxal 5`-phosphate synthase. Glutaminase activity of Pdx2 is only observed in the presence of the Pdx1. Pdx2 has a kcat of 0.11 s-1 and a KM of 0.56 mM for glutamine. Pdx1 uses either ribose 5-phosphate or ribulose 5-phosphate as pentose and glyceraldeheyde 3-phopshate or dihydroxyacetone phosphate as triose sugar. The specific activity of the complex is 92.1 pmol min-1 mg-1 using ribose 5`-phosphate and glyeraledeyde 3-phosphate as substrates (3).
(ii) Complex formation of P. falciparum PLP synthase was characterized by titration calometry. The presence of glutamine increased the tightness of interaction by about 30-fold and changed the thermodynamic signature of the complex. While the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characterisitics concerning the relative contribution of apolar/polar surface area (4, 5).
(iii) In addition, the importance of the Pdx1 N-terminus for complex formation was proved by mutational analysis (4).
Referring to 5: A putative interaction partner was identified in a yeast 2hybrid screen and is currently further characterized.
Referring to 6: (ii) The following structures were determined in course of the project: Pdx2 from P. falciparum (1.6 Å), Pdx1 from Bacillus subtilis (to 2.0 Å), Pdx2 from B. subtilis in free (1.7 Å) and inhibitor complexed state (2.2 Å) and the ternary complex of B. subtilis Pdx1:Pdx2 with substrate glutamine (2.1 Å) (3, 6). The B. subitils structure of the ternary complex was the first structure of a PLP synthase being solved. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers arranged in two hexameric rings, which are on top of each other.
(ii) Based on the ternary complex and using the Pdx2 structure from P. falciparum, a homology model of the plasmodial PLP synthase was constructed and tested with experimental data on complex formation, using isothermal calorimetry (see above) (4).
(iii) An activation mechanism of the glutaminase - initially based on the structure of PfPdx2 and later on supported by the B. subtilis structures - has been proposed: (a) The side chain of Cys87 of the catalytic triad, which was found in two conformations, has to attain the one which allows for catalysis. (b) The "oxyanion hole", which is obstructed in the Pdx2 structure, has to be formed by a peptide bond isomerization of the peptide bound Gly51-Gly52 likely occuring by binding of the synthase partner. (c) The synthase domain contributes to the glutaminase active domain (3).
Antimalarial and possibly antiapicomplexan and/or antibacterial drug development