Malaria currently remains one of the most worrisome global infectious diseases, causing more than 400.000 deaths annually, with non-lethal infections still having a stark negative impact on the populations of countries where the disease is widespread. Severe malaria is predominantly caused by the protozoan Plasmodium falciparum, with strains of the organism resistant to established antimalarial drugs appearing in recent years. This development strengthened the incentive to discover novel, species-selective drugs to inhibit the growth of Plasmodium falciparum. One of the most promising targets for potential inhibitors is the biosynthetic pathway for pyrimidine nucleotides, whose rate-limiting step is catalysed by dihydroorotate dehydrogenase. To date, researchers have identified several classes of potential small-molecule dihydroorotate dehydrogenase inhibitors as well as solved the crystal structures of some of these inhibitors in complex with the enzyme. A recently discovered inhibitor class are the bicyclic pyrazolidinones, whose crystal structures with the enzyme have yet to be elucidated.
The aim of our research was to prepare a construct for the expression of a recombinant variant of Plasmodium falciparum dihydroorotate dehydrogenase capable of forming diffraction-quality crystals as was previously described. This would aid future research aiming to study the crystal structures of the three most promising pyrazolidinone inhibitors bound to the enzyme. The recombinant protein contains a deletion of a disordered loop that would otherwise prevent crystallisation of the protein. The construct was successfully prepared through mutagenesis by overlap extension PCR and can further be ligated into an expression vector of choice. We also performed an in silico docking study using AutoDock Vina to predict the binding modes of these pyrazolidinones. The 3D-structures of the inhibitors were docked into a partially flexible binding pocket. As a result, we obtained more informative data compared to a recent similar docking study. Our results were also more in line with existing kinetic data. We postulate that the binding of pyrazolidinones relies predominantly upon hydrophobic interactions with the binding pocket, with little possibility for classic hydrogen bond formation, which distinguishes this inhibitor class from most of the other inhibitor types. The results also suggest that the substituted phenyl group, present on each of the docked inhibitors, plays a non-negligible part in lowering their binding energy by π-stacking and similar interactions.
|
