Discovery of protein aggregation in plasmodium parasites and development of a combinational antimalarial therapy at the nanoscale

Laburpena

Malaria is a vector-borne disease caused by protist parasites of the genus Plasmodium and transmitted by the bites of Anopheles mosquitoes. With approximately 200 million cases and 400,000 deaths during 2019, Malaria represents one of the major health problems of humanity. Moreover, the emergence of resistant parasites endangers the progress achieved until today. Therefore, research on novel therapeutic targets and efficient drug delivery systems capable of optimizing the drug dosage is essential to eradicate this disease. Current antimalarial therapy is based on the utilization of artemisinin in combination with a partner drug. The rationale is to ensure the effectivity of the treatment as artemisinin quickly kills the majority of parasites while the partner drug eliminates the rest of them. However, if the two drugs have different pharmacokinetic profiles, resistant parasites can be easily selected. To solve this, the first part of this Thesis consisted on the investigation of a glycophorin Atargeted nanovector capable of co-encapsulating the anti-malarial drugs atovaquone and pyronaridine tetraphosphate for delivering them into Plasmodium-infected red blood cells and gametocytes (the parasite’s stage responsible for the transmission from the human to mosquitoes). The obtained results showed that these co-immunoliposomized drugs had significantly higher activities compared to their free forms when tested into P. falciparum cultures. On the other hand, many hydrophobic antimalarials present a low water solubility and reduced oral absorption, which hamper their clinical implementation. For this reason, the second part of this Thesis consisted on the investigation of the utilization of hybrid lipid/polymer and polymeric nanoparticles for the oral delivery of the hydrophobic compound curcumin. The results showed that the utilization of the gastro-resistant polymers Eudragit® and Nutriose® enhanced the oral antimalarial activity of curcumin-bearing liposomes in a malaria murine model. However, when curcumin was encapsulated in zwitterionic mucopenetrating polymeric nanoparticles, no improvement of oral its performance was observed, although parasitized-red blood cells specifically accumulated these polymeric nanoparticles. Finally, the role of protein aggregation in Plasmodium proteins was investigated in the third part of the Thesis as Plasmodium proteins are extraordinarily rich in intrinsically unstructured regions and stretches of asparagine repeats; features commonly found in proteins capable of forming amyloid fibrils. Using the amyloid-specific dye PROTEOSTAT®, the presence of abundant protein aggregates in live P. falciparum blood stages and P. yoelii mosquito stages was discovered. Thereafter, a proteomic approach was used to purify aggregating polypeptides from P. falciparum blood stages that showed to be enriched in nuclear-import processes. Five peptides from this protein subset were selected and Thioflavin-T and transmission electron microscopy assays were carried out, which indicated that 4 out of the 5 peptides aggregated with different pharmacokinetics. Together, these results indicate that protein aggregation is a generalized process in the pathophysiology of malaria. Therefore, strategies based the unbalancing of the parasite’s proteostasis either through the induction or inhibition of protein aggregation could lead to the development of a novel antimalarial therapy.