Targeting Plasmodium falciparum protein aggregation as a new antimalarial design strategy

Abstract

[eng] Malaria caused 241 million cases and 627,000 deaths worldwide in 2020, representing one of the biggest threats for global health nowadays. The currently available arsenal of antimalarial drugs is insufficient to progress towards eradication of the disease, a scenario that is worsened by the rampant evolution of resistance by Plasmodium, the causative agent of malaria. Protein aggregation in malaria parasites is prominent during their whole life cycle. Aggregative proteins are distributed throughout the parasite’s cytosol, especially in the endoplasmic reticulum adjacent areas, where protein translation and folding take place. In this thesis, we intended to target the aggregative features of the Plasmodium falciparum proteome with the final objective of developing an effective antimalarial strategy. Firstly, based on in silico and in vivo data, we selected a group of aggregative peptides present in parasite proteins. Those peptides formed aggregates in vitro; however, attempts to further increase the high aggregation propensity of the P. falciparum proteome by delivering them to in vitro cultures did not significantly decrease the viability of the pathogen. To confirm the lack of activity of the peptides on P. falciparum viability, their entrance inside the parasite was improved combining two different methods: their tagging with cell-penetrating peptides and their encapsulation inside ghost red blood cells. Despite the significantly enhanced entrance of the peptides inside parasites using these two approaches, P. falciparum growth was not affected. To test the alternative hypothesis, i.e. if inhibiting protein aggregation in the parasite might impair its development, we treated in vitro cultures with amyloid pan-inhibitors, which are molecules able to prevent amyloid fibril formation. All of these compounds showed some extent of antiplasmodial activity. Particularly one of them, the double pyridinium salt YAT2150, exhibited potent antimalarial activity with an in vitro IC50 of 90 nM. This drug was also effective on the sexual forms of P. falciparum and on the hepatic stages of P. berghei. In relation with its mode of action, YAT2150 is a powerful inhibitor of the aggregation of the amyloid β peptide fragment 40 in vitro and it reduced in P. falciparum cultures the amyloid content and the quantity of ubiquitinated proteins, as well as the amount in aggregative proteins detected with thioflavin T. Thus, YAT2150 antimalarial mode of action is the inhibition of protein aggregation in the parasite. Moreover, we observed that YAT2150 resistance emergence is not easily developed by P. falciparum cultures and that already acquired resistances to other antimalarial compounds do not affect YAT2150 activity. In this thesis we show that targeting P. falciparum protein aggregation is a valid antimalarial strategy and that YAT2150, belonging to a chemical family with no other antimalarials described, acting through a new antiparasitic mechanism not shared by other currently used drugs, and targeting many gene products, is a good candidate to significantly contribute to malaria eradication.

[eng] Se estima que en el año 2020 se produjeron en todo el mundo 241 millones de casos de malaria. Esta enfermedad, causada por parásitos del género Plasmodium, sigue siendo una gran amenaza para la salud global. Sin embargo, los antipalúdicos usados actualmente son insuficientes para combatir la malaria, especialmente porque se han detectado parásitos resistentes a la gran mayoría de ellos. Por otro lado, sabemos que la agregación de proteínas en Plasmodium es prominente, de hecho aquí mostramos que se detecta en todas las fases del desarrollo del parásito. Esta tesis se centra, precisamente, en el uso de las proteínas agregativas de P. falciparum como dianas terapéuticas para el desarrollo de nuevas estrategias antipalúdicas. Exploramos dos hipótesis complementarias: el aumento de los niveles basales de agregación proteica en el parásito mediante la administración de péptidos agregativos presentes en el proteoma de P. falciparum, y la inhibición de la agregación de las proteínas agregativas del parásito mediante el tratamiento con compuestos inhibidores de la formación de amiloides. Mientras la primera estrategia no reduce de manera efectiva la viabilidad del parásito, la segunda sí lo hace. Especialmente, uno de los compuestos testados, YAT2150, tiene un efecto nocivo tanto para las formas asexuales como sexuales de P. falciparum y para las hepáticas de P. berghei. Además, este compuesto inhibe la agregación proteica en P. falciparum in vivo (reduce el contenido de proteínas amiloides, de proteínas ubiquitinadas y de proteínas detectadas por tioflavina-T) y no permite el desarrollo de resistencias en cultivos in vitro del parásito.