Identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial efficacy.

Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system. Here, we investigate Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified seven scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages.

High-content imaging as a tool to quantify and characterize malaria parasites.

In 2021, Plasmodium falciparum was responsible for 619,000 reported malaria-related deaths. Resistance has been detected to every clinically used antimalarial, urging the development of novel antimalarials with uncompromised mechanisms of actions. High-content imaging allows researchers to collect and quantify numerous phenotypic properties at the single-cell level, and machine learning-based approaches enable automated classification and clustering of cell populations. By combining these technologies, we developed a method capable of robustly differentiating and quantifying P. falciparum asexual blood stages. These phenotypic properties also allow for the quantification of changes in parasite morphology. Here, we demonstrate that our analysis can be used to quantify schizont nuclei, a phenotype that previously had to be enumerated manually. By monitoring stage progression and quantifying parasite phenotypes, our method can discern stage specificity of new compounds, thus providing insight into the compound's mode of action.

A Proteasome Mutation Sensitizes P. falciparum Cam3.II K13C580Y Parasites to DHA and OZ439.

Artemisinin-based combination therapies (ACTs), the World Health Organization-recommended first-line therapy for uncomplicated falciparum malaria, has led to significant decreases in malaria-associated morbidity and mortality in the past two decades. Decreased therapeutic efficacy of artemisinins, the cornerstone of ACTs, is threatening the gains made against this disease. As such, novel therapeutics with uncompromised mechanisms of action are needed to combat parasite-mediated antimalarial resistance. We have previously reported the antimalarial activity of Plasmodium falciparum-specific proteasome inhibitors in conjunction with a variety of antimalarials in clinical use or in preclinical investigations and of proteasome mutants generated in response to these inhibitors. Here, we discover that despite harboring K13C580Y, which has conventionally mediated artemisinin resistance in vitro as measured by increased survival in ring-stage survival assays (RSA), the Cam3.II strain parasites of Cambodian origin that have acquired an additional mutation in the proteasome display increased susceptibility to DHA and OZ439. This discovery implicates the proteasome in peroxide susceptibilities and has favorable implications on the use of peroxide and proteasome inhibitor combination therapy for the treatment of artemisinin-resistant malaria.

Plasmodium falciparum Artemisinin Resistance: The Effect of Heme, Protein Damage, and Parasite Cell Stress Response.

Despite a significant decline in morbidity and mortality over the last two decades, in 2018 there were 228 million reported cases of malaria and 405000 malaria-related deaths. Artemisinin, the cornerstone of artemisinin-based combination therapies, is the most potent drug in the antimalarial armamentarium against falciparum malaria. Heme-mediated activation of artemisinin and its derivatives results in widespread parasite protein alkylation, which is thought to lead to parasite death. Alarmingly, cases of decreased artemisinin efficacy have been widely detected across Cambodia and in neighboring countries, and a few cases have been reported in the Guiana Shield, India, and Africa. The grim prospect of widespread artemisinin resistance propelled a concerted effort to understand the mechanisms of artemisinin action and resistance. The identification of genetic markers and the knowledge of molecular mechanisms underpinning artemisinin resistance allow prospective surveillance and inform future drug development strategies, respectively. Here, we highlight recent advances in our understanding of how parasite vesicle trafficking, hemoglobin digestion, and cell stress responses contribute to artemisinin resistance.