Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA: Blocking invasion and inactivating extracellular merozoites.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.

Advances in Plasmodium research, an update: Highlights from the Malaria in Melbourne 2021 conference.

The Malaria in Melbourne 2021 conference was held online in October. This conference aims to provide a platform for students and early career researchers to share their research and develop new collaborative networks. The program covered a broad range of topics including antimalarial drug development, epidemiology, immunology, molecular and cellular biology, and other emerging technologies. This article summarises recent advances in Plasmodium research presented at the Malaria in Melbourne 2021 conference.

Red Blood Cell BCL-xL Is Required for Plasmodium falciparum Survival: Insights into Host-Directed Malaria Therapies.

The development of antimalarial drug resistance is an ongoing problem threatening progress towards the elimination of malaria, and antimalarial treatments are urgently needed for drug-resistant malaria infections. Host-directed therapies (HDT) represent an attractive strategy for the development of new antimalarials with untapped targets and low propensity for resistance. In addition, drug repurposing in the context of HDT can lead to a substantial decrease in the time and resources required to develop novel antimalarials. Host BCL-xL is a target in anti-cancer therapy and is essential for the development of numerous intracellular pathogens. We hypothesised that red blood cell (RBC) BCL-xL is essential for Plasmodium development and tested this hypothesis using six BCL-xL inhibitors, including one FDA-approved compound. All BCL-xL inhibitors tested impaired proliferation of Plasmodium falciparum 3D7 parasites in vitro at low micromolar or sub-micromolar concentrations. Western blot analysis of infected cell fractions and immunofluorescence microscopy assays revealed that host BCL-xL is relocated from the RBC cytoplasm to the vicinity of the parasite upon infection. Further, immunoprecipitation of BCL-xL coupled with mass spectrometry analysis identified that BCL-xL forms unique molecular complexes with human µ-calpain in uninfected RBCs, and with human SHOC2 in infected RBCs. These results provide interesting perspectives for the development of host-directed antimalarial therapies and drug repurposing efforts.

Structure of the Pf12 and Pf41 heterodimeric complex of Plasmodium falciparum 6-cysteine proteins.

During the different stages of the Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites.

A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum-infected red blood cell proteome.

BACKGROUND: Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry-based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility. RESULTS: Here, we present a comprehensive P. falciparum-infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites. CONCLUSION: A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Eryptosis and Malaria: New Experimental Guidelines and Re-Evaluation of the Antimalarial Potential of Eryptosis Inducers.

Erythrocytes possess an unusual programmed cell death mechanism termed eryptosis, and several compounds have been previously claimed to induce eryptosis in vitro. Malaria parasites (genus Plasmodium) reside in erythrocytes during the pathogenic part of their life cycle, and the potential of several eryptosis inducers to act as antimalarials has been tested in recent years. However, the eryptosis-inducing capacity of these compounds varies significantly between eryptosis-focused studies and malaria investigations. Here, we investigated the reasons for these discrepancies, we developed a protocol to investigate eryptosis in malaria cultures and we re-evaluated the potential of eryptosis inducers as antimalarials. First, we showed that eryptosis read-out in vitro is dependent on culture conditions. Indeed, conditions that have consistently been used to study eryptosis do not support P. falciparum growth and prime erythrocytes for eryptosis. Next, we defined culture conditions that allow the detection of eryptosis while supporting P. falciparum survival. Finally, we selected six eryptosis-inducers based on their clinical use, molecular target and antimalarial activities, and re-evaluated their eryptosis inducing capacities and their potential as antimalarials. We demonstrate that none of these compounds affect the viability of naïve or P. falciparum-infected erythrocytes in vitro. Nevertheless, three of these compounds impair parasite development, although through a mechanism unrelated to eryptosis and yet to be elucidated. We conclude that careful consideration of experimental set up is key for the accurate assessment of the eryptosis-inducing potential of compounds and their evaluation as potential antimalarials.

Manipulating Eryptosis of Human Red Blood Cells: A Novel Antimalarial Strategy?

Malaria is a major global health burden, affecting over 200 million people worldwide. Resistance against all currently available antimalarial drugs is a growing threat, and represents a major and long-standing obstacle to malaria eradication. Like many intracellular pathogens, Plasmodium parasites manipulate host cell signaling pathways, in particular programmed cell death pathways. Interference with apoptotic pathways by malaria parasites is documented in the mosquito and human liver stages of infection, but little is known about this phenomenon in the erythrocytic stages. Although mature erythrocytes have lost all organelles, they display a form of programmed cell death termed eryptosis. Numerous features of eryptosis resemble those of nucleated cell apoptosis, including surface exposure of phosphatidylserine, cell shrinkage and membrane ruffling. Upon invasion, Plasmodium parasites induce significant stress to the host erythrocyte, while delaying the onset of eryptosis. Many eryptotic inducers appear to have a beneficial effect on the course of malaria infection in murine models, but major gaps remain in our understanding of the underlying molecular mechanisms. All currently available antimalarial drugs have parasite-encoded targets, which facilitates the emergence of resistance through selection of mutations that prevent drug-target binding. Identifying host cell factors that play a key role in parasite survival will provide new perspectives for host-directed anti-malarial chemotherapy. This review focuses on the interrelationship between Plasmodium falciparum and the eryptosis of its host erythrocyte. We summarize the current knowledge in this area, highlight the different schools of thoughts and existing gaps in knowledge, and discuss future perspectives for host-directed therapies in the context of antimalarial drug discovery.