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. 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.II^R539T) and artemisinin-sensitive (Cam3.II^rev) parasites. 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.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite’s lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.

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-x_L 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-x_L is essential for Plasmodium development and tested this hypothesis using six BCL-x_L inhibitors, including one FDA-approved compound. All BCL-x_L 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-x_L is relocated from the RBC cytoplasm to the vicinity of the parasite upon infection. Further, immunoprecipitation of BCL-x_L coupled with mass spectrometry analysis identified that BCL-x_L 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.

New antimalarials are needed due to the threat of emerging resistance against existing antimalarial therapies. A phenotypic screen uncovered the N-aryl acetamide class that inhibits the development of P. falciparum asexual ring-stage parasites. The structure-activity relationship of this class was investigated, and key modifications were introduced that produced WEHI-326 with potent antimalarial activity. Enhancing the metabolic stability of this class will be a future challenge to achieve efficacy in a malaria mouse model. WEHI-326 was found to have a moderate barrier to resistance and a moderate rate of asexual kill, potently inhibited gametocyte and gamete development, and in turn, blocked the transmission of parasites to the mosquito. Forward genetics and cross-resistance profiling determined that parasites resistant to N-aryl acetamides had mutations in rhomboid protease 8 (ROM8) and the putative cation channel, CSC1. WEHI-326 will be an important tool in unraveling the role of ROM8 and CSC1 in P. falciparum development.

With resistance to current frontline antimalarials spreading globally, new drug candidates need to be discovered to populate the antimalarial drug development pipeline. We previously screened the Medicines for Malaria Venture Pathogen Box for compounds that prevent Plasmodium falciparum parasites from exiting and invading human erythrocytes, steps essential for the proliferation of parasites in the blood, which causes disease. Compound MMV020512 (M-512) was identified in this screen and live cell imaging here established that it does not specifically inhibit invasion but likely inhibits intraerythrocytic parasite growth. M-512 resistance selection in parasites led to the identification of mutations in the membrane protease Pf ROM8 and the cation ion channel Pf CSC1. Pf ROM8 was validated as a target of M-512 when a L562R putative resistance mutation was engineered into wildtype parasites reproducing the resistance phenotype. Knockdown of wildtype Pf ROM8, the L562R mutant and CSC1 reduced parasite growth, indicating the proteins are functionally important. Counterintuitively, the Pf ROM8 and Pf CSC1 knockdown parasites became more resistant to M-512 suggesting that the compound is an agonist of both proteins which may form a functional complex and that dysregulation of this complex is deleterious to parasite growth.