The emergence of Plasmodium falciparum resistance raises an urgent need to find new antimalarial drugs. Here, we report the rational repurposing of the anti-hepatitis C virus drug, alisporivir, a nonimmunosuppressive analog of cyclosporin A, against artemisinin-resistant strains of P. falciparum . In silico docking studies and molecular dynamic simulation predicted strong interaction of alisporivir with Pf Cyclophilin 19B, confirmed through biophysical assays with a K _d value of 354.3 nM.

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca²⁺ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13^R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13^R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

The pore forming Plasmodium perforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease. Author Summary: Malaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages of Plasmodium spp. life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria.

Artemisinin resistance in Plasmodium falciparum remains a major public health challenge for the ongoing malaria elimination programs. Till now, artemisinin resistance is linked with the variations in the genome of P. falciparum which are mainly clustered in the Pfkelch13 gene. Several studies reported artemisinin resistance in the malaria endemic areas without finding any evidence of genetic variation in parasite for this occurrence. This study interrogated whether host genetic variations have any association with artemisinin sensitivity of malaria parasite. We investigated the relationship of 1) genetic variations in the human ATP2B4 gene and 2) the resultant surface expression of plasma membrane calcium ATPase (PMCA4b) with the intraerythrocytic levels of calcium, reactive oxygen species (ROS) and the resistance towards artemisinin in the intraerythrocytic parasite. This study found negative correlation of PMCA4b expression level with intraerythrocytic calcium and ROS levels. Further in-vitro growth assays revealed that artemisinin sensitivity is reduced in the parasites growing within the RBCs having low PMCA4b and high oxidative stress. Overall, this study highlights the strong association of host PMCA4b in cellular calcium mediated redox imbalance, which significantly contributes to artemisinin resistance in malaria parasite. Hence, this is the first study to document the effect of host variations on artemisinin sensitivity of the parasite. We further emphasize that many redox modulating RBC polymorphisms that are prevalent in malaria endemic areas could influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response. Thus, detailed population specific research may provide new insights for personalized malaria treatment and may inform future drug development strategies. Significance: The human host and malaria parasite share a closely interconnected relationship hence host variations could influence parasite drug resistance. This study demonstrates that reduced PMCA4b expression, the primary calcium efflux pump in RBCs, significantly increases the erythrocytic calcium and reactive oxygen species (ROS) levels, thereby decreasing the P. falciparum growth as well as artemisinin sensitivity. Many ROS inducing RBC polymorphisms, such as sickle cell, thalassemia, G6PD deficiency, PMCA4b variations etc. have emerged as strong malaria protective traits in several studies in malaria endemic areas. But these traits could also influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response and highlights the importance of host erythrocytes oxidative microenvironment surveillance in monitoring antimalarial drug resistance.