The human malaria parasite, Plasmodium falciparum possesses unique gliding machinery referred to as the glideosome that powers its entry into the insect and vertebrate hosts. Several parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with the glideosome motor complex in the inner membrane complex of the merozoite. Using an experimental genetics approach, we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5, and an uncharacterized protein—referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade host RBCs. Additionally, the downregulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites showed abrogation of reorientation/gliding. However, initial attachment with host RBCs was not affected in these parasites. Together, the data presented here show that proteins of the PhIL1-associated complex play an important role in the orientation of P. falciparum merozoites following initial attachment, which is crucial for the formation of a tight junction and hence invasion of host erythrocytes.

Phylum apicomplexan consists of parasites, such as Plasmodium and Toxoplasma. These obligate intracellular parasites enter host cells via an energy-dependent process using specialized machinery, called the glideosome. In the present study, we used Plasmodium falciparum GAP50, a glideosome-associated protein, as a target to screen 951 different compounds from diverse chemical libraries. Using different screening methods, eight compounds (Hayatinine, Curine, MMV689758 (Bedaquiline), MMV1634402 (Brilacidin), and MMV688271, MMV782353, MMV642550, and USINB4-124-8) were identified, which showed promising binding affinity (KD < 75 μM), along with submicromolar range antiparasitic efficacy and selectivity index > 100 fold for malaria parasite. These eight compounds were effective against Chloroquine-resistant PfINDO and Artemisinin-resistant PfCam3.1^R359T strains. Studies on the effect of these compounds at asexual blood stages showed that these eight compounds act differently at different developmental stages, indicating the binding of these compounds to other Plasmodium proteins, in addition to PfGAP50. We further studied the effects of compounds (Bedaquiline and USINB4-124-8) in an in vivoPlasmodium berghei mouse model of malaria. Importantly, the oral delivery of Bedaquiline (50 mg/kg b. wt.) showed substantial suppression of parasitemia, and three out of seven mice were cured of the infection. Thus, our study provides new scaffolds for the development of antimalarials that can act at multiple Plasmodium lifecycle stages.

Amoebiasis, a widespread disease caused by the protozoan parasite Entamoeba histolytica, poses challenges due to the adverse effects of existing antiamoebic drugs and rising drug resistance. Novel targeted drugs are in need of the hour to combat the prevalence of this disease. Given the significance of cysteine for Entamoeba survival, the rate-determining step in the serine (the sole substrate of cysteine synthesis) biosynthetic pathway, i.e., the conversion of 3-phosphoserine to l-serine catalyzed by phosphoserine phosphatase (PSP), emerges as a promising drug target. Our previous study unveils the essential role of EhPSP in amoebas' survival, particularly under oxidative stress, by increasing cysteine production. The study also revealed that EhPSP differs significantly from its human counterpart, both structurally and biochemically, highlighting its potential as a viable target for developing new antiamoebic drugs. In the present study, employing in silico screening of vast natural and synthetic small chemical compound libraries, we identified 21 potential EhPSP inhibitor molecules. Out of the 21 compounds examined, only five could inhibit the catalytic activity of EhPSP. The inhibition capability of these five compounds was subsequently validated by in silico binding free energy calculations, SPR-based real-time binding studies, and molecular simulations to assess the stability of the EhPSP-inhibitor complexes. By identifying the five potential inhibitors that can target cysteine synthesis via EhPSP, our findings establish EhPSP as a drug candidate that can serve as a foundation for antiamoebic drug research.

Malaria remains one of the leading causes of morbidity and mortality worldwide, mainly because of the emergence of drug resistance against current antimalarials. The Plasmodium falciparum food vacuole (FV) proteins, P. falciparum chloroquine (CQ) resistance transporter (PfCRT), PfMDR1 and the cytosolic protein PfKelch13 have been linked to CQ and artemisinin resistance, respectively. Here, we aimed to identify the associations of these resistance markers with mutations in other FV transporters in several field isolates. In this study, we isolated intact P. falciparum FVs and carried out detailed proteome analysis to identify new FV transporters. Furthermore, we carried out co-existing mutational analysis for these transport proteins identified in the FV-enriched fraction with known PfKelch13 and PfCRT polymorphisms via single-nucleotide polymorphism (SNP) data from the Pf3K and MalariaGEN databases. Proteome analysis identified 16 transporter proteins in Plasmodium FVs. Comparative amino acid analysis of these transporter proteins revealed a coassociation of mutations in several transport proteins identified in the FV-enriched fraction with mutations in the PfKelch13, PfCRT, and PfMDR1 proteins. SNP data analysis of the Pf3K and MalariaGEN databases for 2,517 samples revealed the coassociation of six mutations in four transporter genes, PfCRT, PfNT1, PfCTR2, and PfMDR2, with the PfKelch13 polymorphisms ( P < 0.0001), suggesting the contribution of additional parasite transporters to the evolution of CQ and artemisinin resistance. Furthermore, functional complementation with the wild-type PfNT1 and PfMFR5 proteins and their mutant forms (PfNT1-F394L, PfMFR5-S278T, and PfMFR5-Y570F) in Saccharomyces cerevisiae resulted in resistance to mutant phenotypes in the presence of dihydroartemisinin, suggesting a possible role of these mutations in the acquisition of drug resistance. Together, the genome sequence data from field isolates and yeast complementation analysis of the mutant phenotypes identified novel loci related to PfKelch13-mediated antimalarial resistance and revealed unexplored contributions of transporters to artemisinin resistance.