In the current study, the 3380 essentially reported proteins of P. falciparum strain 3D7 were analyzed to address potent novel druggable targets. These proteins were analyzed based on their non-homology with the human host as well as human gut microbiome proteome. The targets were additionally shortlisted based on strict threshold criteria of basic druggability features. Among the shortlisted targets (Table 2), the protein, Bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) (C0H4F3_PLAF7) also known as asymmetrical diadenosine 5′,5″-P1,P4-tetraphosphate hydrolase (PfAp4A) (EC 126.96.36.199) is an enzyme belongs to the hydrolase family . This enzyme participates in pyrimidine and purine metabolism . The PfAp4A hydrolase exhibited high-temperature stability even at 60°C . Previously in few studies, the PfAp4A is also tested as a potential drug target against P. falciparum [43, 44].
The protein serine/threonine-protein phosphatase (Q8I2N2_PLAF7) was also found among finally shortlisted target that involve in regulation of many cellular signaling pathways by catalyzing the removal of phosphate group from target enzymes. This enzyme plays a central role in the functional regulation and control of different genes related to the cell cycle . The phosphorylation regulates several primary steps in P. falciparum's diverse life cycles. Many of the kinases and phosphatases as well as their substrates are specific to parasites, making eventually the phosphorylation event as a viable target for anti-parasitic action . The protein phosphatase-1, a type of PfPPP, involve in the mitotic division of P. falciparum and plays an important role in the liberation of merozoites. Prior studies on P. falciparum revealed that the activity of PfPP1 is more important as compared to protein phosphatase 2A (PP2A) . This also verified by transcriptomic analysis, where the PfPP1 transcript levels reported higher than PP2A after 24 hours of RBC infection . The okadaic acid (OA), a toxin initially isolated from a marine sponge, i.e. Halichondria okadai has been identified as a selective inhibitor of serine/threonine protein phosphatases (PPPs) and reported to strongly inhibits PP1, 2A, and 2B in-vitro . Out of 30 examined protein phosphatase, the 16 protein phosphatases along with PP1 and putative phosphatases seem to be important for blood-stage parasites . Moreover, some studies also showed that PfPP1 is indispensable for blood-stage parasite survival . Many phosphatases play key roles in the pathological pathways, and their inactivation may help to prevent or postpone the emergence of human diseases. Therefore, the potent inhibitors for such phosphatases might be of great therapeutic benefit.
The enzyme cytochrome b5 Reductase (cb5r) (Q8I599_PLAF7) plays a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics . This protein has been thoroughly studied in mammals, but still need to be characterized in microorganisms, such as fungi and parasites, including P. falciparum. There is a close phylogenetic relationship between the plant and P. falciparum cb5r proteins. The plant cb5r has been identified as a novel herbicidal target . This protein reported essential for P. falciparum survival and was found human host non-homolog and possibly a potent therapeutic target, thereby might be a worthy candidate for drug development against malaria.
The vacuolar protein sorting-associated protein 29 (VPS29) (Q8IM27_PLAF7) is involved in the essential metabolic process of proteins translocation to the subcellular organelles. The P. falciparum sort and traffic newly synthesized proteins to target intracellular organelles as well as beyond the plasma membrane into the host cell in some cases . The P. falciparum VPS29 (i.e. PfVPS29) is the functional component in the assembly of the retromer complex . During the PPI analysis, the pfVPS29 shows direct interactions with other retromer complex components i.e. PfVPS26, VPS9, VPS10 as shown in Fig. 2. The PfVPS29 is located in the cytosol and highly expressed in early trophozoite and schizont stages . Inhibiting the activity of PfVPS29 may lead to the disassembling of the retromer complex and possibly halt the protein sorting function of the P. falciparum.
The multifunctional methyltransferase subunit (Q8IM19_PLAF7) have methyltransferase activity during post-translational modifications, chromatin remodeling and protein heterodimerization activity . The protein methyltransferases (PMTs) have been linked to the pathogenesis of a variety of diseases, including human cancers, inflammatory diseases, metabolic diseases, and neurodegenerative diseases. The PMTs are highly attractive among the histone-modifying enzymes and act as drug targets [55, 56]. However, to date no study been conducted about the targeting or inhibition of P. falciparum methyltransferase.
The RuvB-like helicase (Q8ID85_PLAF7) also shortlisted as therapeutic target in the current study. The RuvB-like helicase function like ATP- dependent helicases. It has a vital role in the cell cycle and transcription [57, 58, 59]. The RUVBL proteins (RUVBL1 & 2) are known to regulate various essential cellular processes in different organisms like S. cerevisiae, drosophila and C. elegans [60, 61, 62]. Three types of RuvB, i.e., PfRuvB1, PfRuvB2, and PfRuvB3 are present in the P. falciparum. The PfRuvB1 possesses ssDNA-stimulated ATPase activity and function as a helicase that unwind the DNA in a 5' to 3' direction . The PfRuvB2 works similar to PfRuvB1, however, its helicase activity is comparatively weak. The PfRuvB3 function only as ATPase with no helicase activity during schizont/merozoits or interaerythocytic mitosis . During the developmental stages of the parasite, the PfRuvB1 and PfRuvB2 are expressed in the asexual phase, while the PfRuvB3 expresses only during the schizont stage, where intraerythrocytic mitosis of P. falciparum occurs . The PfRvuBL3 protein is a true homolog of yeast RuvBL2. Since in yeast, RuvBL proteins are shown to be extremely essential for survival and known to regulate the transcription of almost 5% of yeast genes . The RuvB- like helicases are suitable drug targets to control malaria due to their essentiality for pathogen and non-homology with human host proteome. It is reported that helicases are needed for the proliferation of bacteria, viruses and plasmodium, and inhibiting the DNA unwinding activity reduces the replication of these pathogens in cell cultures and animal models [65, 66, 67]. The PfRuvB1 ATPase activity is formerly reported to be inhibited by actinomycin, novobiocin, and ethidium bromide .