Analyzing the Essential Proteins Set of Plasmodium Falciparum PF3D7 for Novel Drug Targets Identi cation Against Malaria

Fawad Ali Abdul Wali Khan University Mardan Hira Wali Abdul Wali Khan University Mardan Saadia Jan Abdul Wali Khan University Mardan Muneeba Aslam Abdul Wali Khan University Mardan Imtiaz Ahmad Abdul Wali Khan University Mardan Sahib Gul Afridi Abdul Wali Khan University Mardan Suliaman Shams Abdul Wali Khan University Mardan Asifullah Khan (  asifullah111@gmail.com ) Abdul Wali Khan University Mardan https://orcid.org/0000-0002-1444-4249


Human host non-homologous and virulent proteins identi cation
The P. falciparum essential proteins non-homolog to human host were identi ed by comparative sequence analyses via BLASTp tool [19]. The threshold values of 35% query coverage and sequence identity were set during this analysis [20]. The proteins having signi cant similarity with human proteome were discarded and the remaining non-homologs were shortlisted for further analysis. The non-homology search against human gut microbiota proteins sequences was also carried out with a threshold cutoff, i.e. E value 0.001 [21,22]. The Vectors database was screened for P. falciparum 3D7 virulent proteins annotation. The Vectors database contains 5304 virulent proteins data from various parasites including P. falciparum [23].

Drugbank database scanning
The shortlisted essential proteins of P. falciparum from above analyses were scanned against the Drugbank database to identify novel drug targets with 60% query coverage and percent identity threshold of BLASTp [24].

Structure homologs search
The proteins data bank (PDB) was screened to identify the homologous 3D structures of pathogenic proteins [25]. The pathogen proteins were BLAST against the entire PDB database entries with 60% percent sequence identity and query coverage [26,22]. The pathogen sequences having < 60% homology were modeled with Swiss Model [27] and veri ed by ERRAT [28] and RAMPAGE [29].

Druggablity analysis
The prioritized list of essential proteins shortlisted from above analyses were tested for druggability potential. The drug-like molecules binding pockets of the targets were identi ed by PockDrug-server [30]. The subcellular localization was performed with CELLO v.2.5 [31].
The protein-protein interaction (PPI) and molecular weight analysis were performed [32]. The PPI analysis was performed by STRING database and the Hub proteins were identi ed based on node degree (K ≥ 5) that represent the number of interactions [33].

Subtractive proteomic analyses
Essential genes perform key cellular functions for the survival of pathogens [34]. The P. falciparum strain 3D7 essential genes information were obtained from the recently published articles [16,17] and total 3380 essential protein were identi ed. These proteins sequences were retrieved from Uniprot. Seven paralogous protein sequences were excluded by CD-HIT analysis and the remaining 3373 sequences were considered for downstream analysis (Supplementary Table S1). The non-paralogous protein sequences were subjected to BLASTp against human proteome as well as human gut microbiome proteome data with threshold parameters. The gut ora is helpful to the host in many ways like vitamins biosynthesis and absorption of short chain-fatty acids [35]. The unintended inhibition of gut microbe leads to a decrease in gut ora and colonization of pathogenic bacteria in the host gut [36]. The comparative sequences analyses identi ed total 183 P. falciparum strain 3D7 essential proteins non-homologous to human as well as human's gut ora proteins (Supplementary Table S2). The homology screening against Drugbank repository for these 183 proteins inferred no homology with already reported drug targets deposited in drugbank database. The ve among these 183 were annotated as P. falciparum 3D7 virulent proteins during screening of Vectors database (Table 1).

Druggability analyses
The Drugbank non-homologous proteins were prioritized for downstream druggability analyses. The subcellular localization is one of the key aspect of druggability and the cytoplasmic proteins are considered as suitable drug targets [37,38]. The 15 P. falciparum proteins among shortlisted prioritized targets were annotated as cytoplasmic proteins. The proteins 3D structures identi ed by Swiss model were validated with the ERRAT tool with quality factor score of > 50, which is accepted as high quality model [39]. Ramachandran plot identi ed 80-90% of modeled proteins residues in the allowed region assuring good quality structure modeling of the target proteins (Table 2). Finally, six (06) proteins were prioritized on the basis of (i) pockdrug probability score 0.5 [30], (ii) ERRAT quality factor ≥ 90 [28], and (iii) protein-protein interaction node degree i.e. K ≥ 5 [33] (Table 2) (Fig. 2). These six prioritized targets are speculating to hold promising druggable pockets to anchor small drug-like molecules and act as indispensable hub proteins in P. falciparum metabolic network.

Discussion
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 protein serine/threonine-protein phosphatase (Q8I2N2_PLAF7) was also found among nally 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 [45]. 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 speci c to parasites, making eventually the phosphorylation event as a viable target for anti-parasitic action [46]. 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) [47]. This also veri ed by transcriptomic analysis, where the PfPP1 transcript levels reported higher than PP2A after 24 hours of RBC infection [48]. The okadaic acid (OA), a toxin initially isolated from a marine sponge, i.e. Halichondria okadai has been identi ed as a selective inhibitor of serine/threonine protein phosphatases (PPPs) and reported to strongly inhibits PP1, 2A, and 2B in-vitro [49]. Out of 30 examined protein phosphatase, the 16 protein phosphatases along with PP1 and putative phosphatases seem to be important for blood-stage parasites [47]. Moreover, some studies also showed that PfPP1 is indispensable for blood-stage parasite survival [50]. 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 bene t.
The enzyme cytochrome b5 Reductase (cb5r) (Q8I599_PLAF7) plays a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxi cation of xenobiotics [51]. 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 identi ed as a novel herbicidal target [51]. 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 tra c newly synthesized proteins to target intracellular organelles as well as beyond the plasma membrane into the host cell in some cases [52]. The P. falciparum VPS29 (i.e. PfVPS29) is the functional component in the assembly of the retromer complex [53]. 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 [54]. 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 modi cations, chromatin remodeling and protein heterodimerization activity [40]. The protein methyltransferases (PMTs) have been linked to the pathogenesis of a variety of diseases, including human cancers, in ammatory 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 [58]. 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 [63]. 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 [64]. 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 [60]. 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 [68].

Conclusion
We took advantage from the recently published essential proteins of P. falciparum and employed the comparative subtractive proteomics in couple with in silico druggability approaches to identify novel and suitable drug targets against P. falciparum. The study based on comparative sequence analysis, updated biological databases scanning and multi-direction druggability analyses. This ultimately prioritized and addressed several novel druggable targets against P. falciparum infection not highlighted before. Additional consideration of these shortlisted targets in future drug discovery projects may worthy to combat the anti-malarial drug-resistant issues.

Consent for publication
All authors read and agreed to publish the study.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions FA, HW and S.J performed the analysis and prepared the initial draft. MA, IA, SS and SGA reviewed the critical analysis and helped in draft nal version preparation. AK nalized the draft and supervised the overall study. All authors have read and agreed to the published version of the manuscript.

Figure 1
The stepwise work ow adopted for novel anti-malarial drug targets identi cation.