In silico characterization of human-malarial parasite species based on their DHFR and GST targets leading to a change in binding conformations of anti-malarial drugs

In this study, we mainly focused on three anti-malarial drugs which were analyzed against the two malarial targets. Chloroquine, Me loquine and, Proguanilwas chosen as anti-malarial drugswhile DHFR andGST targets from human malarial parasites like Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax were considered for the study. This study was conducted to understand the sequence and structural similarity between protein DHFR and GST among four Plasmodium species as well as to ind out there in silico interactions with above-stated drug candidates. There were many bioinformatics databases, tools, and software’s were run to bring out research. Our data showed not many structural differences between Plasmodium sequences but yet other characteristics of them that make them different from each other. Hence that variation has shown a difference in the binding patterns of drugs with target proteins.


INTRODUCTION
Malaria worldwide is a substantial public health burden, and the countries where it is endemic exhibit economic crux (Ondeto et al., 2017;Tse et al., 2019). This results in the emergence of novel drug activity versus malaria pandemic (Escalante and Ayala, 1994;Cui et al., 2015). There are four Plasmodium species utterly responsible for invoking parasitic infection in humans viz P. falciparum, P. malariae, P. ovale, and P. vivax. Signi icantly, P.vivax being obligated for the extensive spread of the malarial infection worldwide. P. falciparum, however, is the most dangerous and causes the most fatalities. P. malariae and P.ovale are less widely distributed (Ramasamy, 2014). There are many contrasting characteristics among them yet these four human parasitic species are closely related Oaks et al., 1991;Coulson et al., 2004).
Diverse reviews were published in the past few years stated the eventual future of anti-malarial drugs. In 2014, combinations of the anti-malarial drug were scrutinized with three drug candidates. In 2017, numerous reviews about currently used anti-malarial drug discovery and designing and all the other aspects of malaria in-depth primer were also published. A review emphasizing numerous novel anti-malarial drug candidates in drug discovery and designing was also published in early 2018 (Tse et al., 2019). In this study, we are mainly focusing on the action of quinines -Chloroquine, Me loquine, and Proguanil on proteins Dihydrofolate reductase (DHFR) and Glutathione S-transferases (GST) of four human Plasmodium species. These quinines compounds are not used as front line treatment for malaria because of their side effects except for in severe cases of malarial treatment purpose where there is artemisinin inadequateness. However, quinines are still on WHO's Model List of Essential Medicines (MLEM). Chloroquine (Figure 1a) was directed to treat all forms of malaria and with few side effects amid 1940. In 1950, chloroquine resistance was irstly reported and over many years the other malarial strainshave also developed resistance. In the regions where resistance has developed against P. vivax, Chloroquine is administered as it's on MLEM. Me loquine (Figure 1b) was developed by the United States Army in the 1970s. Me loquine is also on MLEM and is still being used as a drug today. It was originally innovated to treat chloroquine-resistant malaria and it was given as both a prophylactic and curative drug in malaria. In 1986, me loquine resistance was irstly reported. It acts on the blood-stage of the parasite by disrupting hemoglobin digestion. Proguanil (Figure 1c) was reported as one of the irst antifolate anti-malarial drugs in 1945. In 1991, Atovaquone was irst reported for the treatment of malaria. Usually, a combination of these two drugs is used with the name of Malarone TM . In 2000, these two drugs are showing an effective synergistic effect in the treatment of malaria was proven. Proguanil when administered as a single drug exploit as DHFR inhibitor by disrupting the synthesis of deoxythymidylate (Tse et al., 2019).
DHFR is said to be a substantiated drug target for the treatment of parasitic disease-malaria (Anderson, 2005). Frequent drug resistance of Plasmodium species results in the evolution of novel drug candidates with a new mode of action. To maintain a high rate of replication, malarial parasites also require DHFR/folate and they are also capable of synthesizing or scavenging folate newly (Zhang and Rathod, 2013;Tse et al., 2019). In addition to drug resistance, GST is likewise involved in numerous diseases counting malaria (Westling et al., 1997;Sohail et al., 2010). In recent years, many researchers have distinguished the role of free radicals in malarial pathogenesis and other parasitic infections. P. vivax is known as an oxidative stress-sensitive strain of Plasmodium.
Hence GST an oxidative enzyme fascinated bene its in the diagnosis and monitoring ields of malarial complications. There are several diseases including malaria linked with GST de iciency in humans, thus there is a booming perception about the role of GST target (Sohail et al., 2007).

MATERIALS AND METHODS
In this study, we have investigated four human Plasmodium species listed in Table 1 (Antinori et al., 2012;Ortiz-Ruiz et al., 2018). The complete sequence of target protein DHFR and GST from the above four Plasmodium species sequences were retrieved from the UniProt database. For protein structural analysis, only P. falciparum structure with two genes A and B was retrieved from PDB whereas the rest three species protein structures were obtained from homology modeling (Yang et al., 2012) using Phyre 2.0 since their structures are not available in PDB. These Plasmodium sequences were subjected to MSA using Clustal O (1.2.4) program so that the utmost number of residues are matched up on the basis of a particular scoring function.
Phylogenetic relationship was derived by the neighbor-joining (N-J) method, which is a distancebased method that allows calculation of the evolutionary distance between sequences. In the N-J method, the branch length represents the amount of inferred evolutionary changes taken place (Gascuel and Steel, 2006;Pavlopoulos et al., 2010).
These antimalarial drugs ( Figure 1) were further docked using AutoDock vina and binding af inity was calculated in kcal/mol. The interactions of them were studies using Discovery studio visualizer.

RESULTS
MSA of DHFR for amino acid residue ranging from 60-637 ( Figure 2) and GST for amino acid residue ranging from 60-211 ( Figure 5) among P. falciparum, P. malariae, P. ovale and P. vivax was performed using Clustal Omega (1.2.4) that showed sequence similarity between them. The distance based method i.e. N-J method was also used to build a phylogenetic tree of tree data received from MSA. In MSA result amino acids resembling at various positions were denoted with symbols that give graphical interpretation at that respective residue. Amino acid residues with the "*" sign show identical residues, ":" sign shows highly similar whereas "." shows less similar residues at that particular site. Regions with no signs symbolize no similarity at that site. The N-J method was applied to create a unique parsimonious tree from the distance or guide tree data obtained from MSA. The DHFR protein from P. malariae and P. vivax was found to be the most closely related species among four of them. However, P. vivax was found to be the most distant species (Figure 3). The GST protein from P. ovale and P. malariae were found to be the most closely related species among four of them. However, P. fal ciparum was found to be the most distant species among them ( Figure 6).
The 3D structure of protein DHFR and GST from four Plasmodium species were superimposed using PyMol. Superimposed structure of DHFR protein ( Figure 4)  Molecular docking studies were executed using the AutoDock vina tool. Numerous drug interactions with proteins were observed using Discovery studio visualizer and binding af inity were also calculated in kcal/mol (Tables 3 and 4) which is as follows:-Docking analysis showed interactions between Chloroquine and DHFR protein target in four Plasmodium species ( Figure 8)  Docking analysis showed the interactions between Chloroquine and GST protein target in four Plasmodium species (Figure 11)  There was also one unfavourable donor-donor interaction was seen with Tyr 30 and no conventional hydrogen bond was observed in this docking analysis.
Docking analysis showed the interactions between Me loquine and GST protein target in four Plasmodium species (Figure 12)      Docking analysis showed the interactions between Proguanil and GST protein target in four Plasmodium. species (Figure 13)  Multiple sequence alignment was done to study how closely related the malarial species are ancestral. The DHFR protein sequence of all the four Plasmodium species, when aligned with MSA, showed incorporation of gaps and less similarity between sequences whereas in the case of GST proteins highest sequence similarity with many * identical residues was seen. The phylogenetic tree which is drawn based on the of N-J method showed P. malariae -P. vivax and P. malariae -P. ovale is the most closely related species whereas P.ovale and P. falciparum is found to be most distantly related or divergent species in the case of DHFR and GST respectively. Not much structural divergence was seen in 3D structures on superimposing using PyMol.

DISCUSSION
Previous studies involving the analysis of the gene encoding P. falciparum DHFR from resistant parasites suggest that antifolate resistance arises from point mutations in the DHFR domain, mainly at posi-  16, 51, 59, 108, and 164 (Chusacultanachai et al., 2002;Yuthavong, 2002). It has also been demonstrated that parasites with mutations at 16 and 108 develop resistance to cycloguanil, with a thousand-fold drop in the Ki compared with the wild type, whereas the Ki of pyrimethamine is almost unaffected (Kasam et al., 2009). On the contrary, there is cross-resistance between the drugs when multiple mutations at position 51, 59, 108 and 164 occur (Kasam et al., 2009;Rastelli et al., 2003Rastelli et al., , 2000. Combined homology modeling and molecular dynamics simulations studies indicate that how pyrimethamine, cycloguanil and WR99210 (a third-generation antifolate) bind to wild type and resistant mutant P. falciparum and P. vivax DHFRs (Rastelli et al., 2003(Rastelli et al., , 2000. WISDOM-I, the irst large scale deployment of molecular docking application, which took place from August 2005 to September 2005, has seen 42 million dock-ings (Kasam et al., 2009).
Virtual screening of 500,000 chemical compounds was performed by using FlexX against different plasmepsins (aspartic protease implicated in haemoglobin degradation). Experimental results have proved that the some of the compounds selected from WISDOM-I function as sub-micromolar inhibitors against plasmepsin. The main goals of WISDOM project were to identify inhibitors to be tested in the experimental laboratories (Kasam et al., 2009). Drug targets from malaria were chosen initially, however, this could be expanded to determine ligand binding to any target protein. The crystal structure of DHFR enzyme from P. vivax was published by Kongsaeree et al. (2005), where they indicated that the principal difference between DHFR wild type and mutant, implicated in the antifolate resistance, is a structural change in the chain of Asn-108, and this steric con lict is not present in P. falciparum. Glutathione S-transferase of the malarial parasite P. falciparum (PfGST) represents a novel class of GST isoenzymes, Since the architecture of the PfGST substrate binding site differs signi icantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development (Hiller et al., 2006). P. falciparum parasites are increasingly drugresistant, requiring the search for novel antimalarials with distinct modes of action. Enzymes in the glutathione pathway, including glutathione S-transferase (GST), show promise as novel antimalarial targets. By using reverse genetics, Colon-Lorenzo et al. provided evidence that GST is essential for survival of P. berghei intra-erythrocytic stages and is a valid target for drug development (Colón-Lorenzo et al., 2020). Researchers have also studied Glutathione-S-transferases (GSTs) from chloroquine-resistant (CQR, K1) and -sensitive (CQS, T9/94) strains of P. falciparum (Harwaldt et al., 2002;Al-Qattan et al., 2016). The enzymes from both strains exhibited the optimal pH for enzyme catalysis, at pH 7.5, and were stable at temperatures below 60 degrees C. They therefore proposed that GSTs from both malarial strains are identical in their functional domain but different in level of gene expression (Harwaldt et al., 2002;Al-Qattan et al., 2016). Yadav MK et al. analysed the possibility of using variable surface proteins as a common drug target in both the Plasmodium species (Yadav and Swati, 2016). Sequence analysis of variable surface proteins showed a low-level conservation within as well as between the species. Amino acid composition analysis revealed higher frequency of hydrophilic amino acids as compared with that of hydrophobic residues. Structural alignment of variable surface proteins by superimposing them showed less conservation. The noted existence of structural differences showed that the variable surface proteins could not be used as a common drug target in both the malarial species (Yadav and Swati, 2016). The authors concluded that species-speci ic strategy may be followed for drug targeting against variable surface proteins of P. falciparum and P. vivax. Forlemu N et al. studies the effects of DHFR-TS mutations on interactions with antimalarial agents and sulfonamide ligands using AutoDock 4.2 molecular docking (Saitou and Nei, 1987;Mukinay and Forlemu, 2017). Three sulfonamides (SulfaC, SulfaH, and sulfaE) showed better or comparable interaction af inity (-7.0 to -9.5 kcal/mol) with PfDHFR-TS isoforms to antifolate drugs (artemisinin, primaquine, pyrimethamine). The active site was the preferred binding mode in all isoforms studied, with conserved polar residues (D54, S108, R122, Y170) stabilizing complexes formed. The af inities for all isoforms studied had similar magnitudes with each ligand. The indings indicated that mutations do not signi icantly impact binding.
Another study was conducted using molecular docking on 28 compounds belonging to 2,4diaminoquinazoline and 2,4-diaminopteridine analogs (Bharatam and Adane, 2010). The authors used Glide, FlexX and GOLD programs and the X-ray crystallographic structures of the quadruple mutant (1J3K:pdb) and wild type (1J3I:pdb) P. falciparum dihydrofolate reductase enzyme. The authors found that the bound ligand WR99210 was precisely reproduced by the docking procedures as demonstrated by low (<2.00 Å) root-meansquare deviations (Bharatam and Adane, 2010). The results indicated that most of the compounds dock into the active sites of both the wild type and quadruple mutant P. falciparum dihydrofolate reductase enzymes.

CONCLUSION
There is a need to obtain new antimalarial drugs as malaria is one of the most important World health problems. This can be possible only by achieving a deep insight knowledge of Plasmodium species mechanism. To gain comprehension of genomics and proteomics aspects of Plasmodium is very crucial. Bioinformatics tools were used in our study to gain knowledge of evolutionary variations held on DHFR and GST proteins of different Plasmodium species. Me loquine exhibited the highest binding af inity (kcal/mol) with DHFR in all four Plasmodium species whereas with respect to GST it exhibited the highest binding af inity in three Plasmodium species except for P. malariae. Docking analysis of receptor-ligand complex demonstrated that both me loquine and chloroquine are capable of binding and inhibiting the DHFR and GST proteins of Plasmodium species.
Hence, further modi ication in Me loquine and chloroquine may make them unique and potent inhibitor against DHFR/ GST among all four Plasmodium species which will further help in controlling the worldwide spread of malaria.

Funding Support
The authors declare that they have no funding support for this study.

Con lict of Interest
The authors declare that there is no con lict of inter-est for this study.