Implication of serine-decarboxylase-phosphoethanolamine-methyltransferase Pathway and phosphoethanolamine-methyltransferase in Malaria abolition

Introduction

Malaria is a mosquito borne disease in humans caused by unicellular microorganism called parasitic Plasmodium protozoa [1] . According to the fact sheet 2015, cases of malaria (88%), were estimated African Region, 10% in South-East Asia Region and the 2% in Eastern Mediterranean Region. 438 000 peoples were killed by malaria in 2021 among these were 306 000 children and the malaria was found the fourth highest cause of death [1,2] . Antimalarial drug resistance is greatest challenge for malaria control today responsible for the malaria in new areas and re-emergence and the war against resistance problem led WHO to establish strategies against drug resistance [3, 4,]. The plasmodium spps (P. falci and vivax) has been reported resistant against antimalarial drugs where P. falciparum has been reported resistant against nearly all the available antimalarial drugs [5,6] . WHO recommends artemisinin and its derivatives as first choice of drug in combination of other antimalarial, Artemisinin combination therapy (ACT) [7] . The artemisinin drug resistance was reported in Peru 2008 against plasmodium falciparum [8] but still ACTs is first choice of treatment for uncomplicated malaria [9] . Chloroquine which prevents hemozoin formation in food vacuoles within the parasite has been reported to be expelled out at too large amount to reach level of its action [10,11] . Pfcrt which is a drug exporter gene has found to be responsible for chloroquine resistance [12,13] . Sulfadoxine and pyrimethamine antifolate combination drugs inhibit folate synthesis by inhibiting dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) respectively have shown resistance due to mutations DHFR and DHPS and their alleles were present in resistant malaria parasite [14,15] . Since, plasmodium has shown resistance against most commonly used antimalarial. Hence, there is need of novel and unique pathway to find new potent antimalarial drug. It is very clear that in all unicellular and multicellular membrane systems, phospholipid bilayers are very important for formation and maintenance of the structure and functionality of the system. Among the important phospholipids phosphatidylcholine (PC) is the most abundant phospholipid found in membranes of bacteria, plants, eukaryotes and even in plasmodium. The aim of review is to focus on importance of SDPM pathway and phosphoethanolamine methyltransferase enzyme for the synthesis of PC for development of membrane throughout the plasmodium life cycle within the host for survival and transmission. The phosphoethanolamine methyltransferase absent in human, catalyzes the trimethylation of phosphoethanolamine a rate limiting step makes it a novel potential drug target to overcome the problem of drug resistance [16, 17] .

Significance of PC for Growth and Development

PC is most frequently needed and also an essential eukryotic membrane phospholipid for survival of many organisms such as Leishamnia major, bacteria and eukaryotes [18,19] . PC and phosphatidylethanolamine synthesis occurs mainly by three routes, de novo (CDP-choline) and CDP-ethanolamine (kennedy pathway) and CDP-diacylglycrol pathway where CDP-diacylglycerol route is primary route for PC synthesis in yeast and mammalian hepatocytes [20,21,22] . In de novo CDPcholine route the choline is the precursor for the synthesis of PC via CDP-choline route. In the CDP-ethanolamine (kennedy pathway), PC and PE both get synthesized from choline and ethanolamine respectively though both CDP-choline and CDPethanolamine routes.

In CDP-ethanolamine route phosphatidylethanolamine is formed gets trimethylated by phosphatidylethanolamine methytransferase to synthesize the phosphatidylcholine [23] . In bacteria, PC a typical eukaryotic membrane phospholipid is present in only about 10% of all bacterial species in particular in pathogenic bacteria, revealed that PC plays a fundamental role in symbiotic and pathogenic microbe and host interactions [24, 25] . Bacteria, synthesizes PC either through methylation pathway or the phosphatidylcholine synthase pathway or through both the pathways [26,27] . The methylation pathway involves a three-step methylation of phosphatidylethanolamine by phospholipid N-methyltransferase. These biosynthetic pathways have significance on plant-microbe interactions [28,29] . In Plants, Phosphatidyl-N-methylethanolamine Nmethyltransferase (PLMT) enzymes use phosphatidyl mono methyl ethanolamine (PtdMME) and phosphatidyl dimethyl ethanolamine (PtdDME) and are homologous of the rat PLMT [30]. Presence of two different N-methyltransferases was confirmed in spinach for catalyzing methyl transfer to pethanolamine to form p-choline [31,32] . The inhibition of enzymes diminished the growth of Haemonchus contortus and the gene silencing of HcPMT1 and HcPMT2 implied the essentiality of HcPMT1 and HcPMT2 for the survival of H. contortus [33,34] . Eukaryotes synthesize PC through two major biosynthetic pathways, phospholipid N-methylation pathway and through CDP-choline pathway and their relative's requirement diverges from organism to organism and even from tissue to tissue within the same organism [35] . The enzymes involved in the CDP-choline biosynthesis in euckryotic and P. falciparum are different , Choline, an important nutrient primarily provided from diet is regulated by both the CDP-choline biosynthetic pathway and choline metabolism [35,36] . Choline is phosphorylated into phosphocholine by choine kinase (CK) which finally gets coverted into PC via CDP-choline [37] .

Phosphatidylcholine

formed is converted into phosphatidylserine by enzyme phosphatidylserine synthase1 which further gets converted into phosphatidylethanolamine by phosphatidylserine decarboxylase enzyme but the enzyme phosphatidylserine decarboxylase is absent in humans and plants [38,39]. Plasmodium parasite also requires PC for rapid membrane development important for the survival and multiplication of the parasite within the host [40] . So, the serine-decarboxylasephosphoethanolamine-methyltransferase (SDPM) unique pathway absent in humans becomes the alternative pathway in Plasmodium falciparum for PC biosynthesis. In the SDPM pathway P. falciparum synthesizes phosphatidylcholine using host serine [41,42] .

Sdpm Pathway (Kennedy Pathway)

Normally, plasmodium parasite synthesizes PC from choline through CDP-choline route but when the parasite infects host and need to develop membrane at faster rate for growth and development. Availability of PC phospholipid abundantly is must for faster membrane development. Parasite use alternative pathway SDPM to meet. This pathway is unique in the plasmodium falciparum which makes it as essential target. In SDPM pathway serine from host blood is utilized for PC synthesis. The P. falciparum biosynthesizes PC from serine via CDP-Choline route as demonstrated in Figure 1.The serine of host and get converted into ethanolamine with the help of serine decarboxylase (SD) enzyme which is then converted into phosphoethanolamine using ethanolamine kinase (EK). The role of phosphoethanolamine methyltransferase (PfPMT) absent in human host becomes essential since, itcatalyzes the slowest step of the pathway, i.e., the conversion of phosphoethanolamine into phosphocholine through triple methylation of phosphoethanolamine. Once the phosphocholine is formed it is used as substrate for CDP-choline formation. The (CPT) enzyme completes the last step of the pathway by converting the CDP-choline in the phosphatidycholine [43] . The key enzyme of the SDPM pathway found is PfPMT for triple methylation. When the PfPMT was inhibited or knocked out, the fatal growth of P. falciparum was observed and also greatly affected the gametocytes formation [44] .

Significance of PFPMT in SDPM Pathway PC Synthesis

For the rapid multiplication of the parasite within human erythrocytes it requires an active production of new membranes at very fast rate [44] . PC an important component of membrane is synthesized abundantly from serine decarboxylation pathway during not only intraerythrocytic cycle (with maximum expression at trophozoite stage) but also during gametocyte development cycles of Plasmodium falciparum. Hence, the gene is crucial for intraerythrocytic growth as well as malaria transmission. So, the work done on the PfPMT gene has demonstrated the essential role of this gene in the survival and transmission of P. falciparum parasite [44] . The knock out study also revealed that when the PfPMT was disrupted the PC biosynthesis from serine was completely abrogated and caused serious defects in survival and multiplication of plasmodium falciparum. Loss of PfPMT severely distorted the asexual replication and gametocyte development which revealed that PfPMT plays crucial role in intraerythrocytic asexual replication and gametocyte development. Gametocytes were not produced even when the choline was present but when PfPMT was reintroduced in knockout, gametocyte differentiation was again restored to wild type level. The expression of PfPMT throughout the intraerythrocytic cycle as well as gametocyte development is very important for survival and transmission of parasite that makes it an excellent target for antimalarial drug designing and moreover absence of phosphoethanolamine methyltransferse in human makes this enzyme an excellent drug target for development of new antimalarial [45,46,47] . Phosphatidylserine decarboxyase enzyme is absent in both the P. falciparum and plants. PfPMT and plant methyltransferase (PEAMT) are homologues but plants PEAMT have two catalytic domains [43,48] . PfPMT has one catalytic domain for three methylation steps this implies that same catalytic domain is used for all three methylations. Hence, the inhibition of catalytic domain of PfPMT can lead to complete inhibition of SDPM pathway. Therefore, PfPMT is an essential enzyme for survival of Plasmodium falciparum moreover absence of phosphoethanolamine methyltransferase in human makes this enzyme an excellent drug target for development of new antimalarial. Since, PfPMT is the only enzyme in SDPM pathway which catalyzes the triple methylation of phosphoethanolamine. Hence it represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Enzyme Kinetics of PFPMT

An SAM-dependent triple Methylation coupling assay system for PfPMT activity using of two enzymes SAH nucleosidase (SAHN) and adenine deaminase (BsAda) was developed. As SAH and phosphocholine are products of PfPMT mediated activity using phosphoethanolamine and SAM as substrate and methyl donor respectively. So, enzyme SAHN added is responsible for the conversion of SAH into adenine and Sribosylhomocysteine. Where, BsAda converts adenine into hypoxanthine showed absorption at 265 nm decreases [49] . Activity assay was done spectrophotometrically by coupling the formation of S-adenosyl-L-homocysteine to the reaction catalyzed by PvPMTand S-adenosyl L-homomethionine in the presence of phosphoethanolamine. Assay was performed in 96well UV-transparent plates (acrylic, non-sterile, costar) at 37 0 C. Reaction mixtures were prepared by adding 1 nM MnSO 4 , 1X HEPES assay buffer (100mM HEPES-KOH, pH 7.5), 200 µM SAM, 200 µM phosphoethanolamine, 0.5 µM BsAda, 4.72 µM SAHN. And mixture was first incubated at 37°C for 10 min and then reaction was started by addition of 2.5 µM PfPMT in a total volume of 200 µl. Absorbance at 265 nm was continuously recorded. Activity was determined from the slope of the linear part of the curve obtained by plotting decrements in absorbance at 265 nm with time (dA/dt) [49] . When concentrations of the P-EA and SAM were increased PfPMT activity was increased proportionally and activity was optimal at 200 μM. Concentration more than 200 μM, activity of PfPMT was slightly affected by increasing conc. of P-EA whereas activity was inhibited on increasing conc. more than 200 μM. There a proportional increase in activity was noticed on increasing the concentration of PfPMT and decrease in absorbance over time was also reproduced. Above a particular range, the activity of PfPMT enzyme was noticed to deviate from Linearity. The little effect of different buffers at pH 7, 7.5 and 8 on PfPMT activity was found over time [49] .

Structural Aspects and Inhibitor Study of PFPMT

According to the structural comparison and binding mode, no variation found in position of amino acids interacts with AdoMet, AdoCys and AdoMet analog sinefugin within the PfPMT-AdoMet binding site as shown in Figure 2. Critical interaction of AdoMet with Asp-85 and Ile-86 residues within the AdoMet site since site directed mutation of either residue (D85N/D85A or I86F/I86A) greatly affected the binding of AdoMet and abrogated activity [50] . binding, this residue found to get shift away from the active site [50] .

Fig 3:

Binding mode of (A) SAM (Blue color) and (B) sinefugin (Blue color) with their interacting amino acids within the same binding pocket of crystal structures PDB: 3UJ6 and 3UJ8 respectively. Hydrogen bonds are in green dashed lines [50] According to the Mutant studies of residues in the phosphobase site it was found that the Arg-179 and Lys-247 are required for activity as the alanine and methionine mutants of Lys247 were found inactive. But Alanine and lysine mutants of arginine179 showed activity between 1and 43%, respectively. It was also confirmed that each tyrosine (Tyr-27, Tyr-160, Tyr-175, and Tyr-181) residues within the binding site were also crucial for PfPMT activity. As Methylation of pEA was greatly disrupted when either hydroxyl or phenol moieties were removed [50] shown in Figure 4. vivax and P. knowlesi. Hence Phosphoethanolamine methyltransferase enzyme may be the important drug target as inhibitor of this enzyme can inhibit PMTs of P. vivax and knowlsi [51] . Few countable compounds such as sinefugin (AdoMet analog), miltefosine (Choline analog) amodiaquine (4-aminoquinoline) as given in Table .1 had been identified to inhibit the PMTs of Plasmodium and nematode [49,52] . Crystal structure of phosphoethanolamine methyltransferase has also been solved in plasmodium vivax (Pv PMT) and knowlsi (Pk PMT). The general structural folds of PvPMT as well as PkPMT were found very similar to those folds of already solved crystal structure of Pf PMT with all atom RMSD 0.452 A˚.

Structural comparison Pv PMT and PkPMT with Pf PMT showed that the secondary structural elements where are almost similar except some local changes. The general structure of PkPMT is almost identical to that of Pv PMT and PfPMT with an all-atom alignment RMSD of 0.80 A. The active site residues are well conserved amongst enzymes from P. falciparum, P. vivax and P. knowlesi. Hence Phosphoethanolamine methyltransferase enzyme may be the important drug target as inhibitor of this enzyme can inhibit PMTs of P. vivax and knowlsi. Few countable compounds such as sinefugin (AdoMet analog), miltefosine (Choline analog) amodiaquine (4aminoquinoline) as given in Table . 1 had been identified to inhibit the PMTs of Plasmodium and nematode [51,52] . Miltefosine and its analogs had been validated for antitumor and antileishmanial activities. Miltefosine have been previously identified as PfPMT inhibitor in vitro with a radioactivity-based assay. Miltefosine inhibited PfPMT by 60% at 100 uM but at 150 uM the activity of PfPMT was completely lost . Hexadecyltrimethylammonium bromide (HDTA) was also found as an inhibitor of PfPMT. Inhibitory (app. 50% at 100 uM) activity of was found similar to mitefosine and HDTA inhibited the plasmodium falciparum parasite in culture with an IC50 of 2.1 μM [53]. Dodecyltrimethylammonium (DDTA) a potent antimalarial activity had shown inhibitory activity for PfPMT [54,55] . Besides the antimalarial activity, amodiaquine is a potent inhibitor of the human histamine N-methyltransferase (HNMT) [56] . But no other potent HNMT inhibitors such as SKF91488, tacrine diphenhydramine and chlorpromazine [57,58] were found not to inhibit PfPMT. It was observed that aminoquinolines and amino alcohols such as quinacrine, quinidine and quinine had no PfPMT inhibitory activity at concentrations up to 200 μM. Chloroquine and primaquine were also found to inhibit PfPMT in millimolar. The two compounds ZINC12882412 and ZINC02103914 showed the good schozonticidal activity at µM concentration 3.0 µM and 2.1 µM respectively, that might be reason for the better docking affinity towards PfPMT occupying the crucial catalytic dyad between Tyr19 and His132 to prevent its biological function. These compounds may be used structure-based drug development against multi drug resistance [59] . The three compounds from Asinex database were also identified through computer aided drug designing approaches as potent inhibitors for the PfPMT with better ADMET and the docking affinity. The identified inhibitors showed the better activity against schizonts and gametocytes of Plasmodium falciparum. Two inhibitors ASN.1 and ASN.3 inhibited the PfPMT competitively at IC 50 1.49µM and 2.31µM respectively along with the promising reduction of parasite growth in mice (orally and intravenous). These compounds may be used for structurebased drug developmentagainst multi drug resistance [60] Table . 3.

Future Prospective

The review will emphasize the current scenario of urges the elucidation of novel pathways either absent in human or nonhomologous of human. The unique and alternate SDPM pathway alike plants in plasmodium essential for the progression and rapid membrane generation; late stages and gametocyte development in intra erythrocytic forms of malaria offers an attractive molecular drug target for the development of novel inhibitor-based antimalarial therapies. The rate limiting step, conversion of phosphoethanolamine into phosphocholine by plasmodium phospho-ethanolamine methyl transferase (PMT) plays an important role in membrane development for growth and transmission of parasite met the urge of novel drug target for antimalarial development. Since, SDPM and PMT is absent or different from methyltransferases found in humans, a better understanding of molecular and Structural analysis of PMT enzyme in plasmodium may lead to the development of a novel class of antimalarial inhibitor compounds to be used as drugs using rational drug designing. Inhibitor library of compounds designed and identified by in silico structural modeling and in-vitro studies to inhibit target PMT enzyme may provide novel broad-spectrum antimalarials. The PMTs of plasmodium have conserved domain and shares significant structural identity which suggest that the common PMT inhibitor as antimalarial may be development. In this review the importance of the SDPM pathway in the development and transmission of parasite has been explored along with the inhibition portfolio of PfPMT inhibitors till date will not only advance our knowledge about SDPM and PMT among plasmodium but may also provide the structural lead for the computer aided drug designing.

Acknowledgement

AcknowledgementWe would like to acknowledgement National Institute of Malaria Research, Dwarka-8, New Delhi for providing working facilities. We also thank laboratories colleaguefrom MDU, Rohtak and NIMR, New Delhi for their support throughout the completion of this review.

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