Antimalarial antifolates have been the central drugs for prophylaxis and treatment of malaria. Plasmodium falciparum readily develops resistance to the antifolates pyrimethamine and proguanil through a particular set of mutations in the dihydrofolate reductase (DHFR) gene that results in the less competitive drug binding at the site of the enzyme. Similar mutations can be found in the DHFR gene in Plasmodium vivax (DHFR) also. The aim of this research work is to reveal the interactions of the DHFR inhibitors with the target proteins and to investigate the effect of mutations computationally. Commonly used antifolate drugs against malaria such as trimethoprim, pyrimethamine, proguanil and WR99210 have been considered for the analysis. Proteins which are targeted by the antifolate drugs in malarial organisms have been identified and characterized. Interaction studies of the wild and mutated DHFR proteins with the known drugs have been made. It has been found that the ligand binding to mutated proteins is considerably low compared to that of the wild proteins in most of the cases except WR99210 drug. This discloses the inadequacy of the antifolate drugs and the need to improve the antimalarial antifolate in vivo effectiveness and to recognize powerful novel antifolate agents. From the interaction studies of antifolate drugs against the target DHFR in malarial parasites Plasmodium vivax and Plasmodium falciparum it has been identified that these parasites show resistance to the chemotherapeutic antifolate drugs. A combination of antifolate drugs can be effective against malarial parasites or a designing of a new antifolate drug.
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Keywordsââ‚¬" Antifolate drugs, Malaria, DHFR, Drug Resistance, Interactional Studies.
Antifolate drugs are category of drugs which impair the functions of folate. Antifolates are primarily DHFR inhibitors which are mainly used in the cancer treatment. Since antifolate action specifically targets rapidly dividing cells, antifolates are also used as antiprotozoal agents. Drug resistance in microorganisms is referred as the mechanism by which the organism acquires resistance against the effect of the drug. The resistance is offered by different mechanisms like altering target sites, pumping of the drug out of the cell or by some gene mutations. Chemotherapy is the most important way to control malaria which is one of the dangerous contagious parasitic diseases in the world. Folates are water-soluble form of vitamin B9 , which is necessary for the production and maintenance of a new cell and for nucleotide biosynthesis. Folate after conversion to dihydrofolate undergoes metabolism with the help of dihydrofolate reductase (DHFR) enzyme to give tetrahydrofolate (THF), which is a coenzyme in metabolism of amino acids and nucleotides . The recognition and advancement of antifolates as drugs evolved from the understanding of the task of folate derivatives in humans.
The development of antifolate drugs originated from tasks to treat leukemia. The inhibition of the presence of folic acid could result in anti-proliferative property, by developing a folate deficiency condition. This important fact led to the development of the first antifolate drug, aminopterin or 4-aminopteroyl-glutamic acid. The clinical examination of aminopterin produced temporary improvement in some of the patients with acute leukemia . This revelation was a milestone in cancer chemotherapy as it gave, for the first time, proof that an antimetabolite could be a useful antineoplastic drug. Currently, the interference of the folate metabolic pathway is one of the major targets for the treatment of tumors diseases in humans. [4, 5] The achievement of antifolates in the treatment of tumors also led to the utilization of this group of drugs in the treatment of other quickly dividing cells such as bacteria and parasites . Antifolate drugs mainly used against malaria are the inhibitors of DHFR .
Each year, there are approximately 350ââ‚¬"500 million cases of malaria. Resistance has complicated the treatment of malaria and threatened the control and elimination of the disease.
Drug resistance in microorganism is the phenomenon of neutralizing the negative effect imparted by the drug to the organism. Drug resistance is becoming an increasingly important factor in the effective treatment of malaria. Due to the antifolate action the amount of THF in the malarial parasites decreases considerably. In response to this more DHFR gets transcribed and the competitive inhibition is tackled. This is one approach of drug resistance towards antifolate drugs. Point mutations in the DHFR allele of the malarial parasites end up in producing mutated proteins which have less binding affinity towards the drug. This has been the major idea which is being described in this study.
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Resistance to the antifolate drugs is attributed by the point mutations in proteins have been identified in plasmodium falciparum and Plasmodium vivax [8, 9]. Interaction studies of the wild and these mutated DHFR proteins with the known drugs have been conducted to analyze the metabolic consequences of the mutation in the wild proteins. A possibility of combination therapy using multiple antifolate drugs was also analyzed.
The target enzymes of the antifolate drugs, wild and mutated DHFR from both Plasmodium falciparum and Plasmodium vivax were identified and collected from Protein Data Bank (PDB) . The protein molecules taken were subjected to detailed sequence analysis, structural analysis and computational modeling for characterization. Multiple sequence alignment of the proteins from both the organisms was performed using CLUSTALW tool . The functional domains and families of the proteins were determined using Prosite tool . The parameters like number of amino acids, atomic composition, extinction coefficient, instability index, aliphatic index, grand average hydropathy (GRAVY), molecular weight and estimated half life were identified from ProtParam tool .
The secondary structural details were found out using Self-Optimized Prediction Method (SOPMA)  and using the Computed Atlas of Surface Topography of protein (CASTp) Tool  the binding pockets and the surface characteristics of the proteins were analyzed.
All the modeling and simulations were performed using Accelrys Discovery studio [16, 17] with ââ‚¬Ëœsmart minimiserââ‚¬â„¢ algorithm and CHARMm force field. The input for the modeling was the PDB file collected from the repository Protein Data Bank. The collected protein structures were subjected to geometry optimization and the corresponding minimum interactional potential energy, RMS gradient, van der Waals energy, electrostatic energy and kinetic energy values were computed. Most of the structures attained convergence at about 2000 steps of iteration.
The antifolate drugs pyrimethamine, trimethoprim, Proguanil, and WR99210 were taken from Drug Bank . The drug molecules were modeled and optimized using Gauss View and Gaussian 03W tools . Electrostatic energy of the proteins was computed using the APBS module  in MGL (Molecular Graphics Laboratories) python molecule viewer (pmv) .
To analyze the interaction between the proteins and the drugs docking analysis were carried out using the CDOCKER tool of discovery studio . CDOCKER is a grid-based molecular docking method that employs CHARMm forcefield. The receptor is held rigid while the ligands are allowed to flex during the refinement. It allows running a refinement docking of any number of ligands with a single protein receptor. Each protein has numerous active sites and these are docked with the ligand. For each final pose, the CDOCKER energy (interaction energy plus ligand strain) and the interaction energy are noted. The poses are sorted by CHARMm energy and the top scoring one which is found to be exothermic (thus favorable for binding) is retained.
Results And Discussions
The results obtained from the multiple sequence alignment amongst the wild and mutant showed the presence of point mutations which concrete the idea described in the introduction. The common functional sites, protein domains and families of these proteins have been identified using Prosite tool. Both the wild and mutant varieties of proteins have been subjected to sequence and structural analysis using Expasy tool ProtParam. The instability index values of the proteins have been calculated and it has been found that the mutant proteins were found to be highly stable compared to the wild proteins. The aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains. It may be regarded as a positive factor for the increase of thermostability of globular proteins. Hydropathicity of these proteins were also calculated. From the results, it has been observed that all the proteins are hydrophilic in nature.
From the secondary structure analysis of the proteins from both Plasmodium falciparum and Plasmodium vivax, the percentages of random coils were more compared to other secondary structures in mutated proteins. The conformational entropy associated with the random coil state significantly contributes to its energetic stabilization and accounts for much of the energy barrier to protein folding. CASTp analysis results have shown that the number of binding pockets was more in the wild protein of Plasmodium falciparum and it decrease for mutated proteins implying the stability. Similar observation was also seen in Plasmodium vivax.
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From the modeling of the protein structures it was found that the interactional potential energy for most of the proteins was highly negative implying high thermodynamic stability. The Van der Waals energy was also found to be negative for most of the proteins, which indicates high possibility of secondary bonding. The RMS gradient value is almost close to 0, indicating the stability of the protein structures.
Electrostatic analysis has been performed for the protein molecules using MGL pmv APBS module. Electrostatic interactions are important for both protein stability and function. For this reason, they play a critical part in protein-protein interactions, ligand binding, and catalysis. As protein modeling and design is important in these areas, an electrostatic energy analysis is of great value. In particular, effects due to the aqueous environment such as polar solvation and solvent screening need to be evaluated as well as protein-protein interactions such as hydrogen bonding.
Electrostatic energy decreased as the mutation increases for both the Plasmodium falciparum DHFR (PmDHFR) and Plasmodium vivax (PvDHFR) proteins. This will readily affect the ligand binding towards the proteins and it can be assumed that this mainly constitutes for the antifolate drug resistance in malarial organisms
Interactional analysis between the antifolate drugs and the wild as well as different levels of mutated DHFR proteins has been carried out. While analyzing the results, interaction energy was found to be very low for the interactions between drugs and mutated proteins when compared to interaction with wild proteins (Table 1) of Plasmodium falciparum. WR99210 has been found to be highly effective against wild as well as mutated DHFR proteins in Plasmodium falciparum strain. The interaction energy in mutated proteins was found to be slightly less than wild variety.
Interaction Analysis Results for Plasmodium falciparum Proteins
Interactional Energy (kcal/mol)
WR99210 has been found to be a novel inhibitor of DHFR, effective even against the most pyrimethamine-resistant Plasmodium falciparum strains also which has been verified by the computational analysis performed.
Similar results were obtained for the proteins from Plasmodium vivax also (Table 2). DHFR inhibitors have not been considered for cure of vivax malaria, as the initial lab experiments showed comparatively low efficiency against Plasmodium vivax. From the interactional analysis carried the drug WR99210 has been found to be an effective drug for treating Plasmodium vivax malaria. Double and quadruple mutations in proteins were identified in Plasmodium vivax. It has been found out that the quadruple mutated proteins have interaction energy less compared to double mutated proteins for the drugs Pyrimethamine, Proguanil and Trimethoprim. But the drug WR99210 has shown a good interaction with mutated proteins indicating the efficacy of the drug.
Interaction Analysis Results for Plasmodium vivax Proteins
Interactional Energy (kcal/mol)
Certain new drugs also have shown high interaction towards all the wild proteins compared to the common antimalarial drugs. But from the interaction analysis it has been found out that the mutation in these proteins has resulted in the resistance by the organisms for these drugs also.
Antifolate drug treatment is the primary mean to treat the malarial infection. In this research work antifolate drugs and target DHFR wild and mutated proteins have been collected and characterized. The interaction studies of antifolate drugs against the target DHFR in malarial parasites, Plasmodium vivax and Plasmodium falciparum shown that, they were showing resistance to the chemotherapeutic antifolate drugs due to the point mutations it acquired in the protein level. The interactions between drugs and proteins are found to be very less for mutated proteins compared to the non-muted proteins or the wild proteins. The drug WR99210 has been found to be an efficient drug against the resistant strains of Plasmodium. But a chance of developing resistant strains for this drug is also very high. The interaction analysis carried out indicates the above fact. A combination of antifolate drugs can be effective against malarial parasites. From the above results Improvement of antifolate drugs is absolute necessary for the cure of malaria, since it is the best way to cure it.