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Identification of Novel Dihydrofolate Reductase Inhibitor as Potential Antimalarial Drug: In silico Studies.

Byline: Zeeshan Iqbal Mubashir Hassan Jawaria Munir Irfana Mariam Farah Rauf Shakoori Abdul Rauf Shakoori and Nasir-ud-Din


Advancement in computational biology leads to improve the efficacy for new compounds to cure the diseases. Malaria is the most virulent diseases and causing millions of deaths annually especially in developing and under-developed countries. Plasmodium falciparum dihydrofolate reductase (PfDHFR) is one of the most important drug target for different antifolates. Pyrimethamine with sulphadoxine complex is the most recommended and efficient antifolate prescribed against PfDHFR. But malarial parasites have developed resistance against this drug due to the point mutations in PfDHFR. This study focus to design a novel antimalarial drug (analog) against mutated PfDHFR by considering the in silico approaches. The new antimalarial drugs were designed by the addition/substitution of different functional groups and molecules in parent compound of pyrimethamine.

The docking studies of newly designed compound and pyrimethamine with mutated receptor protein of PfDHFR were performed by using different docking servers. Various in silico therapeutic calculations for novel antimalarial compound and pyrimethamine were executed using computational approaches. The basic of ligand properties docking results energy calculations and drug score favor indicated that the new antimalarial drug compound have potential to show better efficacy than pyrimethamine. This designed analog could be used for preclinical test and have the potential to eradicate P. falciparum.

Key words: In silico drug designing malaria PfDHFR molecular docking pyrimethamine


Malaria is a lethal disease causing one million deaths annually in the world (Muerhoff et al. 2010). Plasmodium falciparum is the most lethal amongst all Plasmodium species. P. malariae and P. vivax are the most virulent in Pakistan but P. falciparum has more lethality in rest of the world (Omonuwa and Omonuva 2002; Gupta et al. 2009). The life cycle of Plasmodium species involve two alternating hosts such as insect vector and vertebrate host (Florens et al. 2002; Zakeri et al. 2010). The viability of malarial parasite depends upon folate metabolism. This pathway is important for purine and pyrimidine production in DNA replication (Gregson and Plowe 2005). In folate pathway mechanism deoxythymidine monophosphate (dTMP) (dTMP) is produced from deoxyuridine monophosphate (dUMP) by oxidation of tetrahydrofolate to dihydrofolate catalysed by dihydrofolate reductase (DHFR) (Hyde 2005; Abali et al. 2008).

PfDHFR-TS is a homodimeric protein comprising DHFR domain junction region (JR) and TS domain. The JR associates the DHFR and TS domains in most of the parasitic protozoa (Yuvaniyama et al. 2003). In P. falciparum JR acts as a bridge and assembles DHFR domains by making a strong interaction between these domains. This JR interaction is also significant in the conformation of DHFR domain which helps in the development of novel effective anti-malarial drugs (Chaianantakul et al. 2013). Antifolates prevent malaria by inhibiting the activity of dihydropteroate synthase (DHPS) and DHFR enzymes.

Mutations in PfDHFR (3D7) at different residues like Ala16 Asn51/Cys59 Ser108 and Ile164 causes resistance against antifolates (Lynch et al. 2008). In P. falciparum clone Ser108Asn is the most effective mutation at DHFR domain (Cowman et al. 1988; Sirawaraporn et al. 1997; Shallom et al. 1999; Basco 2003; Kamchonwongpaisan et al. 2004). These mutations cause resistance to antifolates like pyrimethamine in P. falciparum. It has been observed that the increased number of mutations in DHFR protein inhibit the binding of pyrimethamine with DHFR (Sirawaraporn et al. 2002).

Several antimalarial drugs have been successfully used against PfDHFR (Plowe et al. 1997; Nzila 2006). The DHFR is an effective target for various antimalarial drugs (Peterson et al. 1990; Yuthavong et al. 2005) such as proguanil (FArnert et al. 2002) cycloguanil (Khan et al. 1997) and chloroguanil/chlorproguanil (Fidock and Wellems 1997; Fidock et al. 1998; Maitarad et al. 2009).

Chloroguanil and cycloguanil both were derived from proguanil. The most important antifolate is pyrimethamine which belongs to the 2 4- diaminopyrimidine families. The pyrimethamine (5- (4-chlorophenyl)-6-ethyl- 24-pyrimidinediamine) is a well reported antifolate drug which has been utilized against malaria to target DHFR protein. The designing of novel antimalarial drugs against mutated PfDHFR are required to cure the malaria. In silico drug designing approaches have played significant role in developing novel compounds against resistant proteins (Sehgal et al. 2013). In current study novel antimalarial compounds were designed against mutated PfDHFR. The pyrimethamine was considered as a parent compound and various modifications by addition/deletion and/or substitution of different functional groups. The designed analogs were characterized analyzed by computational models and finally docked with receptor protein of PfDHFR.

The analog drug scoring values and ligand protein docking results suggested the superiority of newly designed analog over pyrimethamine.


Receptor protein selection

The crystal structure of the PfDHFR was retrieved from the PDBSum (Laskowski 2001). Various 3D structures are available against PfDHFR with different PDBIDs. The selected structure for PfDHFR has PDBID IJ3J which is the most recently reported crystal structure and has two mutations at C59R and S108N. It exists in dimeric form with four chains (A B C and D). The chains A and B are for the DHFR domain and chains C and D for TS domain. The chains A C and D were removed from the original pdb file with the help of edit tools of Discovery Studio 3.5 Visualizer for docking analyses. Additionally co-crystallized ligands were also removed by using Discovery Studio 3.5 Visualizer. The finally prepared coordinate file was used for docking studies.

Designing of novel analog

To design a novel antimalarial drug compound various drug databases such as Drug Bank ( PubChem (http:// Chembank (http:// were screened to check the basic properties of available antifolates and its mode of action against malaria. The pyrimidinediamine was considered as the basic skeleton to design new analog by considering different functional groups benzene rings and long chains. The biochemical activity and molecular properties like molecular weight (MW) hydrogen bond acceptor (HBA) hydrogen bond donor (HBD) pH drug likeness values volume chemical structure and optimal docking area (ODA) of each designed analog were calculated by Molsoft ( and Molinspiration ( The finally selected compound was 3-(4-hydroxy-2-{3-[(246- triaminopyrimidin-5-yl)oxy]propoxy}phenyl)propa- noic acid (Fig. 1) and was used for further computational studies.

Molecular docking

Molecular docking of finally selected analog and pyrimethamine with DHFR protein were performed separately by utilizing two different docking programs PatchDock and Molecular Docking Server (Schneidman-Duhovny et al. 2005). Several complexes were generated by these docking servers but the highest scoring complexes were selected for further post-docking analyses.

Post docking and comparative analyses

Docked complexes of both ligand and pyrimethamine with DHFR were analyzed on the basis of energy minimization values. The lowest binding energy values of docked complexes reflect the stability and efficacy of analog molecule. Docked complexes were visualized by Raswin Discovery Studio 3.5 Visualizer and Pymol softwares. Drug ScoreONLINE tool was used for further evaluation of docked complexes like drug score (Spyrakis et al. 2007) and pseudo binding energy calculations (Gohlke and klebe 2001).


The PfDHFR is associated with TS domain and acts as a bifunctional enzyme (Franca et al. 2004). The DHFR plays an important role in folate biosynthetic pathway in protozoans and humans (Gregson and Plowe 2005). In P. falciparum the DHFR and TS exist as a single polypeptide with amino and carboxy terminals respectively (Ivanetich and Santi 1990). Different protozoans have different lengths of DHFR and TS but in P. falciparum the DHFR domain has 231 amino acids and TS domain has 288 amino acids. The DHFR and TS domain are separated by a JR that consists of 89 amino acids (Chaianantakul et al. 2013).

The PfDHFR protein used as a receptor molecule has various clinical isolates of wild type and mutants (single double triple and quadruple). The selected crystal structure (1J3J) has two mutations (C59R and S108N) and UniProt code of the protein is P13922 (DRTS_PLAFK). The development of the resistance for pyrimethamine against PfDHFR is mainly due to the mutation at 108 position (S108N) (Funanage et al. 1984; Das et al. 2012). The binding of the pyrimethamine with receptor protein and above described mutations are observed at the B chain of the selected structure. The binding of the pyrimethamine with receptor protein and above described mutations are observed at the B chain of the selected structure. The topology of B chain secondary structure comprises alpha helices (22.6%) and beta sheets (27.6%).

Most of mutated residues lie in helices and interaction of our analog with S108N was also confined in helix six (H6) region which may be responsible for minimizing the resistance against antifolates (Fig. 2). These mutated residues induce some conformational changes in its binding pocket that cause hindrance for pyrimethamine binding which favors in the prevalence of malaria (Choowongkomon et al. 2010).

Pyrimethamine is a well reported and clinically approved drug compound from Food and Drug Administration (Gutman et al. 2012). In this study pyrimethamine was taken into account as a parent molecule to design new analog. The basic mechanism of pyrimethamine against malaria is to target on folic acid which produces tetrahydrofolates by inhibiting the enzyme dihydrofolate reductase (DHFR) (Yuvaniyama et al. 2003). This tetrahydrofolates are the potent machinery for DNA and RNA synthesis in protozoa (Yuthavong 2002). Pyrimethamine is a most renowned drug that targets B-chain of our selected protein structure. Drug databases such as Drug Bank PubChem and ChemBank were used to identify the pyrimethamine characteristics which would help to design the analog molecules.

The best modified analog structure was designed by adding specific functional groups as amino group (NH2) carboxyl group (COOH) hydroxyl group (OH) and two oxygen molecules embedded in a long polar chain in between two benzene rings (Fig. 1). The NH2 (basic) and COOH (acidic) group will favor the analog structure because it provides more chances in peptide bond formation by H+ association and dissociation respectively. Similarly OH group also has the affinity in hydrogen bond with different amino acids by hydrolysis or condensation. All these newly introduced functional group properties in analog structure support more chances of good binding against mutated residues as compared to pyrimethamine. The basic molecular and drug likeness properties on the bases of Lipinski rule of five were calculated by utilizing Molsoft and Molinspiration tools.

These two web-based software's have the capacity to calculate the molecular formula molecular weight number of hydrogen bond acceptor (HBA) number of hydrogen bond donor (HBD) molecular polar surface area (MolPSA) MolLogP MolLogS and MolVol. The molecular volume and the molecular weight for the novel analog were 322.38 A3 and 363.15 kD respectively. Both these values are higher than those of pyrimethamine (Table I). MolPSA justifies the implication that more polar surface area favors and enhances the chances of binding affinity between ligandprotein interactions.

The MolPSA values for pyrimethamine and analog were 211.74 A3and 248.08 kD respectively which also emphasize the importance of this analog over pyrimethamine. The MolPSA value for the pyrimethamine was 60.88A2 and for the newly designed analog it was 143.46 A2. Likewise the drug likeness score for designed analog was 1.19 whereas for pyrimethamine was 0.98. The calculated values for both molecules showed that this novel compound had better response as drug candidate (Table I).

Table I.- Molecular properties and drug likeness values by Molinspiration Molsoft and PEARLS.


Molecular formula###C12H13ClN4###C16H21N5O5

Molecular weight###248.08kD###363.15kD

Molecular volume###211.74 A3###322.38 A3

Drug-likeness score1###0.98###1.19

Binding Energy2###-2.24###-2.80

MolPSA###60.88A2###143.46 A2

After the selection and preparation of receptor protein and drug molecules PatchDock and Molecular Docking Server were used for the prediction of docking complexes. Both pyrimethamine and newly designed analog docked with mutated DHFR. Several docking complexes were predicted by both docking servers but the selection of the docked complexes were performed on the basis of lower binding energy values and the bindings of both compounds with receptor proteins essentially at mutated residues (Figs. 3 4). The binding distance between receptor and ligand molecules should be less than the binding distance between receptor and pyrimethamine. The distances between docked molecules and receptor protein were measured by Raswin visualization tool. The binding distance calculated between pyrimethamine and Asn108 was 7.81A while it was 3.9A with the analog (Fig. 5 6). Novel analog molecule interacts with mutated residue (Asn 108) of PfDHFR which showed better efficacy compared to pyrimethamine.

The bindings of analog with mutated residues of DHFR stretch the advantage towards the novel analog that may give potential results against malaria caused by P. falciparum.

Docking analyses depend on binding energy values between the ligand and receptor molecule. Gibbs free energy method was used to calculate energy and to predict the binding efficacy between ligand and receptor molecules. The lowest energy values imply better results and help in evaluating docking complexes. Bioinformatics and Drug Design (BIDD) (Chen 2006) and Program of energetic analysis of receptor ligand system (PEARL) (Han et al. 2006) softwares were utilized to evaluate the binding energies of selected docking complexes. The binding energies calculated by PEARL for novel analog (-2.80 kcal/mol) were less compared to that of pyrimethamine (-2.24 kcal/mol). Furthermore Drug ScoreONLINE and drug scoreCDS (Gohlke et al. 2000) tools were used for estimation of drug score values. The calculated values of drug score for pyrimethamine (0) was higher than novel analog (-7). The drug-likeness score 1.19 of designed ligand and for pyrimethamine (0.98) was analyzed by Molsoft tool.

These values indicated that the novel analog has high potential therapeutic values to target the PfDHFR. Choowongkomon et al. (2010) have shown that Ala16 Leu40 Gly44 Leu46 Asp54 Phe58 Ser108 Ser111 Ile112 Ilu164 Ser167 Tyr 170 Thr 185 and Val 195 are the interacting residues of PfDHFR with pyrimethamine. The novel designed analog shown interaction with different residues such as Ile14 Cys15 Ala16 Leu40 Leu46 Trp48 Asp54 Meth55 Phe58 Asn108 Ser111 Ilu164 and Tyr170. These interacting residues indicate the binding pocket of DHFR protein and its vicinity for the analog molecule. The analog molecule that interacts with these residues may have the potential to minimize the resistance against malaria. The mutation of Ser108Asn in DHFR eliminates the resistance of pyrimethamine with DHFR (Sirawaraporn et al. 2002).

This mutation inhibits the binding of pyrimethamine with receptor molecules whereas designed analog structure binds with Asn108 which might enhance the efficacy of drug binding with target site.


In this study a novel antimalarial drug has been proposed against the mutant PfDHFR. In this new drug compound different functional groups and molecules were attached in the parent compound (pyrimethamine). The assessment of this novel antimalarial drug compound was performed by utilizing different computational tools. The results generated by utilizing computational tools suggested that the novel drug compound have potential to show better efficacy than the previously reported drugs. This in silico study will be useful for designing an efficient antimalarial drug compound.

Furthermore the current study will also lead to design an experimental protocol for new drug molecules.


Nasir-ud-din acknowledges financial support from Pakistan Academy of Sciences for amino group studies.


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Author:Iqbal, Zeeshan; Hassan, Mubashir; Munir, Jawaria; Farah, Irfana Mariam; Shakoori, Rauf; Shakoori, Ab
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Oct 31, 2014
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