Malaria is the most dreaded parasitic disease of man and it is still a major health problem in tropical countries1. About 216 million cases of malaria were reported in the year, 2016 with an increase of about 5 million cases as compared to year, 2015. Plasmodium falciparum is the most prevalent malaria parasite in the WHO African Region, accounting for 99.7% of estimated malaria cases in 2017, as well as in the WHO regions of south-east Asia (62.8%), the eastern Mediterranean (69%) and the Western Pacific (71.9%). Estimated cases of falciparum malaria grew from 211 million in 2015-216 million in 2016 with an increase1 of 2.4%.
Malaria is caused by hemoprotozoa of the genus Plasmodium. These parasites are transmitted to the human by the bites of infected female anopheles mosquitoes. Six malaria species are commonly known to cause human malaria: P. falciparum, P. vivax, P. ovale curtisi, P. ovale wallikeri, P. malariae and P. knowlesi 2. The majority of malarial deaths are caused by the intracellular protozoan parasite Plasmodium falciparum3. The commonly used classes of antimalarial compounds include the quinolines (chloroquine, quinine, mefloquine, amodiaquine, primaquine), the antifolates (pyrimethamine, proguanil and sulfadoxine), the artemisinin derivatives (artemisinin, artesunate, artemether, arteether) and hydroxynaphthaquinones (atovaquone)4. Drug resistance has been reported to almost every known anti-malarial agent, underscoring the case by which parasite population can adapt and survive5.
The P. falciparum relies exclusively on de novo pyrimidine biosynthesis to supply precursors for DNA and RNA biosynthesis6. In contrast, the human host cells contain the enzymatic machinery for both de novo pyrimidine biosynthesis and for salvage of performed pyrimidine bases and nucleosides7. Plasmodium purine and pyrimidine metabolic pathways are distinct from those of their human hosts. Thus, targeting purine and pyrimidine metabolic pathways provides a promising route for novel drug development8. Dihydroorotate dehydrogenase (DHODH) is a flavin mononucleotide (FMN) dependent mitochondrial enzyme that catalyzes the oxidation of L-dihydroorotate (L-DHO) to produce orotate as part of the fourth and rate-limiting step of the de novo pyrimidine biosynthetic pathway9.
PfDHODH is essential for parasite growth and has been validated as an antimalarial drug target for development of new antimalarial agents10. Several derivatives of triazolopyrimidine, benzamide, naphthamide and urea have been reported to inhibit PfDHODH11. But due to the resistance to these existing drugs against malaria much more efforts are required to develop new anti malarial which overcomes the plasmodium resistance to clinically available drugs. Therefore, considering the high mortality, morbidity, emergence of resistance to existing drugs against malaria, the present study was undertaken for designing and optimization of these putative molecules in order to generate new drug candidates likely to act against malaria.
MATERIALS AND METHODS
Location and time duration of study: The present study was carried out during July 2017-April 2018 at Laboratory of Bioinformatics, School of Biotechnology, IFTM University, Delhi Road (NH-24), Moradabad 244102, Uttar Pradesh, India.
Preparation of protein structure: The 3D coordinates of the crystal structure of Plasmodium falciparum dihydroorotate dehydrogenase with a bound inhibitor (PDB id: 1TV5) was retrieved from PDB and taken as the receptor model in flexible docking program. Plasmodium falciparum dihydroorotate dehydrogenase was optimized by chimera tool for removal of all heteroatom (A26, FMN, N8F, ORO and Sulphate ion) and water molecules from PDB file of PfDHODH (PDB id: 1TV5) and further polar hydrogen atom were added to protein to make the receptor molecule suitable for docking.
Active site analysis: The active site residues of Plasmodium falciparum dihydro-orotate dehydrogenase were obtained from the PDBSUM entry of 1TV5 having binding site residues LEU531, PHE227, GLY535, VAL532, PHE188, MET536, TYR528, HIS185, LEU172, CYS184, ARG265, GLY181, CYS175, PHE171 and ILE263 for inhibitor A26 (2-cyano-3-hydroxy-N-(4-trifluoromethyl-phenyl)-butyramide).
PubChem compound database screening: A total of 25 analogues of benzamide were screened using the criteria (Compounds having similarity value>= 95%) for docking studies (Table 1). Twenty five benzamide derivatives were docked with PfDHODH and validated in two parts: (i) Prediction of docking energy between the docked compounds with PfDHODH and (ii) Hydrogen bond details of the best-ranked docked pose using Python Molecular Viewer.
Molecular docking: Obtained benzamide derivatives against Plasmodium falciparum dihydroorotate dehydrogenase structure were docked using molecular docking program AutoDock12. Gasteiger charges were added and maximum six numbers of active torsions were given to the lead compounds using AutoDock tool, Kollman charges and the salvation term were then added to the protein structure using the same.
|Table 1:||List of benzamide derivatives screened from Pubchem compound database
The spacing parameters of grid points were adjusted to cover the entire active site residues of the PfDHODH and the default value 0.375Å was set between grid points. The Lamarckian genetic algorithm was implemented and docking parameters were set as follows: 30 docking trials, population size of 150, maximum number of energy evaluation ranges of 25,0000, maximum number of generations is 27,000, mutation rate of 0.02, cross-over rate of 0.8, while Other docking parameters were set to the software’s default values.
RESULTS AND DISCUSSION
The retrieved crystal structure of Plasmodium falciparum dihydroorotate dehydrogenase with a bound inhibitor (PDB id: 1TV5)13 from PDB was modification using chimera tool and the above modified molecule was docked using Autodock software tool to study its interaction with ligands. The docked molecule was further analyzed through Python Molecular Viewer14.
Molecular docking: The docking results of 25 screened compounds with PfDHODH are shown in Table 2 After screening the results based on docking energy, it was predicted that the compound CID 867491 has least docking energy (-4.82 Kcal Mol1) among the 25 docked compounds, which inhibits PfDHODH.
Results of hydrogen bond details: Details of hydrogen bond formation between each compound and PfDHODH with atoms involved and their respective bond lengths are shown in Table 3.
Screening and molecular docking studies of benzamide analogues followed by hydrogen bonding formation suggested compound CID 867491 (N-(4-bromo-3-methylphenyl)-2-methyl-3-nitrobenzamide) as a potent compound for targeting Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). The novel findings based on an in silico approach may be significant for potent drug design against malaria.
The PfDHODH is a protein drug target for drug discovery to combat malaria15. Flexible Molecular docking of ligands from chemical database to receptor target is an emerging approach and is widely used in drug discovery to reduce cost as well as time pertaining to wet laboratory experiments16.
Molecular docking studies are used to determine the interaction of two molecules and to find the best orientation of ligand which would form a complex with overall minimum energy17.
|Table 2:||Docking results of benzamide derivatives against PfDHODH
|Table 3:||Hydrogen bonds and amino acid position of 25 benzamide derivatives PfDHODH
Auto Dock enables us to understand these molecular interactions between a ligand and corresponding protein in terms of binding and docking energy values, the lowest docking value is used to identify a possible drug candidate against target protein18. Therefore, optimal interactions and the best autodock score were used as criteria to interpret the best conformation, generated by AutoDock program19. The present study predicted the compound CID 867491 with least docking energy (-4.82 Kcal Mol1) which inhibits PfDHODH (Fig. 1).
|Fig. 1:||Chemical structure of the best compound CID 867491
Docked complex of Plasmodium falciparum
dihydroorotate dehydrogenase with compound CID 867491. One H-bond was formed between amino acid
HIS185 and compound CID 867491. Compound is represented by yellow lines and amino acid
as sticks and ball. Hydrogen bond is represented by green dotted spheres
Hydrogen bonding plays an important role in the inhibition of a complex molecule by providing structural and functional stability20. The details of atoms in the formation of hydrogen bonds with the bond lengths may provide useful information for in-depth understanding binding mechanism of the compound to the active site of the protein21. The Compound having CID: 867491 was found to have hydrogen bond formation with HIS185 residue.
A close view of the Docked complex of Plasmodium falciparum dihydroorotate dehydrogenase with compound CID 867491 was analyzed through Python Molecular Viewer shown in Fig. 2. Compound is represented by yellow colour lines, amino acid as sticks in and ball in blue colour while hydrogen bond is represented by green colour dots.
On the basis of present study the molecule CID 867491 (N-(4-bromo-3-methylphenyl)-2-methyl-3-nitrobenzamide) was found to have lowest docking energy (docking energy = -4.82 Kcal mol1) and hydrogen bond formation of compound CID 867491 with active site residues HIS 185 of PfDHODH, these in silico findings validates the structural and functional stability of ligand and receptor protein complex and suggested CID 867491 molecule to be a suitable antimalarial drug candidate.
Data obtained from the present study provide new insights into the identification and validation for a new specific antimalarial drug candidate. However, it is required to test and validate the identified target in relevant wet (Biochemistry and Molecular Biology) laboratories prior to be successfully brought into practice in view as an active antimalarial drug molecule.
The Plasmodium falciparum dihydroorotate dehydrogenase is a drug targeting protein for the drug discovery to combat against malaria. Auto-Dock is a popular non-commercial docking program and an emerging approach widely used in drug discovery for docking ligand from chemical database with target protein which helps in reducing the cost and time for drug discovery process which otherwise takes many years. Information acquired from the present in silico study give new bits of knowledge for the identification and validation of a new inhibitor against a specific drug target. However, other preclinical , in vitro and in vivo testings, with other important relevant wet laboratories experiments are needed to be performed to test and validate the computationally identified molecule prior, to be successfully brought into practice in view as an active antimalarial drug agent.
Drug design and development is not only a costly procedure but also time-consuming. Therefore, computational approaches and methodologies can be of significance for pharmacophore generation in a drug-discovery procedure. The present study revealed potent antimalarial drug candidate employing computational tools. Besides, this molecule can be validated and tested in wet labs prior to be successfully brought into practice in view of active antimalarial drug compound application and in vivo drug trials on mammalian system followed by approaching the relevant drug for human system.
The authors are grateful to Prof. (Dr.) A.K. Ghosh, Vice Chancellor, IFTM University, Moradabad for providing a platform and financial support in terms of Research Promotion Grant (IFTMU/SBT-RP-003) to carry out the current research work.