Malaria is a common and life threatening disease in many tropical and subtropical areas. It is a mosquito borne infectious disease of humans and other animals caused by parasitic protozoan belonging to the genus Plasmodium [1]. The disease causes about one to two million deaths each year, which accounts for about one hundred and fifty (150) to three hundred (300) deaths each hour [2]. Malaria causes symptoms that typically include fever, fatigue, vomiting and headaches. In severe cases, it can cause yellow skin, seizures, coma or death [3].

The disease is transmitted most commonly by an infected female Anopheles mosquito. The mosquito bite introduces the parasites from the mosquito's saliva into a person's blood [1]. The parasites travel to the liver where they mature and reproduce. Five species of Plasmodium can infect and be spread by humans [3]. Most deaths are caused by Plasmodium falciparum because Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae generally cause a milder form of malaria [1,3]. The species Plasmodium knowlesi rarely causes disease in humans [1].

Triterpenoids are naturally occuring and are used against several types of human diseases. Triterpenoids have chemopreventive and anticancer potentials. This is achieved by regulating various transcription and growth factor. [4]. Lonchocarpus cyanescens is a species of shrub from fabaceae family. It is commonly known as elo and anunu by Igbo people, in Yoruba, as talaki, in Hausa, sauru in Tiv and as ebelu in Edo people. Various studies has been carried out on the bioactivity, phythotherapeutic, anti-psychotic properties [4,5-16] of Lonchocarpus cyanescens. Experimental studies on Lonchocarpus cyanescens triterpenoids showed that they posses antimalaria effect Moronkola and Oladosu [17]. Bioassay investigation carried out on the leaf extract for anti oxidant activities revealed that the anti oxidant properties correlated with its phenolic and flavonoid contents [18]. Shuaibu et al. [19] used microfluorometric method by fluorometric assay using picogreen for activity-guided isolation of anti-plasmodium compound from different plant extracts, the in vitro anti-plasmodial activity of methalonic extracts of these plants showed that the IC₅₀ of Lonchocarpu cyanescens as determined by the parasite DNA concentration ranged from 20-200 μg/ml for Plasmodiun falciparium. It was reported that the extract and the isolated compound did not affect the integrity of human erythrocyte membrane at the observed IC₅₀. It was also stated that the adverse effects manifest in a concentration dependent fashion from IC₅₀ ≥ 500 μg/ml. Direct study of the compounds on the parasites such as inhibition of the hemozoin polymerization on the parasite [20,21] were carried out by the introduction of a new layer into a pre-existing sequence of the DNA (intercalation with DNA) of the parasite [22] and inhibition of the lactase dehydrogenase of the Plasmodium falciparium (an essential enzyme used to generate energy within the parasite) was carried out using a disquiterpene extracted from seeds of Gossypium species [23,24]. Inhibition of the formation of microgamates by introducing an interference into the formation of the mitotic spindles and the assembly of microtubules into typical axonemes in the gamates were also carried out [25,26]. Studies on the molecular mechanism of anti malaria action of Lonchocarpus cyanescens triterpenoids has not been described. Therefore the goal of this work is to present the docking analysis of these triterpenoids using three different malaria proteins with PDB codes 3QS1, 1LS5 and 1SME.

Computational Procedures

The pdb files 3QS1, 1LS5 and 1SME for the three malaria receptors were obtained from the Protein Data Bank. The initial preparation of the pdb files to select the needed chains, delete multiple ligands and non-protein parts using Discover Studio 4.1 visualizer and ligand explorer, OpenBabel GUI version 2.3.2a was used to convert the pdb file format, Spartan 14 version 1.18 was used for geometry optimization and geoemtric analyses of the ligands. AutoDock Tools 1.5.6 and AutoDock Vina version 1.1.2 was used for molecular docking process (downloaded from http://autodock.scripps.edu) for molecular docking process. EduPymol version 1.7.4.4 was used for analysing docking results. The exhaustiveness which determines how comprehensive the software search for the best binding mode was set to the default value of 8 Angstrom which accompanied the ligands from the list of protein residues reported in the literature to characterise their binding sites for all the docking runs. Before the docking runs, Autodock tools (ADT) was used to add polar hydrogen to the prepared receptor. The three-dimesional affinity and electrostatic grid boxes were generated to cover the entire active site using autogrid. The number of grid points in the x, y, z axes 40 × 40 × 40 mm each separated by 1.000 Angstrom. The grid center x, y and z were also specified and it varied from one receptor to another (Table 1). The protein was prepared by adding Gasteiger charges. The Authodock vina were the analysed with Edu Plymol version 1.7.4.4. The receptor in the pdbqt file format as well as the different binding modes of the ligand obtained after the calculation was opened with EduPlymol to check the best binding mode that fitted well with the protein binding site. Two derivatives (OH and OCH₃) of the tripterpenoids of L. cyanescens were used as ligands in the docking. The ¹H-NMR and ¹³C-NMR analyses, (Moronkola and Oladosu, 2013) showed that they are triterpenoids that have the sturctures as shown in Figure 1. The converted structures were energetically minimized and then optimized geometrically using Molecular Mechanics Force Field (MMFF) in Spartan’14.

Table 1: Grid centers (X, Y and Z) used for the docking runs.

Figure 1: Structures of the Ligands.

Docking Studies

The docking studies were carried using AutoDock Vina programme. The grid dimension used for all the three (3) proteins are 40 × 40 × 40 Å (grid size) with point separated by 1.000 Å (grid-point spacing). The X, Y, and Z coordinates (grid centres) were also specified and it varied from one receptor to another (Table 1). Also, the exhaustiveness was set to the default value of eight (8) for all the docking runs. The binding energy/affinity of the ligand to the protein was computed using the AutoDock Vina software package by search Algorithm. After the successful completion of the docking runs, different conformations of the ligands known as Binding modes were obtained with their respective binding energy/affinity and the stable one which happens to be the one with the lowest binding energy/affinity was picked as the pose and was employed in the post-docking analysis using Edupymol The inhibition constants K_i were calculated using the equation:

ΔG=RTlnK_i

K_i=exponential(ΔG/RT)

where ΔG is the binding affinity/energy in Kcal/mol, R is gas constant, 1.987 cal/mol/K and T is absolute temperature, assumed to be room temperature, 298.15 [27].

Post-docking analysis

The AutoDock Vina results were analysed using Edu Pymol version 1.7.4.4. The receptors in the PDBQT file format as well as the different binding modes of the ligands obtained after running AutoDock vina which were also in PDBQT file format were opened with Edu Pymol to check the best binding mode that fitted well with the binding site cavity. Also, the protein-ligand hydrogen interactions were also viewed and the various atomic distances were measured using the same software.

Docking score results

The results in Table 2 showed that the synthesized ligands compared favourably well with the standard ligands. The OH and OCH3 derivatives triterpenoids gave binding affinities values (-8.2 Kcal/mol for 3QS1, -7.5 Kcal/mol for 1LS5 and -7.8 Kcal/mol for 1SME receptors) and (-8.1 Kcal/mol for 3QS1, -7.0 Kcal/mol for 1LS5 and -7.7 Kcal/mol for 1SME receptors) respectively against the standard ligands value (-9.3 Kcal/mol for 3QS1, -8.1 Kcal/mol for 1LS5 and -8.0 Kcal/mol for 1SME receptors). Also, the results showed that the OH derivative of the triterpenoids gave better binding interaction than OCH3 derivative (Figures 2a, 3a and 4a for redocking and Figures 2b, 2c, 3b, 3c, 4b and 4c for OH and OCH₃ respectively) which implies that the OH derivative of the synthesized triterpenoids will be more effective than OCH₃ derivative in the treatment of the disease. The binding affinity and inhibition constant for the OCH₃ are very high. This is as a result of the inductive (electron releasing) effect of the methyl group. Furthermore, the residues involved in the binding of the ligands to 3QS1 receptors are Gly-217, Ile-287, Ser-287, Tyr-189, Val-76, Asp-215, Thr-222, Ser- 77, Asn-292, Lys-296 while Gly-78, Ser-79, Gly-216, Asp-34, Tyr-192, Asp-214, Leu-290 and Asp-214, Thr-217, Val-219, Ala-351, Gly-216, Asn-92, Val-349 are also involved in the binding of the ligands to 1LS5 and 1SME receptors respectively. The result is in agreement with the result obtained from previous study on a triterpenoid isolated from the stem of Ganoderma sinense (ganoderiol-F) which showed higher affinity towards HIV-1 protease (binding energy=-11.40 kcal/mol and K_i=4.68 nM) than to plasmepsin I (binding energy=-9.96 kcal/mol and K_i=50.94 nM). Computational studies of G. lucidum triterpenoids with aspartic protease enzymes of HIV 1 and plasmepsin I, performed using Nelfinavir and KNI-10006 as the standards for HIV-1 protease and plasmepsin I, respectively. It was observed that the four triterpenoids are able to interact with both enzymes. Ganoderat acid-B showed the best affinity to HIV-1 protease (binding energy=-7.49 kcal/mol and K_i=0.001 mM) which is better than nelfinavir. Furthermore, the best affinity to Plasmepsin I is showed by ganodermanondiol (binding energy=-7.14 kcal/mol and Ki=0.005 mM which is better than KNI- 10006 [28].

Figure 2a: Re-docking Analysis of 3QS1 Receptor (Surface Representation).

Figure 2b: Docking Analysis of 3QS1 Receptor VS OH derivative ligand (Surface Representation).

Figure 2c: Docking Analysis of 3QS1 Receptor VS OCH3 derivative ligand (Surface Representation)..

Figure 3a: Re-docking Analysis of 1LS5 Receptor (Surface Representation)..

Figure 3b: Docking Analysis of 1LS5 Receptor VS OH derivative ligand (Surface Representation).

Figure 3c: Docking Analysis of 1LS5 Receptor VS OCH3 derivative ligand (Surface Representation).

Figure 4a: Re-docking Analysis of 1SME Receptor (Surface Representation).

Figure 4b: Docking Analysis of 1SME Receptor VS OH derivative ligand (Surface Representation).

Figure 4c: Docking Analysis of 1SME Receptor VS OCH3 derivative ligand (Surface Representation).

The Lipinski properties (Table 3) such as Molecular Weight (358.610 for OH ligand derivative and 372.637 for OCH3 ligand derivative), Partition coefficient value (Log P) (3.20 for OH ligand derivative and 3.42 for OCH3 ligand derivative) which indicates the Lipophilicity of the ligand; number of Hydrogen Bond Donor (1 for OH ligand derivative and 0 for OCH3 ligand derivative) and number of Hydrogen Bond Acceptor (1 for OH ligand derivative and1 for OCH3 ligand derivative) for the ligands were obtained (Table 2). Based on the Lipinski’s rule of five, the drug must have Molecular Weight value of ≤ 500, Hydrogen Bond Donor ≤ 5, Hydrogen Bond Acceptor ≤ 10 and Partition coefficient (Log P) value ≤ 5. From the results in Table 2, the synthesized ligands passed the Lipinski’s rule of five.

Table 2: Docking score results for malaria receptors.

Table 3: QSAR properties of the ligands.

Also, the Polar Surface Area (PSA) which is an indicator of the ligand hydrophilicity plays an important role in shaping the protein- ligand interaction by affecting the non-bonded contribution to the binding energy. Molecules with PSA greater than 140 Å2 are usually believed to be poor at penetrating cell membranes. For molecules to penetrate the blood-brain barrier, PSA should be less than 60 Å2 [29] which the ligands also fulfilled (Table 2).

Structure-based methods remain one of the most logical approaches in drug discovery. In the present work, the studies revealed that the ligands docked well with the targets suggesting that the ligands are efficacious in the treatment of malaria.