Molecular Docking of the Inhibitory Activities of Selected Phytochemicals in Artemisia Afra Against NADH-Ubiquinone Oxidoreductase of Plasmodium Falciparum (PfNDH2)

Molecular Docking of the Inhibitory Activities of selected Phytochemicals in Artemisia Afra Against NADH-Ubiquinone Oxidoreductase of Plasmodium Falciparum (


Introduction
Malaria is one of the most dangerous infectious diseases and the development of drug resistant parasites only exacerbate the situation.The advent of drug-resistant malarial strains has increased continuously in recent times and has led to a great challenge for health globally.Malaria, a mosquito borne disease that is life threatening has been receiving innovative attention for effective anti-malaria drugs.The latter unfolding of multi drug resistant strains of malaria pathogen Plasmodium falciparum persist to cause deaths in the torrid and semiarid region, but malaria vaccine are not yet available for efficient and effective treatment.As Against NADH-Ubiquinone Oxidoreductase of Plasmodium Falciparum (PfNDH2) a result, it dictate for new and effective anti-malaria drugs are increasing rapidly.So, new anti-malaria drugs with innovative approaches to targets are absolutely required to address drug-resistant malaria.
One of the most commonly used plants in traditional medicine in South and West Africa is Artemisia afra. it is an aromatic shrub used to treat various disorders including coughs, colds, influenza, and malaria.The treatment of a wide ranging and unrelated list of health conditions and symptoms indicates that A. afra can, or should be considered a panacea.Infusion or decoction often made into syrup for bronchial treatment is the usual preparation.A. afra is used to treat coughs and colds, chills, dyspepsia, loss of appetite, stomach-ache and other gastric derangements, colic, croup, whooping-cough, gout and as purgative, flu, headaches, inflammation, gout, sore throat, malaria, diabetes, bladder and kidney disorders, asthma, constipation as well as numerous other health problems [1] It is also used by insertion of fresh leaves into the nostrils to clear clogged nose [2].
Molecular Docking Studies of Lonchocarpus cyanescens Triterpenoids as Inhibitors for Malaria showed that Structurebased methods remain one of the most logical approaches in drug discovery.[3] Docking analysis of Proguanil and its analog with Human Dihydrofolate Reductase receptor carried out using HEX and Argus lab docking software showed that some of the modified drugs are better than the commercial drugs available in the market [4].
The computational method showed the advantage in saving time, cost and resources.It is feasible to block the interaction of NAD-dependent protein deacylases protein from selected compounds using virtual screening based on pharmacophore and molecular docking studies to serve as a preliminary study of designing an inhibitor against Plasmodium falciparum [5].The predicted structure of Pftk in molecular-docking study of malaria drug target enzyme transketolase in Plasmodium falciparum was reported to serve first hand in the future development of effective Pftk inhibitors with potential antimalarial activity [6].
The high binding energy obtained in molecular docking, synthesis and in vitro antimalarial evaluation of certain novel curcumin analogues showed that the designed compounds have good affinities for the PfATP-6 protein and that this could be responsible for its antimalarial activity against the Plasmodium falciparum species [7].
Synthesis, molecular docking and antiplasmodial activities of Tetrahydro-β-Carbolines was reported as a promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity.In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism [8].
An in-silico approach to Molecular docking analysis of apigenin and quercetin from ethylacetate fraction of Adansonia digitata with malaria-associated calcium transport protein showed that it could lead to further development of potent calcium transporter inhibitors for the prevention and treatment of malaria and related conditions [9].Docking analysis obtained from quantitative Structureactivity relationship and molecular docking of Some Pyrrolones as antimalarial agents against Plasmodium Falciparum revealed that three of the studied compounds with binding affinity values of -10.7 kcal/mol, -10.9 kcal/mol and -11.1 kcal/mol possess higher potency than standard antimalarial drugs with binding affinity values of -8.8 kcal/mol, -9.5 kcal/mol and -9.0 kcal/mol.This showed that the information obtained by the QSAR and molecular docking results will offer important structural insights for the design of novel and highly potent antimalarial from the pyrrolones [10].
Molecular docking and QSAR studies for modeling of antimalarial activity of hybrids 4-anilinoquinolinetriazines derivatives with the wild-type and mutant receptor pf-DHFR showed that the quantitative structureactivity relationship (QSAR) and docking studies performed for previously reported 4-anilinoquinoline and 1,3,5-triazines based molecular hybrids.The generated model also showed good correlation coefficients (R 2 = 0.70) and test set prediction coefficient (R 2 = 0.74).These outcomes showed the good predictive ability of the conventional QSAR model [11].
Series of methods have been used by the researchers in finding efficient and effective drugs for curing various diseases which involves long time laboratory works, too much capital and energy but this present work focused on the use of one of the tools of Computer aided drug design (CADD) called molecular docking which has also been used by various researchers to discover the potent among the selected phytochemicals isolated from Artemisia afra.
Molecular Docking is a computational modeling method of Drug Discovery mostly used to assist in understanding protein-ligand interaction [12][13][14][15][16] and forecasting the proteinligand binding mode.Relative to the traditional method, it is efficient, effective, fast and minimizes cost compared to the traditional method.Therefore, this current work targeted to check the inhibitory activities of selected phytochemicals in Artemisia afra against NADH-Ubiquinone Oxidoreductase of Plasmodium falciparum (PfNDH2) for anti-Malarial drug discovery in order to give scientific evidence for their conventional uses.
NADH-ubiquinone oxidoreductase of Plasmodium falciparum (PfNDH2) appeared to be a viable target for antimalarial drug development.

Ligands Preparation
There were 50 selected Compounds from Artemisia afra used against the target receptor (NADH-Ubiquinone Oxidoreductase of Plasmodium falciparum (PfNDH2)).Those compounds were downloaded from a drug database called PubChem, an open chemistry database, and a drug bank consisting of substance, compound, and bioassay (https://pubchem.ncbi.nlm.nih.gov/).[17] The following compounds were downloaded: Endo-Borneo-CID_120151.These compounds served as the ligand molecules used in determining their potency against NADH-Ubiquinone Oxidoreductase of Plasmodium falciparum (PfNDH2).The downloaded compounds were converted to 3-dimensional structures saved in .pdbformat for the effective virtual examination exercise employing SMILES and Online Translator) and Discovery studio then later minimized to acquire lowest energy and most stable conformer before docking.

Target Receptor Preparation
Crystal structure of Nicotinamide Adenine Dinucleotide Hydrogen (NADH)-ubiquinone oxidoreductase of Plasmodium falciparum (PfNDH2) describe a possible target for anti-malarial drug discovery (Protein ID: 5jwa) was downloaded [18] and It undergoes further treatment using BIOVIA Discovery Studio Software (version 19.1) [19] to remove the unwanted molecules and prevent molecular interaction during virtual screening.

Molecular Docking Simulations
All atoms and complexes including water molecules attached with our target receptor (PDB ID: 5jwa) were detached using [19] in order to have a clean and accurate virtual screening.Pyrx-virtual screening tools (Autodock Vina and Open Babel,) were used for the docking process while Autodock tool-1.5.6 program [21] was used to determine the grids, the dimension and binding centre of 5JWA (-5.825,-58.985,22.747)and (103.170,100.056, 129.571 Å) for (x, y, z) respectively with 1.000 Å spacing, then the docking scores and other calculations were carried out using AutoDock Vina, MGL tools-1.5.6,PyMOL Console Edu and BIOVIA Discovery studio 4.5.

Pharmacokinetics (ADMET) and Drug-Likeness Properties Evaluation
ADMET properties related to Chemical absorption, distribution, metabolism, excretion, and toxicity were evaluated [22,23].The drug-likeness of the selected compounds was assessed using Mol inspiration Online Tool (https://molinspiration.com).This is important in drug discovery and development and a high-quality drug candidate should have sufficient efficacy against the therapeutic target and also display suitable ADMET properties at a therapeutic dose level.

Density Functional Theory (Quantum Mechanics)
Density Functional Theory of Quantum mechanical calculations were done for the five key compounds (Hits) obtained from the virtual screening.Structural optimization was done using Spartan'14 software with DFT/B3LYP at 6-311G* levels.This was done to obtain the frontier orbital energies, the highest occupied molecular orbital (HOMO), the lowest occupied molecular orbital (LUMO) and energy gap.Information on the stability and reactivity of the compounds are also provided.

Results and Discussion
Molecular docking is a computer-based method used in drug discovery to discover of new compounds of medicinal nature by predicting ligand-target interactions and the binding affinity of the ligand to the protein on a molecular interaction.Crystal structure of NADH-Ubiquinone Oxidoreductase of Plasmodium falciparum (PfNDH2) with PDB ID 5JWA (Figure 1) was used as the target protein in this research.Docking was performed on Fifty (50) compounds to PfNDH2 target protein with code 5JWA and the binding affinities of the selected compounds are as shown in Table 1 ).Hydrophobic and electrostatic interactions are also reported and for 5jwa-2,4,6-TPD, the hydrophobic interactions include Trp30, Phe77, Pro210, Thr211, Thr435, Gln437, Gly471, Leu473, 5jwa-CHA include Ser309 while for 5jwa-ARO we have Tyr444, Asn448, Ile466, Ser464 and for5jwa-1-EBSP, the hydrophobic and electrostatic interactions include Gly245 as well, that of 5jwa-CMD include Ser309.The molecular interaction is shown in Figure 2.

A-1 A-2 A-3 A-4
A-5   Assessment of the physicochemical parameters and druglikeness of compounds is one of the important step in drug discovery and design, but the potential active, according to Lipinski, an efficient oral therapeutic drug must obey the 'rule of five' with only one (1) violation and according to the rule, an orally bioavailable drug must possess molecular weight (MW) ≤ 500Da, hydrogen bond donor (HBDs) ≤ 5, hydrogen bond acceptor (HBAs) ≤ 10 and logP (octanolwater partition coefficient) ≤ 5 [24].Descriptors of oral bioavailability are essential as they predict the permeability and absorption of drug across biological membrane such as epithelium cell.Partition coefficient value (LogP) is especially important in predicting intestinal absorption of drugs.Considering all the ligands) binding affinity (Table 1), inhibition constant, (Table 2) efficacy and safety profile using the Molinspiration online (http://www.molinspiration.com/),and ADMET SAR-2 web-server (Table 3) [21], Aromandendrene; Cis-muurola-3,5diene; Chamazulene were qualified as hit compounds for further analysis and are coded A-3, A-4 and A-5 respectively.Drug-likeness of all the ligands were evaluated with Molinspiration online (http://www.molinspiration.com/)as shown in Table 2, it is apparent from the table that none of them had more than one violation of the 'rule of five' which is a good indication for satisfactory oral bioavailability and permeability.

ADMET Analysis of the Selected Compounds
Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profile of a molecule is an essential assay in the early stage of drug discovery.ADMET have been and still are significant reason for attrition in drug discovery and the data obtained compliment the selection and identification of molecules with optimal safety therapeutic profile dose along the discovery process rather than at the final stage, as it helps in avoiding waste of time and precious resource on drug molecules that may eventually be discarded [25].ADMET profile of selected but potent phytochemicals from A. afra as computed using ADMET SAR-2 web-server [5] were as shown in Table 3, as part of the drug ADMET profile, a drug molecule should have good human intestinal absorption (HIA), solubility (log S) range between -1 to -5, and should not be toxic.[25] All the selected hit, A-1, A-2, A-3, A-4 and A-5 are well absorbed in the human intestine and were found to cross the blood-brain barrier, although an oral drug does not necessarily need to cross the blood-brain barrier but for only central nervous system target drugs [26].Prediction shows that the five (5) selected hit compounds were noninhibitor of the cytochrome P450 which is the microsomal enzyme, an indication of a good metabolic activity of the drug in the hepatocytes [27] The Ames toxicity value express the potential of a drug molecule to cause mutation in DNA and could be a major reason for excluding a drug molecule along the discovery process [28].Similarly, the hits compound possesses type III acute oral toxicity values (slightly toxic) which could easily be converted to type IV (nontoxic) during hit-lead optimization.

Bioactivity of Selected Compounds
Table 4 shows the Bioactivity parameters of the five (5) selected hit compounds.The higher the binding energy the lower the inhibition constant as shown in the .inverserelationship between binding Energy and inhibition constant (Equation 1), indicating that the inhibition constant of a potential hit compound is expected to have values ranging between 0.1-1.0µMand not exceed 10nM for a potent drug [26].Inhibition constant value of the hit compounds range from 0.30 to 6.28 µM.This shows that the five (5) selected compounds are qualified drug candidates with 2,4,6-Triphenyl-1,3-dioxane (0.30 µM) being the most potent of all in terms of their binding affinities.It was also observed form the result obtained for Ligand Efficiency (LE), Fit Quality (FQ), and Ligandefficiency-dependent lipophilicity (LELP) according to equation 2-5 that all the hit compounds had values within the recommended fit quality of ≥0.8 [29].
%&'() = 0.873 ././01 ×3.4 − 0.064 (3) HOMO and LUMO energy was calculated for the five top hit compounds (2,4,6-TPD, 1-EBSP, Aro, CMD and Cha) using the quantum mechanical Density Functional Theory (DFT), the result is as shown in Figure 3. Chamazulene (Cha) has the lowest energy gap of 3.16eV with -5.09eV and -1.93eV HOMO/LUMO energy value respectively.In comparison, the values of HOMO and LUMO energies of the other compounds are 2,4,6-TPD (-6.73eV and -0.51eV), 1EBSP (-5.85eV and -0.56eV), Aro (-6.49eV and -0.40eV) and CM3,5D (-5.48eV and 0.58eV) with an energy gap of 6.22eV, 5.29eV, 6.09eV and 4.9eV respectively (Table 4).The chemical species reactivity is described by the frontiers orbitals, the highest occupied molecular orbital (HOMO), and the lowest occupied molecular orbital (LUMO).The HOMO and LUMO energy values describe the ability of the compounds to donate and accept electrons.Energy gap value gives the difference between the LUMO and HOMO energy and this represents the intramolecular charge transfer and kinetic stability.Low chemical reactivity and high kinetic stability are associated with a large energy gap and this is used to predict the strength and stability of the compounds.In contrast, compounds with small energy gaps are more reactive with less kinetic stability [30].

Conclusion
The results obtained from this research analyzing the inhibitory activities of selected phytochemicals in Artemisia afra against PfNDH2 for anti-malaria drug discovery shows that five (5) of the compounds, 2,4,6-Triphenyl-1,3-dioxane (2,4,6TPD); (+)-epi-Bicyclosesquiphellandrene (1EBSP); Aromandendrene (Aro); Cis-muurola-3,5diene (CMD); Chamazulene (Cha) escaped through most of the analysis but two of them aromadendrene and chamazulene were still more potent considering the quantum mechanistic study and they were the potential compounds for anti-malaria drug development and therefore, recommended for therapeutic efficacy investigation and adoption to join the existing drugs for malaria.

Table 1 .
Binding affinity, H-bond interaction, Electrostatic/hydrophobic interactions and inhibition constant of the selected Hit Compounds.

Table 2 .
Druglikeness of the of the selected Hit compounds.

Table 3 .
ADMET analysis of the selected Hit compounds.

Table 4 .
Showing the Bioactivity of the selected Compounds.

Table 5 .
Shows the highest occupied molecular orbital (HOMO), the lowest occupied molecular orbital (LUMO) and the energy gap.

Table 6 .
Shows the downloaded 50 Ligands with their respective CID and Binding energy value.