Kolaviron (Kolaavanone), apigenin, setin as potential Coronavirus inhibitors: In silico investigation

The outbreak of COVID-19 caused by SARS-CoV-2 is increasing with an alarming rate of associated frightening mortality without no known approved therapy. The emergence of new infectious diseases and increase in frequency of drug resistant viruses demand effective and novel therapeutic agents. This study investigated the putative inhibitory potentials of apigenin, setin, kolaavanone, and remdesivir towards SARS-COV2 major protease (6LU7) using in silico methods. Pharmacodynamics, pharmacokinetics and toxicological proles of the compounds were also examined using the pkCSM server. All the compounds showed good anity to the binding pocket of 6LU7. It was observed that kolaavanone exhibited the highest binding anity when compared to apigenin, setin, and remdesivir. The amino acids ASN238, TYR237, LEU286, and LEU287 were showed as the key residues for kolaavanone binding to human SARS-COV2 major protease. The Pharmacodynamics and pharmacokinetics results suggested that all the tested compounds have signicant drug likeness properties and they could be absorbed through the human intestine. Additionally, all the tested compounds except remdesivir are not hepatoxic and also displayed non or relatively low toxic effects in human. Summarily, the results of this study indicated that all the tested compounds are potential putative inhibitors of SARS-COV2 major protease with no or low toxicity effects. However, further experimental and clinical studies are needed to further explore their activities and validate their ecacies against COVID-19.


Introduction
Novel Coronavirus disease 2019 (COVID-19), ranked among the ninth deadliest world pandemic, is a highly infectious and severe acute respiratory disorder caused by a morbi c virus called SARS-CoV-2 which is transmitted to humans via contact with infected persons and/or feeding on infected animals.
The COVID-19 clinical manifestations are very similar to viral pneumonia and include fever, fatigue, cough, shortness of breath, and other complications. According to reports obtained on WHO and NCDC websites as of 27 th June 2020, the coronavirus breakout in Wuhan, a city in Hubei Province of China in November 2019 and has spread to many countries in the world. This global pandemic has forced many nations to lock down their social and economic activities which in turn have adverse effects on the economy. Globally, more than ten million people have been con rmed infected with over 500,000 deaths. Nigeria is one of the countries seriously affected by the virus having over 25,000 cases and more than 500 mortalities [1,2]. Thus, there is an exigent need for effective and non-invasive treatment.
Coronaviruses (SARS-CoV) are non-segmented positive-sense single-stranded RNA viruses with a large viral RNA genome of diameter 80-120 nm ( gure 1). They belong to the family of Coronaviridae, in the subfamily Orthocoronaviridae which consists of four genera namely: Alpha, Beta, Gamma, and Delta coronavirus [3]. Some of the modes of actions of SARS-CoV-2 include hyper-in ammation characterized by a sudden and fatal hyper-cytokinaemia with multi-organ failure [4]; immunosuppression; reduction of Angiotensin-Converting Enzyme 2 (ACE2) to enhance pulmonary vascular permeability and damage the alveoli [5]; and activated by ORF3a, ORF3b, and ORF7a via JNK pathway which induces lung damage [6].
At present, there is no known effective treatment or vaccine that can mitigate/inhibit coronavirus.
Available clinical interventions for COVID-19 are only palliative and limited to support. Therefore, many research groups around the world are currently focusing on developing novel treatments such as vaccines and antivirals. This research investigated the inhibitory potentials of some naturally occurring phytochemicals against COVID-19 major protease (6LU7). These phytochemicals can be repurposed to mitigate the pathogenesis of the SARS-CoV-2 and thus put an end to the frightening associated mortality rate.

Protein preparation
The crystal structure of SARS-COV2 major protease (6LU7) was retrieved from the protein databank (www.rcsb.org). Prior to docking and analysis, the crystal structure was prepared by removing existing ligands and water molecules. Also, missing hydrogen atoms were added using Autodock v4.2 program, Scripps Research Institute. Thereafter, non-polar hydrogens were merged while polar hydrogen was added and subsequently saved into pdbqt format in preparation for molecular docking.

Ligand preparation
The SDF structures of apigenin, setin, kola avanone, and remdesivir were retrieved from the PubChem database (www.pubchem.ncbi.nlm.nih.gov). The compounds were converted to mol2 chemical format.
Polar hydrogens were added while non-polar hydrogens were merged with the carbons and the internal degrees of freedom and torsions were set. The protein and ligand molecules were further converted to the dockable PDBQT format using Autodock tools.

Molecular docking
Docking of the ligands to the targeted protein and determination of binding a nities was carried out using AutodockVina [7]. The PDBQT formats of the receptor and that of the ligands were positioned at their respective columns and the software was run. The binding a nities of compounds for the protein target were recorded. The compounds were then ranked by their a nity scores. The molecular interactions between the receptor and compounds with most remarkable binding a nities were viewed with Discovery Studio Visualizer, BIOVIA, 2016. The respective binding free energy was calculated by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) using HawkDock Server (http://cadd.zju.edu.cn/hawkdock/).

Molecular dynamics simulation
The conformational stability of the protein-ligand interactions was evaluated using molecular dynamics simulations analysis performed through iMODS server (http://imods.chaconlab.org) by normal mode analysis (NMA) predicting properties such as deformability, mobility pro les, eigenvalues, variance, covariance map and elastic network of the protein-ligand interactions [8].

Molecular properties of the phytochemicals
Data in table 2 revealed the results of the molecular properties of the phytochemicals used in this study.
Remdesivir was found to have the highest molecular weight of 602.585, while that of kola avanone is 588.521, setin is 286.239 and apigenin is 270.24. Similarly, the surface area of the compounds: Remdesivir, kola avanone, setin, and apigenin are 242.488, 242.369, 117.519 and 112.519 respectively.
Kola avanone has the highest lipophilicity of 3.7063, apigenin has 2.5768, while setin has the least lipophilicity of 2.2824.

Predicted absorption properties of the phytochemicals
The predicted absorption properties of each of the compounds were reported in table 3. The result showed that kola avanone has the highest water solubility value of -2.9 while apigenin has the lowest valve of -3.329. Apigenin has the highest permeability of 1.007, with setin having the least permeability valve of 0.058. All the compounds are substrate of P-glycoprotein. Only kola avanone and remdesivir are inhibitors of P-glycoprotein-I while kola avanone is the only inhibitor of P-glycoprotein-II.
Predicted in vivo distribution and cytochrome P450 promiscuity of the phytochemicals Table 4 showed the predicted in vivo distribution of the compounds. All the compounds tested have relatively low steady-state volume of distribution. Also, the predicted result revealed that kola avanone has the highest unbound fraction in the human blood. All the compounds have relatively low blood-brain barrier and CNS permeability values. Table 5 displayed the predicted human cytochrome P450 promiscuity of the screened compounds. All the compounds are not substrate of CYP2D6 and CYP3A4 expected remdesivir which is predicted to be CYP3A4 substrate. Similarly, none of the compounds is an inhibitor of CYP2D6 and CYP3A4. Only apigenin is an inhibitor of CYP2C19 and setin as inhibitor of CYP2C9.

Predicted in vivo clearance of the phytochemicals
The predicted clearance of each of the compounds were reported in table 6. Apigenin has the highest total clearance rate of 0.566 while kola avanone has the least clearance rate of -0.359. Likewise, only kola avanone is substrate of renal organic cation transporter.
Predicted toxicological pro le of the phytochemicals Table 7 reported the predicted toxicological pro le of all the tested compounds. None of the compounds has mutagenic potentials against bacteria (AMES toxicity), nor has potential dermal adverse effects and they are human ether-a-go-go-related gene (hERG I) inhibitors. All the compounds have relatively low maximum recommended tolerated dose except setin having valve (0.579) greater than recommended value of 0.477 (log mg/kg/day). Likewise, all the compounds are non-hepatotoxic except remdesivir.

Discussion
Virtual screening and molecular docking are vital methods use in drug repurposing approach to fast track drug discovery and appropriate therapeutic interventions in a pandemic situation. They elucidate the mechanism of ligand binding and discover potent inhibitors for targeted proteins. In this study, kola avanone showed the best docked score (-7.2 kcal/mol) with the SARS-COV2 major protease (6LU7).
The binding energy suggests the a nity of a speci c ligand and the strength by which the compound interacts with and bind to the pocket of the target proteins. The scoring value with a lower binding energy (or high docking score) suggests it as a possible drug candidate.
Kola avanone (kolaviron) is the most abundant Garcinia bio avonoids in Garcinia kola Heckel (Guttiferae) (Farombi et al., 2019). It is popular for the treatment of malaria, hepatitis, neurodegenerative disease, male sexual dysfunction, and immune-destructive diseases [9,10]. It also protects against the oxidation of lipoprotein [11]. Garcinia kola is also used to relieve cold and cure laryngitis [12]. kolaviron has been documented to effectively delay the development of clinical symptoms and and suppresses mortality associated with in uenza virus [13]. Experimental studies have proven the antioxidative, antiin ammatory, anti-apoptotic and modulatory effects of kola avanone which could be helpful to inhibit the pathogenesis of COVID-19.
Apigenin (4',5,7-trihydroxy avone) is one of the most studied phenolics and it is the most widely distributed avonoids in the plant kingdom. Apigenin is chie y present herbs (thyme, oregano, chamomile, basil), in vegetables (celery, parsley, onions), plant-based beverages (beer, tea, and wine) and fruits (oranges). It is also found in abundance in Artemisia, Achillea, Matricaria, and Tanacetum [14]. Apigenin has been reported to have anti-diabetic and anticancer properties, also, its bene cial role in amnesia, Alzheimer's Disease, depression and insomnia treatment has been well documented [14].
The results of the solubility, pharmacodynamics, pharmacokinetics and toxicological pro les of apigenin, setin, kola avanone, and remdesivir are presented in Tables 2-7. The pro les were investigated as a systemic virtual screening of drugs and potential drugs. This is done as alternative to in vivo examinations which are essential complements in drug discovery. The Lipinski's rule is a major criterion to evaluate drug likeliness and to determine if a compound with a particular pharmacological and biological actions has physical and chemical properties that could favour its activities in human. The molecular properties of the compounds based on the computed partition coe cient (log P) showed that the compounds have relatively good lipophilicity as the logP values were less than 5 [17,18]. All the tested compounds could be maintained in the system at appropriate concentrations.
Intestinal absorption and Caco2 permeability are indicators that determine the ultimate bioavailability of the drug candidates. The tested compounds (apigenin, setin, kola avanone, and remdesivir) have relatively low Caco2 permeability potential (<8×10 -6 cm/s) and could be absorbed through the human intestine [19]. However, ADMETSAR1 as predicted that remdesivir is subcellular localization in the lysosome [20]. Furthermore, the observed lipophilicities have an association with Caco2 permeability but correlated negatively with water solubility potentials of the tested compounds. This result is in tandem with the ndings of Yazdanian et al [21] who used the human colon adenocarcinoma (Caco-2) cell line assay to document no correlation between the drug permeability and measured lipophilicity. All the tested compounds were predicted to be substrates of P-glycoprotein, a member of the ATP-binding cassette transporter and an e ux membrane transporter found chie y in epithelial cells. On the other hand, kola avanone and remdesivir were predicted as P-glycoprotein inhibitors. This indicate that they could modulate the normal physiological activities of P-glycoprotein including restricting the active uptake and the distribution of drugs [22].
The volume of distribution calculated using a steady-state volume of distribution (VDss) as predicted showed that kola avanone has the lowest theoretical dose required for uniform distribution in the plasma when compared with other tested compounds (apigenin, setin, and remdesivir). VDss showed the distribution of drug in the tissue and plasma. The degree of diffusing across plasma membrane increases in this order remdesivir <apigenin< setin < kola avanone measured as the fraction that is in the unbound state. The predicted evaluation on the nervous system distribution of the compounds revealed that lipophilicity of the compounds correlates to the degree of permeability across the central nervous system and the blood-brain barrier.
Cytochrome P450 is a group of enzymes that perform crucial functions in drug metabolism. They play a major role in the activation of drugs and also in the toxicity effects of the drugs. Only remdesivir is substrate of CYP3A4, all other tested compounds were neither substrate of CYP2D6 nor CYP3A4. The lipophilicity of the drug appears to correlate negatively to metabolism-related toxicity. Furthermore, only kola avanone was a substrate of renal organic cation transporter, this implies that other tested compounds are possibly cleared through other available routes such as sweat, bile, etc. Also, kola avanone was observed to have the least total clearance while apigenin has the highest. Drug clearance is related to bioavailability and is crucial for determining dosing rates to achieve steady-state concentrations.
The toxicological assessment of the tested compounds revealed that all the tested compounds expect remdesivir are not hepatoxic. Similarly, none of the compounds are skin sensitive (dermal toxic) or bacterial mutagenic potential drugs using the AMES toxicity examination. However, all the compounds showed high level of toxicity to Tetrahymena pyriformis toxicity test. Acute and chronic toxicity were also carried out on the tested compound to determine the safety of the compounds when administered. Exposure to low-moderate doses/concentrations of xenobiotics over long period of time is of signi cant concern in many treatment strategies or interventions. Chronic studies are designed to identify the lowest dose of a compound that can result in adverse effects (LOAEL), and the highest dose at which no adverse effects are observed (NOAEL). None of the compounds is an inhibitor to hERG I but kola avanone and remdesivir may be inhibitors to hERG II. Inhibition of the hERG potassium channel could result in delayed ventricular repolarisation leading to a severe disturbance in the normal cardiac rhythm and disrupt hepatic functions [23].

Conclusion
This study screened apigenin, setin, kola avanone, and remdesivir as potential molecules that could mitigate/inhibit SARS-COV2 using in silico methodology and guide in the development of effective therapeutics to combat COVID-19. Kola avanone was observed to exhibit the highest binding a nity with the SARS-COV2 major protease (6LU7). All the three tested phytochemicals (apigenin, setin, kola avanone) showed more binding a nity with 6LU7 than remdesivir, an established antiviral drug. Binding of these phytochemicals to SARS-COV2 could inhibit or interfere the pathogenesis of COVID-19 thereby preventing its cellular entry and proliferation. The pharmacodynamics and pharmacokinetics properties of the phytochemicals showed that they would be good drug candidates and the toxicological evaluations showed that these phytochemicals have relatively low or no toxic effect in human. However, further experimental and clinical studies are needed to further explore their activities and validate their e cacies against COVID-19.

Declarations
Funding This research was done using authors personal funds without speci c grant from any funding agency in the public, commercial, or not-for-pro t sectors.

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.     VDss: Steady-state volume of distribution, BBB: Blood-brain barrier, CNS: Central nervous system.