Molecular docking study of copaíba oil interacting with the spike protein of Sars-CoV-2

COVID-19 triggered by Sars-CoV-2 has caused hundreds of thousands of deaths worldwide. Organic and inorganic compounds have been tested as potential in-1 hibitors of this lethal virus. For these tests, several techniques are use to design molecules of biological interest for drug composition, in which molecular coupling plays an important role. In the present work, the compounds acids kaurenoic, copalic, and beta-caryophyllene that form the copaiba oil were studied as anti-inﬂammatories and opens the possibility to inhibit Sars-CoV-2. Molecular docking showed alkyl, pi-alkyl, conventional H-bond, unfavorable bump, and Van der Waals interactions. The calculated electrostatic potential maps showed the nucleophilic and electrophilic regions. The negative binding energies obtained for the three acids suggest the stability of the complexes. The minimum energy states for β -caryophyllene are lower than the other compounds analyzed, and it can be predicted that this is the most stable.


Introduction
The first Coronavirus was discovered in the 1930s [1], but severe acute respiratory syndrome (Sars) gained notoriety in the world between the years 2002-2003 [2]. At the end of 2019, Sars-CoV-2 triggered a pandemic that has already caused nearly 3.5 million deaths worldwide [3].
From serological analyzes and genetic studies, it it is possible to classify the coronaviruses into four different genera: α, β, γ and β-CoV [4]. β-CoV type is the cause of the COVID-19 found in Wuhan, China [5]. The Coronaviruses are enveloped, spherical, or pleomorphic viruses and can vary their shape according to the period of the reproductive life cycle or environmental conditions, with typical sizes ranging from 80 to 120 nm [4]. The coronavirus spike protein is a multifunctional molecular machine that initially binds to a receptor on the surface of the host cell through its S1 subunit and then fuses viral and host membranes through its S2 subunit, leading to viral binding [6].
We are investigating the inhibitory potential of Copaiba oil against the COVID-19 virus, and we associated the oil compounds with the binding of the Sars-COV-2 spike protein receptor as a target using molecular docking for these calculations. Molecular docking has been used by pharmaceutical companies to study the Structure-Activity Relationship of drugs for more than three decades. New drugs have been discovered and developed during these years [7].
This method gives the possibility to predict both the binding affinity between ligand and protein and the structure of the protein-ligand complex using the computational method, which is relevant information for optimization [8,9]. Molecular docking techniques aim to predict the best matching binding mode of a ligand to a macromolecular partner. It consists of the generation of several possible conformations/orientations [10]. Docking is a technique of designing drug molecules by simulating the geometry of these molecules and their intermolecular forces [3]. In this way, molecular docking optimizes the ligand bound to the active site of the receptor protein and investigates protein-ligand interactions. Molecular coupling algorithms provide results for quantitative energy binding markers, including a variety of coupled compounds supported by the binding affinity of ligand-receptor complexes with pharmacokinetic properties. From this calculation, we can predict the different interactions between the oil compounds and the spike protein. Ligand-protein interactions are involved in many biological processes with consequent pharmaceutical industry implications [10].
Several studies have been done on Copaiba oils, mainly due to the interest from the pharmacological and food industries around the globe [11]. The Brazilian Amazon concentrates a large number of natural resources species with various therapeutic applications in alternative medicine. Copaiba oil is an oleoresin extracted from the trunk of trees of the Copaifera genus (Fabaceae). Frequently, copaiba grows in tropical regions of South America. The oil-resin is a natural product of the Amazon's biodiversity. Copaiba oil is commonly used in folk medicine to treat multiple diseases, such as ulcers, wounds, syphilis, bronchitis, and inflammation [12].
Besides these properties, some studies suggest that these species are free from toxicity and teratogenic activity during pregnancy [16]. Also, the copaiba oil can be used as an anti-tumor, anti-inflammatory, antimicrobial against a wide range of microorganisms, and healing on different tissues of the human body.
In several animal models, studies demonstrated that copaiba oil has healing and anti-inflammatory effects. Besides, anti-inflammatory and anti-tumor properties of copaiba oils have been described in several works [17][18][19][20].Those results are significant because they can the designed as applications to improve the quality of life in a society.

Computational details
Initially, the constituent compounds of the copaiba oil were accessed in the ChemSpider database. All compounds were subjected to a classical simulation to find the lowest energy geometries, based on the lamarckian genetic algorithm (LGA) algorithm, using the Forcite Code [21,22].
The universal force field (UFF) was selected to perform the calculations. After obtaining the best conformation of the geometries, the structures were submitted to a new optimization at the DFT level using the DMOL3 Code [23,24], where the generalized gradient approximation (GGA) considers all the electrons of the molecules.
Molecular electrostatic potential surfaces were investigated to identify the most reactive nucleophilic and electrophilic regions. We performed molecular docking with the ArgusLab 4.0.1 program. There are two options for docking algorithms, the first one is GA dock (Genetic Algorithm), and the other one is Argusdock (Shape-Based Search Algorithm).
The calculations are from GAdock docking algorithms, which take into account the Lamarckian Genetic Algorithm. The docking location was defined using a box with coordinates 47.25 × 36.00 × 49.75Å, spacing of 0.400Å, and flexible binder coupling mode. Figures 1A, B and C show the optimized structures and the numbering of the atoms forming the acids kaurenoic, copalic, and β-caryophyllene. The H bond represents an interaction between two electronegative atoms. Table 1 illustrates their total energies (ET), binding energy (BE) and maximum cartesian force (MCF) of these acids. The global minimum energies are found to be -922.5966100 a.u (−25105 eV), −924.0927850 a.u (-25145 eV), -580.6642644 (-15800 eV) for kaurenoic, copalic and β-caryophyllene, respectively. The binding energy of the protein spike with kaurenoic acid was −10.56 kcal/mol, with copalic it was −10.77 kcal/mol, and with β-caryophyllene, it was −10.96 kcal/mol. Negative values of binding energies suggest the stability of the complexes.

Optimization and electrostatic interaction
Their maximum Cartesian forces are found to be 0.139052×10 −2 , 0.495639× 10 −3 and 0.196084×10 −2 , as can be seen in table 1. The addition of other atoms in the geometry of compounds influences their stability. We can notice in table 1 that the β-caryophyllene compound is the most stable because the globalminimum energy is the smallest compared to the other acids ones. Fig. 2 shows the maps of molecular electrostatic potential (MEPs) of the copaiba oil-forming acid molecules. The MEP is a tool used to describe the most reactive nucleophilic and electrophilic regions of a molecule against reactive biological potentials and intermolecular interactions [25,26]. The electrophilic site indicates strong attraction and the nucleophilic site indicates strong repulsion. In these regions, the formation of hydrogen bonds occurs. MEPs provide regions of negative, positive and neutral electrostatic potential in terms of color grading and are an indicator in researching molecular structure properties. Atoms in red represent the most electronegative electrostatic potential; atoms in this region tend to attract electrons (electrophilic). Atoms in blue indicates the most electropositive potential atoms in this region tend to repel electrons (nucleophilic). In yellow we can see the forming acids of the copaiba oil (binders). As a result, the surfaces of the MEPs range from -0.100 a.u (deepest red) to 0.100 a.u (deepest blue) for the three compounds. Figures 3A, B, and C show the molecular docking of kaurenoic, copalic, and β-caryophyllene acids interacting with the spike protein of Sars-CoV-2. The purpose of docking is determine the modes of interaction of ligands (copaiba oil-forming acids) while organizing favorable orientations for the binding of a ligand to a receptor [27][28][29][30][31].

Molecular docking and 2D visual representations
The receptor represents the COVID-19 protein that has one or more specific active sites. In this work, before coupling, all native ligands and water molecules were removed from the protein structure. In addition, polar hydrogen atoms are added and Kollman atom charges are assigned to protein atoms. At each step of the calculation, the interactions are affected, and the best orientation of ligands was determined to investigate the different types of interactions between the copaiba oil-forming compounds and the protein. In copalic acid, alkyl interactions were observed surrounded by amino acids HIS:505 and PHE:504 with 1.51632Å and 1.53555Å, respectively. Conventional H-bond interactions are surrounded by amino acids ARG:273 and TYR:515 with 1.53098Å and 1.36785Å, respectively. Van der Waals interactions are surrounded by amino acids ASN:508, SER:128, and TRP:271 with 1.53108Å, 1.52889Å, and 1.54616Å, respectively.

Conclusion
Considering the anti-inflammatory properties of copaiba oil, we studied three compounds that form the oil. We performed the classical optimizations calculations to obtain the most stable geometric conformation, using the conjugate gradient (LGA) and quantum gradient (GGA). The total and binding energies obtained for the three compounds were negative, which shows that the investigated complexes are stable. The β-caryophyllene is the most stable of the compounds, as its total energy was the lowest.
The calculated MEPs showed that regions with positive potentials are favorable to nucleophilic attack, while those regions with negative potentials are favorable to electrophilic attack. The results of molecular docking were discussed based on different interactions between acids (ligands) and proteins (receptors).
From the results obtained, it can be inferred that the acids that form the copaiba oil can be used as an inhibitor of COVID-19. These results encourage further in vitro and in vivo investigations into the pharmacological properties of copaiba oil.

Acknowledgements
The financial support for this research by CAPES (Coordenacão de Aperfeicoamento de Pessoal de Ensino Superior), is gratefully acknowledged W. O. Santos and J. R. da C. Venâncio were supported by a studentship from CAPES.

Declarations
Funding The research developed in this article had no financial support. Conflict of interest/ Competing interests Authors declare any not has financial and personal relationships with other people or organizations that could inappropriately influence in this work. Availability of data and material The article files will be available upon request. Code availability Not applicable. Ethical approval Not applicable. The ethical standards have been met. Authors' contributions Data collection, and analysis were performed by WO Santos, ALF Novais and ERP Novais. The first draft of the manuscript was written by WO Santos, ALF Novais, GC A Oliveira, JRC Venâcio and AM Rodrigues and all authors commented on previous versions of the manuscript. All authors contributed to the study conception and design and approved the final manuscript.
Consent to participate Authors consent to participate in the research project, and they assure this research may not bring commercial benefit to us. Our participation is completely voluntary. Consent for publication The authors declare that they agree with the submission and eventual publication..