Exploring the Binding Interaction of Active Compound of Pineapple against Foodborne Bacteria and Novel Coronavirus (SARS-CoV-2) Based on Molecular Docking and Simulation Studies

Natural resources, particularly plants and microbes, are an excellent source of bioactive molecules. Bromelain, a complex enzyme mixture found in pineapples, has numerous pharmacological applications. In a search for therapeutic molecules, we conducted an in silico study on natural phyto-constituent bromelain, targeting pathogenic bacteria and viral proteases. Docking studies revealed that bromelain strongly bound to food-borne bacterial pathogens and SARS-CoV-2 virus targets, with a high binding energy of −9.37 kcal/mol. The binding interaction was mediated by the involvement of hydrogen bonds, and some hydrophobic interactions stabilized the complex and molecular dynamics. Simulation studies also indicated the stable binding between bromelain and SARS-CoV-2 protease as well as with bacterial targets which are essential for DNA and protein synthesis and are required to maintain the integrity of membranous proteins. From this in silico study, it is also concluded that bromelain could be an effective molecule to control foodborne pathogen toxicity and COVID-19. So, eating pineapple during an infection could help to interfere with the pathogen attaching and help prevent the virus from getting into the host cell. Further, research on the bromelain molecule could be helpful for the management of COVID-19 disease as well as other bacterial-mediated diseases. Thus, the antibacterial and anti-SARS-CoV-2 virus inhibitory potentials of bromelain could be helpful in the management of viral infections and subsequent bacterial infections in COVID-19 patients.


Introduction
Infectious diseases are one of the most dangerous burdens on human health because of the failure of first-line antibiotic-based chemotherapy. Moreover, an increase in resistance against prescribed antibiotics in many bacteria, including Pseudomonas aeruginosa (P. aeruginosa), Enterococcus faecium (E. faecium), Salmonella spp., Campylobacter, Streptococcus pneumonia (S. pneumonia), Neisseria gonorrhoeae (N. gonorrhoeae), etc., has been reported. Bromelain recovered from pineapple fruit is a type of protein-degrading enzyme. The protein bromelain purified from the stem is made in a different way and has a different   [14][15][16][17]. In order to prepare the downloaded 3D crystal structure for docking studies, we have removed the water molecules and HETATM from the published structures and CHARMm force field applied for energy minimization [12,13]. We also analyzed the active site of the pre-bounded ligand molecules in downloaded native structures and obtained the amino acid residue information available in the active site to implement docking analysis by Discovery Studio Visualizer 2020.

In Silico Interaction Analysis
MGL tools 1.5.6, AutoDock 4.2, The Scripps Research Institute, La Jolla, California, USA as used to predict the binding affinity between drug compounds and SARS-CoV-2 Protease and bacterial receptors. Lamarckian genetic algorithm (LGA) was used for interaction studies. Molecular docking methods follow the scoring function by probing the best conformation of receptor-ligand complex on the calculation of binding energy (∆G) by the following equation.
Further, water molecules were removed from the selected 3D native structures before docking, and hydrogen atoms, Gasteiger charge, Kollman united charges, and solvation parameters were added. The values of the Grid box were set to 60 × 60 × 60 • in the X, Y, and Z-axis of a grid point. For the default grid points, spacing was 0.375 Å. Lamarckian Genetic Algorithm (LGA) [21,22] was used for drug-protein molecules flexible docking analysis. The default LGA parameters such as population size 150 (gapop_size), energy evaluations 2,500,000 (ga_num_generation), mutation rate 27,000, crossover rate, 0.02 and step size were set to 0.8 and 0.2 Å. The LGA runs were set at 10 runs. After successful execution of docking steps, obtained conformations of receptor-ligand complexes were analyzed for the interactions and binding energy using Discovery Studio molecular visualization software  [14]. Similarly, the interactions of the bromelain peptide with bacterial pathogens S. aureus TyrRS (PDB:1JIJ), E. coli (PDB:1KZN), and Staph. aureus DHFR (PDB:3FYV) targets were tested to see whether it had antibacterial potential, which was observed as binding energy.

Molecular Dynamics Simulation (MDS) Experimentation
The Bromelain-Protease and S. aureus TyrRS-bromelain complexes docking results need to be further evaluate through an advanced computational technique. Therefore, an MDS environment was set to execute 100 nanosecond (ns) simulation for both complexes; also, we performed simulation for protease and S. aureus TyrRS simulation in water for results comparison using GROningen MAchine for Chemical Simulations (GROMACS) tool 2018 version [23,24] developed by University of Groningen, Netherlands.
pdb2gmx module was used to generate required protease (PDB:6LU7) and S. aureus TyrRS (PDB: 1JIJ) topology file followed by CHARMM27 all-atom force field selection. In the next step, ligand (bromelain) topology files were generated from Swiss Param server [24]. For the solvation step, a unit cell triclinic box filled with water was created. Addition of Na + and Cl − ions were completed for the stabilization of the system followed by energy minimization. The equilibrium setup of the system (protease-bromelain complex) was required and it was completed, followed by two-step ensembles NVT (constant number of particles, pressure, and temperature) and NPT (constant number of particles, pressure, and temperature). Both ensembles provide control over temperature, pressure coupling, resulting constancy, and stabilization of the system through complete simulation [25]. GORMACS contain several packages, for protease-bromelain complex MDS analysis, we used gmx rms for Root Mean Square Deviation (RMSD) [26], gmx rmsf for Root Mean Square fluctuation (RMSF), gmx gyrate for the calculation of Radius of Gyration (Rg) [22,27], and gmx hbond for the calculation of numbers of hydrogen-bond formed during interaction.

Results and Discussion
People who diagnose respiratory tract infections with viruses such as the flu are more likely to develop co-infections, which results in increased disease severity and more deaths. Many different antibiotics, including azithromycin, have been used for the prevention and treatment of bacterial co-infection and subsequent bacterial infections in COVID-19 patients. Antibiotics do not directly affect SARS-CoV-2, but bacterial pneumonia is frequently brought on by viral respiratory infections. Thus, this study was conducted to explore the antibacterial and SARS-CoV-2 antiviral significance of bromelain targeting Gram-positive and Gram-negative bacterial targets and SARS-CoV-2 viral protease [29].

S. aureus TyrRS-Bromelain Docking Analysis
We downloaded the crystal 3D structures of SARS-CoV-2 protease (PDB:6LU7) and bacterial receptors the crystal structure of S. aureus TyrRS in complex with SB-239629 (PDB:1JIJ), the crystal Structure of E. coli 24 kDa domain in complex with Clorobiocin (PDB:1KZN) and Staph. aureus DHFR complexed with NADPH and AR-102 (PDB:3FYV) from Protein Data Bank (PDB) (www.rcsb.org accessed on 5 March 2022). All selected biomolecules already have ligand molecules interacting with active sites. We have analyzed active site amino acid residues information involved in receptor-ligand interaction after visualization in the Discovery Studio Visualization tool. Furthermore, any bounded ligands and water molecules were edited and removed from the published 3D structures before molecular interaction experimentation. After obtaining the docking data, we have analyzed the active site amino acid residues participated in the interaction with the protease-bromelain complex and found that the selected compounds were docked at the same active sites.

S. aureus TyrRS-Bromelain Docking Analysis
We downloaded the crystal 3D structures of SARS-CoV-2 protease (PDB:6LU7) and bacterial receptors the crystal structure of S. aureus TyrRS in complex with SB-239629 (PDB:1JIJ), the crystal Structure of E. coli 24 kDa domain in complex with Clorobiocin (PDB:1KZN) and Staph. aureus DHFR complexed with NADPH and AR-102 (PDB:3FYV) from Protein Data Bank (PDB) (www.rcsb.org accessed on 5 March 2022). All selected biomolecules already have ligand molecules interacting with active sites. We have analyzed active site amino acid residues information involved in receptor-ligand interaction after visualization in the Discovery Studio Visualization tool. Furthermore, any bounded ligands and water molecules were edited and removed from the published 3D structures before molecular interaction experimentation. After obtaining the docking data, we have analyzed the active site amino acid residues participated in the interaction with the protease-bromelain complex and found that the selected compounds were docked at the same active sites.
Tyrosyl-tRNA synthetases (TyrRSs) are ideal cellular sites for therapeutic targets in the management and cure of pathogen attack since they are necessary enzymes as most of the cellular system. Another target for bacteria is dihydrofolate reductase. S. aureus was targeted for control via TyrRS and E. coli was tested to inhibit via dihydrofolate reductase, a key enzyme which catalyses the synthesis of nucleic acid for microbial cells. The docking results of this study showed that bromelain interacted more strongly with Gram-positive bacterial pathogens than with Gram-negative bacterial pathogens.
Plant protease inhibitors in therapeutics focus on cancer therapy. Previous research has also been reported to inhibit the E. coli and L. monocytogenes at the concentration of 4 mg/mL which was much more believed to be a significant therapeutic molecule. For significant therapeutic molecules, a molecule potentially inhibits at the lowest conc. Thus, for dose reduction, the researcher reported a cysteine-rich protease from Bromelia karatas L. that was isolated, characterized by (LC-MS/MS) and reported to inhibit up to 85% of foodborne bacterial pathogens S. Typhimurium and L. monocytogenes at a concentration of 10 µg/mL [33,34]. The proteinaceous molecule has differential results against Grampositive and Gram-negative bacterial pathogens. The binding energy was observed to be more negative with Gram-positive bacterial pathogens as compared to the Gram-negative bacterial pathogens.
Further, the Gram-positive bacterial pathogens were significantly inhibited at low doses as compared to the Gram-negative ones. S. Typhimurium, a Gram-negative bacterial pathogen, inhibited at a conc. of 3.0 mg/mL, while Gram-positive L. monocytogenes inhibited at the conc of 1.65 mg/mL, supporting the results of conducted studies. Gram-negative bacteria's external membrane may be resistant to streptococcal pyrogenic exotoxin A (SPEA), whereas Gram-positive bacteria have a peptidoglycan cell wall. The antiviral potential of the molecules was also checked against SARS-CoV-2 protease.

S. aureus TyrRS -Bromelain Simulation Analysis
We have also simulated S. aureus target in water and it complexes with bromelain for 100 ns.

Protease-Bromelain Docking Analysis
In the search for an effective therapeutic molecule against COVID-19, docking studies with selected molecules with the SARS-CoV-2 virus protease PDB: 6LU7 revealed that bromelain strongly binds to the protease and it can inhibit the virus' entry into the host cell. The docking results analyzed in this study are represented in Figure 4a,b and Table  3's results. The observed binding energy for bromelain with protease was −9.37 kcal/mol. The interacting amino acids were THR25, HIS41, SER46, MET49, PHE140, LEU141, ASN142, GLY143, SER144, CYS145, HIS163, HIS164, MET165, GLU166, EU167, PRO168, GLN189. The observed vdW + Hbond + desolv Energy −8.85 kcal/mol, Inhibition Constant was 15.46 uM and total ten hydrogen bonds formed during SARS-CoV-2 Protease-Bromelain interaction. The analyzed docking features are represented in Figure 4, Table 3.

Protease-Bromelain Docking Analysis
In the search for an effective therapeutic molecule against COVID-19, docking studies with selected molecules with the SARS-CoV-2 virus protease PDB: 6LU7 revealed that bromelain strongly binds to the protease and it can inhibit the virus' entry into the host cell. The docking results analyzed in this study are represented in Figure 4a,b and Table 3's results. The observed binding energy for bromelain with protease was −9.37 kcal/mol. The interacting amino acids were THR25, HIS41, SER46, MET49, PHE140, LEU141, ASN142, GLY143, SER144, CYS145, HIS163, HIS164, MET165, GLU166, EU167, PRO168, GLN189. The observed vdW + Hbond + desolv Energy −8.85 kcal/mol, Inhibition Constant was 15.46 uM and total ten hydrogen bonds formed during SARS-CoV-2 Protease-Bromelain interaction. The analyzed docking features are represented in Figure 4, Table 3.   From recently conducted studies, hydroxyl chloroquine and hydroxychloroquine represented a choice for relief but not for a cure, and were also described as having less  From recently conducted studies, hydroxyl chloroquine and hydroxychloroquine represented a choice for relief but not for a cure, and were also described as having less binding energy (−5.1; −5.7 kcal/mol), but this was more than the binding energy of antiviral drugs Oseltamivir, Ritonavir, and Favipiravir [35,36]. Thus, we selected another antimalarial drug, Artemisinin, to screen its inhibitory potential and found it to have −6.94 kcal/mol binding energy with 8.19 uM and formed three hydrogen bonds using amino acids of SARS-CoV-2 protease HIS163, GLU166 and MET165 and amino acid residues HIS41, PHE140, LEU141, ASN142, GLY143, SER144, CYS145, HIS, HIS163, HIS164, MET165, GLU166, HIS172, GLN189 were involved in hydrophobic interaction (Table 3). It can also be stated that this antimalarial drug could also be potentially inhibited by SARS-CoV-2 protease and could be used in the same way as hydroxychloroquine and remdesivir.

Protease-Bromelain Simulation Analysis
The docking interaction of bromelain was found to be more significant with viral protease (PDB:6LU7). Thus, dynamic simulation studies were conducted only for this target of virus. After the successful run of 100 ns dynamics simulation, analyses were accomplished on the basis of obtained data from RMSD, RMSF, Rg, and the formation of a number of hydrogen bond plots analysis. The deviation and fluctuation of the proteasebromelain complex during the whole simulation period are revealed. The observed results are shown in Figure 5. The average RMSD values observed were between 0.2 and 0.3 nm, while protease simulation in water had RMSD values between 0.15-0.2 nm. Protease molecules remained stable during the whole simulation while, from the starting proteasebromelain complex, they showed little fluctuation until 40 ns. Afterward, they were stable until 100 ns, though a small fluctuation was observed at 25-35 ns (Figure 5a). The RMSF calculation per residue shows values between 0.1-0.20 nm (Figure 5b) for protease and the protease-bromelain complex. Few fluctuations were observed at 10-20, 50-80, 150-200, and 260-280 amino acid residue regions. Amino acid residues present in these regions are also found in the formation of hydrogen bonds in hydrophobic interactions during docking analysis (Figure 4b). The hydrogen bond plot showed the formation of 1-7 hydrogen protease-bromelain bonds during the 1000 ns period (Figure 5c). The radius of gyration analysis is very important for the assessment of the compactness and stability of protein structures during the whole simulation period, due to the presence of ligand molecules. The observed average value of Rg was between 2.2-2.25 nm for the protease in water simulation, while for the protease-bromelain complex it was between 2.1-2.15 nm. (Figure 5d). Overall, Rg analysis indicated that the complex fluctuated at 20,000-250,000 ps.
The adsorption, distribution, metabolism and excretion profiles were estimated. The study drug was not found to absorb through intestine and Skin permeability was −2.735. However, water solubility was observed to be −2.87 and blood-brain barrier crossing capacity and CNS permeability values were −2.889. It was safe for liver enzymes CYP3A4, CYP 1A2, CYP 2C19, CYP 2C9, CYP 2D6, and CYP 3A4. The total clearance value was observed to be 1.686 (Table 4). The obtained data from the pkCSM server (http://biosig. unimelb.edu.au/pkcsm/theory accessed on 23 May 2022) revealed that bromelain has no hepatotoxicity, AMES toxicity, and skin sensitisation properties.  "Let food be thy medicine and medicine be thy food," Hippocrates advised almost 2500 years ago. Nutritional status is influenced by both nutrient consumption and disease occurrence. A well-balanced diet will help you maintain a robust immune system that will help you resist against microbial attacks including viruses. Much research has suggested that strengthening the immune system is the only long-term way to live in the current environment. It is critical to have enough zinc, iron, and vitamins A, B 12, B6, C, and E to keep your immune system in good shape. Except for Vitamin C, there is currently no evidence that any supplement may 'boost' our immune system or treat or prevent viral infections. Vitamin C is one of the most important water-soluble vitamins for maintaining a healthy immune system. Vitamin C has a daily-recommended dietary requirement of 90 milligrams per day for men and 75 milligrams per day for women. In order to tackle COVID-19, it is vital to be aware of the precise forms of food that can strengthen our immune systems [7,8]. Dietary guidelines suggest that fruits be supplemented in diets, such as apples, guavas, bananas, cantaloupe melons, strawberries, grapefruit, papayas, pineapples, lemons, oranges, blackcurrants, etc., during the COVID-19 pandemic. Thus, this evidence suggests that fruits and vegetables have many therapeutic molecules like alkaloids, glycosides, phytosterol and many more that have been used to control many microbial and infectious diseases. Thus, pineapple rich in bromelain has been reported to have many pharmacological activities and it could also be helpful in maintaining the good health of COVID-19 patients [37][38][39][40]. The enzyme has been reported to have potential pharmacological activities including antimicrobial, antioxidant, anti-inflammatory, and anticancer activity. It has good intestinal absorption stability at a wide range of pH (4-9), maintains its maximum concentration after 1 h of its administration and maintains its biological activity with a half-life of 6-9 h, 1 h after administration [40]. Moreover, it is believed that the enzyme complex bromelain also has mucolytic properties and could be beneficial to control respiratory inflammation, including inflammation caused by influenza and asthma allergies [1,7]. It is also thought to have properties that help break up and expel mucus. Many pathological microbial diseases are connected to the involvement of protease in infection of organs such as the mouth, skin, lungs, ears, eyes, nose, and other soft tissues and cavities [10,11]. A virus uses protease to facilitate entry into the host cell and break promising proteins used to pack the new virus particle. Thus, the study reported that bromelain interacted with both target bacteria and viruses significantly, but the observed binding energy of bromelain was more significant as compared to protease, indicating that the significant these interactions with both targets could be helpful to manage bacterial and viral infections [40,41]. Indeed, previously conducted in silico-based studies with the other molecules only reported either antibacterial or antiviral potentialities. In this study, the bromelain molecule has been evaluated for both its antibacterial and antiviral activities. The binding energies of this molecule observed with targets were observed to be more significant as compared to many repurposing drugs and naturally derived molecules. The significant stable interaction with the cellular system has been observed with simulation studies of the molecule, which supports the more significant antimicrobial potentialities of this molecule. until 100 ns, though a small fluctuation was observed at 25-35 ns (Figure 5a). The RMSF calculation per residue shows values between 0.1-0.20 nm (Figure 5b) for protease and the protease-bromelain complex. Few fluctuations were observed at 10-20, 50-80, 150-200, and 260-280 amino acid residue regions. Amino acid residues present in these regions are also found in the formation of hydrogen bonds in hydrophobic interactions during docking analysis (Figure 4b). The hydrogen bond plot showed the formation of 1-7 hydrogen protease-bromelain bonds during the 1000 ns period (Figure 5c). The radius of gyration analysis is very important for the assessment of the compactness and stability of protein structures during the whole simulation period, due to the presence of ligand molecules. The observed average value of Rg was between 2.2-2.25 nm for the protease in water simulation, while for the protease-bromelain complex it was between 2.1-2.15 nm. (Figure 5d). Overall, Rg analysis indicated that the complex fluctuated at 20,000-250,000 ps. The adsorption, distribution, metabolism and excretion profiles were estimated. The study drug was not found to absorb through intestine and Skin permeability was −2.735. However, water solubility was observed to be −2.87 and blood-brain barrier crossing capacity and CNS permeability values were −2.889. It was safe for liver enzymes CYP3A4, CYP 1A2, CYP 2C19, CYP 2C9, CYP 2D6, and CYP 3A4. The total clearance value was observed to be 1.686 (Table 4). The obtained data from the pkCSM server (http://biosig.unimelb.edu.au/pkcsm/theory) revealed that bromelain has no hepatotoxicity, AMES toxicity, and skin sensitisation properties.

Conclusions
Pineapple has bromelain, which has many significant clinical benefits in cancer, diabetes, cardiovascular and lung diseases. The analyzed results of bioactive compounds of bromelain from pineapple show that it has the capability to interact with the viral enzyme Main Protease (Mpro) of COVID-19. Bromelain has shown the best binding energy score of the other studied antibiotics against the main protease enzyme of SARS-CoV-2. The docking score of different antibiotics such as tobramycin, ceftriaxone, piperacillin, and penicillin reported previously have inhibitory potential lower than bromelain, which further confirms its significance. Bromelain may have antiviral activities against SARS-CoV-2. It has been previously proven that bromelain is well absorbed in the body after oral administration and has no major side effects, even after prolonged use. Thus, this study suggests that pineapple bromelain could be used as an effective health supplement to control COVID-19 or to synergize the therapeutic effect of other molecules; however, the mechanism of its action has not been well explored until now. Thus, future in vitro and in vivo research can concentrate on investigating the mechanistic therapeutic intervention.