Introduction. Bacterial biofilm is the colonization of one or more species of bacteria together onto a specific substratum. The adhesion of these bacterial cells is brought up by the extracellular polymeric substances that form matrices favoring high resistance to the embedded microorganisms against adverse conditions [1]. Most of the pathogenic bacteria like Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus viridans and Vibrio cholerae have been found to form biofilms within the host body and in the environment as well [2]. Also, it is quite difficult to treat bacterial infections that have formed biofilm previously.

Vibrio cholerae, a gram negative, comma shaped and motile bacterium has the tendency to easily colonize on marine bodies [3, 4]. It can cause a serious disease called ‘cholera’ in humans when contaminated water and food sources are ingested. Bap1 (Biofilm associated protein 1) protein is considered to be the major factor for biofilm formation in V. cholerae [5]. This protein is abundantly expressed during biofilm formation, enabling adhesion of bacterial cells to the host surface. It has also been reported that the Bap1 protein plays a structural rather than functional role in V. cholerae [5]. The continual use of antibiotics to treat infections may lead to multi drug resistance in bacteria. Hence, there is a need for screening antibiofilm agents that can inhibit further development of biofilm which are naturally available in plants and marine organisms in addition to the synthetic antimicrobial drugs.

The implementation of computational procedures is becoming widely adopted by the researchers for drug discovery, analyzing the protein structures and predicting the stability of mutant proteins from that of the respective wild types [6, 7]. Here, the authors have performed molecular docking techniques in order to screen potential compounds that inhibited the Bap1 protein of V. cholerae as a measure of controlling biofilm production by the bacterium. The docked compounds were also evaluated for druglikeness to treat the disease by reducing the risk of biofilm formation in humans. Along with this, Bap1 protein structure was mutated at several positions that interact more with the test ligand molecules and the mutant protein stability was calculated.

The aim of the study. To target this Bap1 structural protein for screening potent inhibitors of ligands by means of in silico analyses.

Materials and Methods

Protein preparation

The structure of the protein was retrieved from the RCSB-PDB (The Research Collaboratory for Structural Bioinformatics- Protein Data Bank) database. The Bap1 protein with PDB Id: 6MLT bearing the title “Crystal structure of the V. cholerae biofilm matrix protein Bap1” was used as the protein of interest in this study [8]. The protein molecule was retrieved in the ‘.pdb’ format. All the non-standard amino acid residues and water molecules were removed from the Bap1 protein structure and saved the file in the same format using the Chimera software. 

Ligand preparation

A total of 30 compounds grouped into three different groups namely synthetic antimicrobial drugs, phytochemicals [9] and marine compounds which are listed in Table 1 were tested for the antibiofilm activity of V. cholerae bacterium by interacting with the Bap1 protein. The three-dimensional structures of the ligands were retrieved from ChemSpider and PubChem databases in ‘.mol’ and ‘.sdf’ formats respectively. The ligands were also screened for druggability based on Lipinski rule of five simultaneously.

Molecular docking

The retrieved synthetic antimicrobial drugs, phytochemicals and marine compounds were docked against the Bap1 protein structure. The molecular docking was carried out using Chimera software [10] through the AutoDock vina software [11]. The grid parameters were set as x=15.0465, y=30.9405 and z=115.82 and the size x=65.3508, y=58.319 and z=80.6473 for the interaction to be checked. The AutoDock tool provides the results in kcal/mol based on hydrogen bonding, electrostatic energies, desolvation and van der Waals force [10]. The binding poses with maximum binding affinities and lowest binding energies were chosen for all the compounds.

ADME calculation

The Absorption, Distribution, Metabolism and Excretion (ADME) properties for the selected compounds were calculated using SwissADME [12] online server by uploading the canonical SMILES (Simplified Molecular Input Line Entry System) format of the top five ligand molecules from each group (synthetic antimicrobial drugs, phytochemicals and marine compounds). The ADME properties define the druglikeness of the chosen ligand compounds.

Analysis of protein-ligand interactions

Similarly, the ligand molecules interacting with the amino acid residues were determined for the topmost compound showing highest binding affinity from each group by means of Biovia Discovery Studio Visualiser. The hydrogen bonding, hydrophobic interaction, π- interactions and covalent bonds can be visualized between the interacting molecules of the protein.

Protein stability prediction

The stability of the Bap1 protein was predicted by substituting the amino acid residues which frequently took part in forming hydrogen bonds among the best ligand compounds using CUPSAT server [13]. The amino acids were mutated with nineteen other amino acids in the respective positions and the mutant stability was estimated.

Results and Discussion

Molecular docking

The results for molecular docking of all the synthetic antimicrobial drugs, phytochemicals and marine compounds with the Bap1 protein are tabulated in Table 2. In synthetic antimicrobial drugs group, the drug doxycycline shows high binding affinity of -8.5 kcal/mol and the drug with least affinity is metronidazole with an affinity of -5.1 kcal/mol. In the phytochemicals group, ichangin has the maximum binding affinity of about -9.3 kcal/mol and the phytochemical with least affinity was found to be ajoene (-4.1 kcal/mol). Similarly, the compound ageloxime D shows high affinity of -8.8 kcal/mol and butenolide has the minimum binding affinity of about -4.0 kcal/mol in the marine compounds group. Comparatively, natural compounds show greater affinity towards the antibiofilm capability than synthetic chemicals. The drug, doxycycline has shown binding affinity of about -9.8 kcal/mol against ACE2 receptor to prevent the viral entry into the host cells [14]. Amoxicillin showed binding energy of about -7.0 kcal/mol towards Staphylococcus aureus Pyruvate Kinase targeted as a measure of antimicrobial activity [15]. Curcumin binds to the S. aureus biofilm forming protein with an affinity of -6.33 kcal/mol [16]. The marine metabolite hymenidin has shown inhibition of the SARS- CoV2 protease with a binding energy of -6.4 kcal/mol [17]. Likewise, a limonoid phytocompound, ichangin could bind with the same COVID-19 protease exhibiting a binding affinity of about -8.4 kcal/mol docked as a measure of repurposing the plant chemical as antiviral agent [18]. Natural compounds are more capable of inhibiting the biofilm matrix protein due to their efficient quorum sensing (QS) blocking mechanism comparatively higher than synthetic constituents [19]. Further, natural compounds are the best treatment options for bacteria that have developed antibiotic resistance [20].

ADME calculation

The ADME calculation shows that all the top five compounds with maximum binding affinities from each group tend to exhibit druglikeness with no or less violations for a few compounds. The results are given in Table 3.  It can be suggested that almost all the test compounds screened for ADME estimation can be considered for preclinical trials except chelerythrine and polygodial which tend to cross the brain-blood barrier. Both doxycycline and amoxicillin are generally less absorbed in the gastrointestinal tract and hence, the route of administration shall be changed during treatment procedures. The marine compounds screened for ADME estimation did show less solubility in water except hymenidin as predicted by the SwissADME online tool. It has been shown that ichangin can inhibit the biofilms in E. coli at an Inhibitory Concentration (IC₂₅) of 28.3 µM [21]. Ichangin can be used against SARS- CoV2 virus after approving it’s ADME properties [22]. Manoalide exerted anti-inflammatory effects and hence can be administered as a drug considering its druglikeness [23]. Ageloxime D was able to possibly inhibit Cryptococcusneoformans with IC₅₀ value of nearly 5.94 µg/mL proving its ability to act as a drug against bacteria [24]. The natural compounds may be administered in combinations with several antimicrobials which can increase the interactions with their specific targets inside the pathogens [25]. These combinations can reduce the toxicity and minimum inhibitory concentrations of many antibiotics as well to treat severe bacterial infections [26]. The previously developed fatal bacterial biofilms will not get easily vanished since, bacteria are protected within the matrix. In such cases, synergistic combinations of natural antibiofilm compounds can inhibit biofilms therefore, exposing the embedded bacteria to the synthetic antimicrobials that contain bactericidal activities and get killed [27].

Protein-ligand interactions

The interaction between the Bap1 matrix protein of Vibrio cholerae and the compounds- doxycycline, ichangin and ageloxime D are depicted in Figures 1, 2 and 3 respectively. Doxycycline forms five conventional hydrogen bonds with GLN:518, HIS: 520, THR: 677, ASN: 679 and LEU: 690 residues of the protein. In addition to this, ASN: 249, SER: 302, SER: 310, ALA: 311, ASN: 336 ASP: 517, TRP: 675 and HIS: 678 promote van der Waals interactions with this compound. Similar to this interaction, the phytochemical ichangin also forms five conventional hydrogen bonds with ASN: 249, THR: 312, GLN: 518, HIS: 520 and ASN: 679 residues. SER: 302, SER: 310, ASP: 517, TRP: 675, LEU: 676, THR: 677, PRO: 689 and LEU: 690 support van der Waals interaction. Along with this, ichangin forms two carbon-hydrogen bonds with ALA: 311 and HIS: 678 and one π-alkyl interaction with PRO: 307 amino acid. Likewise, the marine compound ageloxime D, exhibiting the highest affinity in the group, comparatively forms only three conventional hydrogen bonds (one with HIS: 678 and two bonds with ARG: 687). The van der Waals’ attractions are also less with this compound. But the π- interactions are remarkably more in ageloxime D involving ARG: 649, PHE: 658, ALA: 663, ALA: 671, ILE: 686 and PRO: 689 (totally six interactions) amino acid residues of the Bap1 protein. In a literature, it is reported that doxycycline was able to form six hydrogen bonds with the main protease of COVID-19 [28]. Ichangin being a limonoid could interact with HIS: 41, GLU: 166 and GLN: 189 residues of the same protein forming three hydrogen bonds [18].

Protein stability prediction

The amino acids that frequently took part in conventional hydrogen bond formation among doxycycline, ichangin and ageloxime D were found to be GLN: 518, HIS: 520 and ASN: 679. Hence, these three amino acids were substituted with nineteen other amino acid residues at the positions 518, 520 and 679 to determine the mutant protein stability which is tabulated in Table 4. Based on the protein stability prediction of Bap1 protein, it can be inferred that destabilization of the protein structure can occur if glutamic acid (∆∆G= -0.39 kcal/mol) gets substituted at the 518^th position. Similarly, valine, leucine and isoleucine are the only three residues which are stabilizing amino acids at the 520^th position. The destabilizing residues at the 679^th position are glycine, proline, tryptophan, threonine, glutamine, tyrosine, cysteine and aspartic acid when ASN: 679 gets substituted due to mutations according to the predictions. It is reported that nearly, ten residues favor instability at the desired positions of mutant CYP3A4 protein using the same server as used in this study [29]. The non-synonymous mutation in the helicase was determined to be ∆∆G= 0.377 kcal/mol by shifting from histidine to tyrosine (H39Y) [30].

Conclusion. The in silico results suggest that targeting the major biofilm forming protein, Bap1 in Vibrio cholerae can potentially inhibit the production of biofilm among the bacterium. Other than synthetic antimicrobial drugs, phytochemicals and marine compounds can also be used as antibiofilm agents [16]. Molecular docking, ADME calculation and protein-ligand interaction analyses of the compounds exhibiting highest binding affinity from each group of ligands used in this study infer that doxycycline, ichangin and Ageloxime D are potent inhibitors of the Bap1 protein. On the whole, natural compounds are capable of inhibiting the biofilm matrix protein than synthetic constituents. These compounds may be administered in combinations with the antimicrobials in order to treat severe infections of the bacterium which may be concluded by performing preclinical experiments before the trials. Also, the use of ichangin and Ageloxime D can work better in the environment as well by preventing the colonization of V. cholerae on land and water bodies too [9]. Thus, screening, production, isolation and purification of natural drugs from plant and marine organisms will have promising effects in the treatment and early management of diseases [31]. Further, the mutant Bap1 protein stability was assessed by inducing mutations at the most interactive amino acid positions. The Bap1 mutants are found to have reduced structural stabilizations which may show deformities during biofilm formation. It could also lead to malformations in the attachment of bacterial colonies onto the desired substrata so that the mutant V. cholerae may not get protected from adverse conditions allowing the bacteria to be easily killed when exposed to physical or chemical agents [27].

Financial support

No financial support has been provided for this work.