Identification of Potential Therapeutics of Mentha Essential Oil Content as Antibacterial MDR Agents against AcrAB-TolC Multidrug Efflux Pump from Escherichia coli: An In Silico Exploration

Multidrug-resistant bacterial pathogens, such as E. coli, represent a major human health threat. Due to the critical need to overcome this dilemma, since the drug efflux pump has a vital function in the evolution of antimicrobial resistance in bacteria, we have investigated the potential of Mentha essential oil major constituents (1–19) as antimicrobial agents via their ability to inhibit pathogenic DNA gyrase and, in addition, their potential inhibition of the E. coli AcrB-TolC efflux pump, a potential target to inhibit MDR pathogens. The ligand docking approach was conducted to analyze the binding interactions of Mentha EO constituents with the target receptors. The obtained results proved their antimicrobial activity through the inhibition of DNA gyrase (1kzn) with binding affinity ΔG values between −4.94 and −6.49 kcal/mol. Moreover, Mentha EO constituents demonstrated their activity against MDR E. coli by their ability to inhibit AcrB-TolC (4dx7) with ΔG values ranging between −4.69 and −6.39 kcal/mol. The antimicrobial and MDR activity of Mentha EOs was supported via hydrogen bonding and hydrophobic interactions with the key amino acid residues at the binding site of the active pocket of the targeted receptors.


Introduction
Worldwide, we are noticing an outstanding increase in bacterial resistance to a wide range of antibiotics due to the indiscriminate use of commercial antimicrobial agents.This forces our attention to search for new antibiotics and/or antibacterial agents to treat infectious diseases [1,2].
Significantly, through the 21st century, severe bacterial infections have become resistant to the frequently used antibiotics [3].Most of the discovered antibiotics so far have become inefficient in overcoming bacterial resistance; new genes and transmission vectors of the bacteria that are identified on a regular basis are encoded antibiotic resistance [4].
Bacterial resistance to antibiotics is achieved via varied and complicated molecular mechanisms; the most common is horizontal gene transfer [5].Besides that, new mechanisms of bacterial resistance have not been identified yet, which led to the term 'superbugs' for multidrug-resistant bacterial strains.The principal factors for such resistance come from misuse and/or overuse of antimicrobial agents [4].
Drug efflux pumps (EPs) have a vital function in the evolution of antimicrobial resistance (AMR) in bacteria [6].Their ability to extrude antimicrobials, prevent the accumulation of toxic levels of antibiotics, and grant pathogen nonsusceptibility to antimicrobial sources among Gram-negative bacteria [7,8].

Protein Preparation
The X-ray crystallographic structure of protein targets (PDB ID: 4dx7 (responsible for transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop in complex with doxorubicin) and 1kzn (Crystal structure of E. coli 24 kDa domain in complex with clorobiocin) were downloaded from the RCSB protein data bank (https://www.rcsb.org/).Non-essential molecules of water, heteroatoms, and co-crystallized ligands bound to the receptors were deleted.Moreover, all hydrogen and missing atoms were added to the receptor molecule's target.Subsequently, Kollman united atom charges were assigned to the receptor atoms [29].Binding pockets with the key amino acids of the selected target proteins were predicted based on their co-crystallized, pounded ligands.Grid boxes were built around the binding sites manually for 4dx7 and 1kzn (Centre: X: 27.974 and 18.411, Y: −37.688 and 25.268, Z: −9.594 and 37.049 Å, respectively, and dimensions: x: 60, y: 60, z: 60), with a grid spacing of 0.5 Å.These dimensions covered the whole binding site and provided enough space for ligand translation and rotation.The corresponding grid center coordinates were set according to the respective binding site residues of the proteins.

Ligands Preparation
The 3D structures of the main essential oil constituents of Mentha spp.(Table 1) were retrieved in (.sdf) format from PubChem (www.pubchem.com),then converted into their (.pdbqt) files using Open Babel software, which is freeware.Subsequently, Gasteiger charges were added to each atom, and the maximum number of rotatable bonds was set according to the torsional bonds in each compound.

Docking of the Receptors with the Ligands
The virtual docking of the selected ligands against target proteins was evaluated by AutoDock 4.2.6 software [29].Firstly, a re-docking process of the original co-crystallized ligand(s), i.e., doxorubicin and clorobiocin of 4dx7 and 1kzn, respectively, was performed for docking validation, which is well reproduced with RMSD values of 0.00 Å and binding energy values of −8.42 and −6.73 kcal mol −1 , respectively.The docking study was performed using the Lamarckian genetic algorithm, with 50 as the total number of GA runs.In each respective run, a population size of 300 individuals with 27,000 generations and 2,500,000 energy evaluations was employed.Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively.The single docked conformation was selected from each docking round based on the clustering RMSD (≤2 Å) and lowest binding energy.The most stable conformations of the ligand molecule were selected based on the lowest binding energy and their binding mode at the active site of proteins, and the 2D and 3D binding interactions of the (.pdbqt) complexes of protein-ligand were analyzed using Discovery Studio Client (Discovery Studio Client is a product of Accelrys Inc., San Diego, CA, USA).

Antimicrobial Exploration of Mentha Essential Oil
Compounds that characterize the different Mentha chemotypes are those commonly occurring as main components of Mentha essential oil (Table 1); their formation reflects differences in biosynthetic pathways, together with a few other compounds that have also been reported sporadically as main Mentha spp.oil components.These variations, of course, accounted for their antibacterial potential with respect to one pathogenic bacteria species; it is well known that EO is not a single compound but a combination of the chemical compounds that carry the specific antimicrobial activity [30,31].
One of the major distinctivenesses of essential oils is their hydrophobicity, which facilitates their penetration into the cell membrane and influences the external envelope of both the cell and cytoplasm, resulting in the cell organelles being affected [15].Mentha EO constituents (Table 1) are characterized by the presence of a free hydroxyl group (oxygenated monoterpenes), which helps in the formation of delocalized electrons, resulting in a proton exchanger, reducing the pH gradient, and destabilizing the cytoplasmic membrane; hence, breakdown of the proton motive force and then drop of the ATP pool, which eventually leads to cell death [28].
The above-recorded findings about the potential antimicrobial activity of Mentha spp.EOs have gained our attention to study their mechanism of action in detail from the side of in silico molecular docking and prove their activity against E. coli MDR.

Molecular Docking Analysis
The crucial role in structure-based drug design (SBDD) is the molecular docking approach, which is used better to understand and estimate the molecular interactions, binding affinities, and energies of ligand(s) within a targeted protein [43].It should be noted that antibiotics target microbial metabolism and restrict their growth by deactivating the vital enzymes involved in cell wall biosynthesis and repair.
Briefly, DNA gyrase is pivotal for bacterial survival as it controls DNA topology by introducing transient breaks to both DNA strands during transcription and replication, so it is essential to exploit bacterial DNA gyrase as a critical target for antibacterial agents.Additionally, bacterial strains develop MDR against antibiotics by effluxing it out through AcrB-TolC.Consequently, a molecular docking study was carried out to examine the binding interactions of the major volatile constituents with the key pockets of regulatory enzymes 4dx7 and 1kzn.

Piperitoneoxid
Moreover, the key amino acids Val43, Val71, Thr165, and Val167 at the binding site of the active pocket formed one H-bond interaction with six ligands, namely carvone (Val167, 2.48 Å), linalool (Val43, 1.94 Å), menthol (Val43, 2.06 Å), menthyl acetate (Thr165, 2.05 Å), piperitoneoxide (Thr165, 2.95 Å), and P-menth-2-en-ol (Val71, 2.08 Å), besides some other hydrophobic interactions.Furthermore, the rest of the list (Table 1) displayed a number of hydrophobic interactions ranging from 4 to 7 (alkyl) with the key amino acids at the active site.Piperitoneoxide (HB, Thr165, 2.95 Å), menthofuran (C-HB, Val71, 2.89 Å), and carvone (HB, Val167, 2.48 Å) (Figure 2) were depicted as the top three binders with the highest molecular interactions (H-bond, C-HB, π-σ, π-alkyl, and/or alkyl) with 8, 7, and 7 interactions, respectively, with binding affinities of −5.17, −5.77, and −5.57.2.05 Å), piperitoneoxide (Thr165, 2.95 Å), and P-menth-2-en-ol (Val71, 2.08 Å), besides some other hydrophobic interactions.Furthermore, the rest of the list (Table 1) displayed a number of hydrophobic interactions ranging from 4 to 7 (alkyl) with the key amino acids at the active site.Piperitoneoxide (HB, Thr165, 2.95 Å), menthofuran (C-HB, Val71, 2.89 Å), and carvone (HB, Val167, 2.48 Å) (Figure 2) were depicted as the top three binders with the highest molecular interactions (H-bond, C-HB, π-σ, π-alkyl, and/or alkyl) with 8, 7, and 7 interactions, respectively, with binding affinities of −5.17, −5.77, and −5.57.On the other hand, as reported by Phan and others [45], the 3D structure of E. coli AcrB-TolC (4DX7) was selected as the target protein to unveil the inhibitory potential of Mentha EOs against E. coli AcrB-TolC MDR.This selection was supported by a previous recommendation by Abdel-Halim and colleagues [46], who examined the sequence alignment of all reported AcrB sequences against the E. coli AcrB sequence and confirmed a high similarity in the overall structure with a high conservation of the residues at the binding site, which is the same in 4DX7.Additionally, 4DX7 was co-crystallized with doxorubicin.Nevertheless, all previous reports supported that the active pocket's binding site of AcrB (Figure 3a) is large and encompasses mainly hydrophobic amino acids (i.e., Ala, Gly, Leu, Ile, and Phe), with some ionized and polar residues (i.e., Gln, Ser, Tyr, and Thr).On the other hand, as reported by Phan and others [45], the 3D structure of E. coli AcrB-TolC (4DX7) was selected as the target protein to unveil the inhibitory potential of Mentha EOs against E. coli AcrB-TolC MDR.This selection was supported by a previous recommendation by Abdel-Halim and colleagues [46], who examined the sequence alignment of all reported AcrB sequences against the E. coli AcrB sequence and confirmed a high similarity in the overall structure with a high conservation of the residues at the binding site, which is the same in 4DX7.Additionally, 4DX7 was co-crystallized with doxorubicin.

Carvone
Nevertheless, all previous reports supported that the active pocket's binding site of AcrB (Figure 3a) is large and encompasses mainly hydrophobic amino acids (i.e., Ala, Gly, Leu, Ile, and Phe), with some ionized and polar residues (i.e., Gln, Ser, Tyr, and Thr).
On the other hand, as reported by Phan and others [45], the 3D structure of E. coli AcrB-TolC (4DX7) was selected as the target protein to unveil the inhibitory potential of Mentha EOs against E. coli AcrB-TolC MDR.This selection was supported by a previous recommendation by Abdel-Halim and colleagues [46], who examined the sequence alignment of all reported AcrB sequences against the E. coli AcrB sequence and confirmed a high similarity in the overall structure with a high conservation of the residues at the binding site, which is the same in 4DX7.Additionally, 4DX7 was co-crystallized with doxorubicin.Nevertheless, all previous reports supported that the active pocket's binding site of AcrB (Figure 3a) is large and encompasses mainly hydrophobic amino acids (i.e., Ala, Gly, Leu, Ile, and Phe), with some ionized and polar residues (i.e., Gln, Ser, Tyr, and Thr).In addition, Nikaido [47] confirmed that this diversity is essential for the binding of various AcrB substrates and/or inhibitors, which should be characterized with hydrophobic groups to form hydrophobic interactions and atoms to create hydrogen bonds (Figure 3b).
Briefly, ligand molecules depicted molecular interactions with the key amino acids at the fragment binding site of the active pocket, with ΔG values in the range of −4.69 to −6.39 kcal/mol.
Briefly, ligand molecules depicted molecular interactions with the key amino acids at the fragment binding site of the active pocket, with ∆G values in the range of −4.69 to −6.39 kcal/mol.

Conclusions
It can be concluded from the current study that Mentha essential oil constituents possess potential antimicrobial activity, which is well documented via wet lab measurements and confirmed by in silico molecular docking of the EO constituents into the active site of DNA gyrase.DNA gyrase is a key to pathogenic DNA topology during transcription and replication.Moreover, the antibacterial MDR activity of Mentha EOs was also evaluated here for the first time against E. coli AcrB-TolC and proved potential inhibition with proper binding affinities and varied molecular interactions at the active site of the hydrophobic pocket.The current study concludes that Mentha spp.EOs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) exert their antimicrobial activity via their ability to inhibit the DNA gyrase of the pathogens and, consequently, interrupt its transcription and replication.Furthermore, Mentha spp.EOs proved their ability to restrain the MDR pathogens through their ability to suppress the AcrAB-TolC

Figure 1 .
Figure 1.Chemical structures of the major EOs of Mentha spp.

Piperitoneoxid 14 ,
x FOR PEER REVIEW 10 of 14

Figure 2 .
Figure 2. Three-dimensional and two-dimensional molecular interactions of piperitoneoxide, menthofuran, and carvone with the key amino acids at the active pocket of DNA gyrase (PDB: 1kzn).

Figure 2 .
Figure 2. Three-dimensional and two-dimensional molecular interactions of piperitoneoxide, menthofuran, and carvone with the key amino acids at the active pocket of DNA gyrase (PDB: 1kzn).

Figure 3 .
Figure 3. (a) 4DX7_Chain-A and its hydrophobic active pocket (in red).(b) Hydrophobic active pocket with the degree of hydrophobicity.

Figure 3 .
Figure 3. (a) 4DX7_Chain-A and its hydrophobic active pocket (in red).(b) Hydrophobic active pocket with the degree of hydrophobicity.

Figure 4 .
Figure 4. Three-dimensional and two-dimensional molecular interactions of menthyl acetate, menthofuran, and piperitenone oxide with the key amino acids at the active pocket of AcrB-TolC (PDB: 4dx7).

Figure 4 .
Figure 4. Three-dimensional and two-dimensional molecular interactions of menthyl acetate, menthofuran, and piperitenone oxide with the key amino acids at the active pocket of AcrB-TolC (PDB: 4dx7).

Table 1 .
Binding free energies, hydrogen bonds, and number of interactions between the amino acid residues and the docked molecules into the binding sites of 4DX7