Optimized Structure, in Silico interaction and Molecular Docking Analysis of Two Benzimidazole-2-Thione Derivatives

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Introduction
Acineto bacter (Gram-negative) is a genus of microorganisms belonging to the proteobacteria family, whose species are found in a wide variety of environments. 1 The bacteria extensively spread in hospitals where they offer the risk of transmitting resistance to other pathogens. 2 Most of the bacterial infections are commonly caused by Staphylococci and it is considered to be the most important source of bacterial diseases found in more than one million patients each year globally. 3,4 Staphylococcus epidermidis is the best known gram-positive bacterium, normally observed on human skin, and mucous membranes. It is a frequent source of infections involving indwelling surgical and medical devices in patients having an impaired immune system. 5 It generates a biofilm to defend itself from the hostimmune system and increase its resistance to antibiotic medicines. 6 Antibiotic resistance is a serious issue throughout the world and develops an urgency to discover an efficient drug for S. epidermidis bacterial infections. Literature survey shows benzimidazole derivatives are used as a potential drug against S. epidermidis bacterial infections. [7][8][9] Since the growth in antibacterial resistance is expected to develop by S. epidermidis, we are on a hunt to study morebenzimidazole derivatives for the cure of bacterial infections. Thus, in view of the wide ranging application of benzimidazole-2-thione core10,11,in silico studies (DFT and HS) on the known crystal structures of 5-Methoxy-1Hbenzo[d]imidazole-2(3H)-thione (M1: CSD refcode WIGYOB)12 and 4, 5-Dimethylbenzimidazolene-2-thione (M2: CSD refcode SINZIY), 13 having close chemical similarity, have been carried out for the computation of their optimized geometry and electronic properties. Both the structures have been optimized by using Gaussian 09 program, 14 while the electrostatic potential maps have been analyzed by using Crystal Explorer 21.5 program, 15 followed by lattice energy frameworks to measure the stability of both molecules toward crystal packing. For the in silico antibacterial activity of these two imidazole derivatives to be an effective lead compounds in S. epidermidis bacterial treatments, the Molecular docking analysis has been made using the crystallographic structure of Staphylococcus epidermidis Tc aR protein (PDB ID : 4EJV) to identify the antibacterial inhibition of both imidazole compounds. The protein (PDB ID: 4EJV) is staphylococcus epidermis TcaR, in complex with chloramphenicol. The S. epidermidis is known to develop a biofilm to guard itself from hostimmune system and enrich their resistance to antibiotic drugs, for more than 60% of the bacterial infections are reportedly produced by the creation of biofilm. The main component involved in the formation of biofilm in S. epidermidis is polysaccharide intercellular adhesin (PIA)16, the production of PIA depends on the expression of the ica ADBC gene and current studies revealed that TcaR acts as a regulatory factor to affect the transcription of ica ADBC. 17 TcaR is a MarR family transcription regulator that is involved in teicoplanin and methicillin resistance. Therefore, it was originally described as a putative transcriptional regulator of the teicoplaninassociated locus (tca). Because of the above reasons we have chosen the Staphylococcus epidermidis TcaR protein (PDB ID: 4EJV) for the present research work. Also from literature survey we came to know that benzimidazole derivatives are good inhibitors of S. epidermidis and hence motivated to dock both ligands into the Staphylococcus epidermidis TcaR protein. The molecular docking outcome shows that both imidazole derivatives M1 and M2 are well bound with the active site amino acids of S. epidermidis receptor, compared to the heteroatom chloramphenicol found in 4EJV.

Computational Details Quantum Chemical Calculation
The chemical structure of M1 and M2 is shown in Figure 1. The theoretical calculations were performed with DFT methods using Gaussian 0914 and visualized through Gauss view 6.0.1618. Both structures were optimized using B3LYP/6-311+G(d,p) level basic set without any symmetrical constraints. Further, the optimized structural parameters were used to calculate the molecular geometry, atomic charges, molecular electrostatic potential map, HOMO-LUMO energy gap and related reactive parameters (electronegativity, chemical potential, hardness, softness, electrophilicity).

Hirshfeld Surface (HS)
Hirshfeld surface (HS) and its associated twodimension (2D) fingerprints plots for M1 and M2 have been constructed using crystallographic information file (CIF), to dissect molecular structures into non-covalent contacts. [19][20][21] High resolution Hirshfeld surface for M1 and M2 were generated using Crystal Explorer 21.5 15 , and functions of curvature and distance, including shape index and normal contact distance (d norm ), were mapped onto the surfaces. 19,21,22 and molecular pair-wise interaction energy of both the structures was estimated from a single-point molecular wave function at B3LYP/6-31G (d, p) level of theory. The calculations were performed by generating a cluster of radius 3.8 Å around the central molecule. The total interaction energy between the pair-wise molecules is the sum of electrostatic energy (E ele ), repulsion energy (E rep ), polarization energy (E pol ) and dispersion energy (E dis ). Further the strength of each energy component is represented by the width of its cylinders. 28,29

Molecular Docking
The crystallographic structure of Staphylococcus epidermidis TcaR 30 was obtained from the RCSB Protein Data Bank server (PDB ID: 4EJV) which consists of 151 aminoacids with resolution of 2.9 Å. The protein is a member of the Multiple Antibiotic Resistance Regulator (MarR) family of transcriptional regulators, named by Escherichia coli MarR. The protein preparation for the docking procedure was carried using Auto Dock 4.2 31 , following the standard protocol. 32 The hetero atoms, water molecules and other cofactors attached with the receptor were removed and polar hydrogens were added and PDBQT format file was saved. The active sites coordinated were found from the position of native ligand chloramphenicol (X= -14.939; Y= -39.3618; Z= -2.9430). Similarly, all the ligand PDBQT files were prepared and saved. The saved files were used in the Auto Dock Vina 1.1.2 software for molecular docking investigations. 33 The docked poses were viewed by Discovery Studio Visualizer 34 and best complexes were selected.

Fig. 1: Chemical Structure of M1 and M2
In 2D fingerprint plots, the combination of d i (distance from point to the nearest atom internal to the surface) and d e (distance from point to the nearest atom external to the surface) provides a summary of intermolecular contacts within both the molecular structures. [23][24][25][26] The position of voids in both the structures has been visualized by constructing (0.002 au)-isosurface of procrystal electron density, which contain more than 98% of the electronic charge of the molecules and is used to identify the empty space in crystal by computing the shape and size of molecules. 27

Energy Framework
For the energy-frameworks analysis, the CIF was used as input to Crystal Explorer 21.5 software 15     9 There exists a π-π interaction between the homo-aromatic rings of the molecule M1 with centroids distance of 3.658 Å (along the

Atomic Charges
Mulliken atomic charges for M1 and M2 were computed at B3LYP methods with 6-311+G (d,p) level basic set as illustrated in Figure 4. The atomic charges have been obtained by the Mulliken population analysis 36   It is generally accepted that the negative region (red) of MEP indicates electrophilic reactivity and positive region (blue) corresponds to nucleophilic reactivity whereas the green colors indicate the neutral potential. The different colors: red, orange, yellow, green and blue in contour map ( Figure 5) represent the increasing orderofelectrostatic potential in terms of electron density. From the contour map of MEP ( Figure 5) the negative region dispersed on the oxygen atom of molecule M1 and sulphur atom in both molecules depicted the electrophilic reactivity (red color) whereas N1 and N2 atoms of both molecules depicted nucleophilic reactivity represent by the blue color range.

Frontier Molecular Orbital Analysis
The analysis of frontier molecular orbital i.e. HOMO (highest occupied molecular orbital) and LUMO (least unoccupied molecular orbital) helps to study the electronic, thermal and chemical properties of the compounds. 37 The HOMO describes the ability to donate an electron while the LUMO has an ability to gain an electron. The HOMO-LUMO gap explains the charge transfer interaction's ability within the molecule. 38,39 Figure 6 represents the HOMO-LUMO energies plot of (M1) and (M2). The positive phase indicated by red color, while negative by green as shown in Figure 6. The HOMO-LUMO energy gaps are about 4.40 eV (M1) and 4.39 eV (M2), respectively, which show that the energy gap of M2 is slightly smaller than that of M1. Theoretical values of HOMO-LUMO and their energy gap in electron-volt help in computing the stability, softness, hardness, electronegativity, chemical potential and electrophilicity index by using Koopman's theorem. 40 Table 2 contains the quantum chemical parameters of both the structures.

Hirshfeld Surface and Crystal Voids Analysis
Hirshfeld surface (HS) analysis is an effective method for gaining further insight into crystal structure by color-coding long (blue) and short (red) interactions. [41][42][43] Figure 7 illustrates the HS of M1 and M2 mapped over d norm surface. In d norm surface the deep red spots on the front and back view (Figure 7) represent the strong hydrogen bonding. Four circular deep red spots in d norm surface of M1 and M2 are due to N-H…S and N-H…O (N1-H1N…S, N2-H2N…S, N1-H1N…O for M1) and N-H…S (N1-H1N…S, N1-H2N…S for M2) whereas the other faint red spots are due to weak interactions. Consequently the intensity of color signifies the intensity of interaction. Figure 8 and 9, respectively, illustrates the shape index contour surface and curvedness plot of M1 and M2. Shape index ( Figure 8) is a dimensionless surface property which is used to identify the complementary red-hollows and blue-bumps pairs in maps where surfaces of molecules joined each other. 44 The π-π interaction in the shape index map of M1 occur between the homo-aromatic rings [symmetry code (1-x, 1-y, 1-z)], visualized by the complementary red and blue triangles (marked in the rectangle, Figure 8). The topography of curvedness maps (Figure9) is a function of the root-mean-square curvature of the surface where low value of curvedness region is relative flat area separated by dark blue boundaries (large curvedness), tends to divide the surfaces into patches related with intermolecular contacts. 45 The large flat green region along the surface indicates the existence of π-π stacking in the structure of M1 (Figure 9) while this feature is absent in M2. value of 2.50 Å. However, the H…H contacts in M1 and M2 accounts for 40% and 46.2% of the total HS area, respectively. Apart from this, all other interactions of M1 and M2 are depicted in donut chart inserted in Figure 10 and 11, respectively.

Energy Frameworks Analysis
The precise interaction energies of M1 and M2 as computed using B3LYP/6-31G (d,p) molecular wave function and their resultant topology is shown using energy frameworks.  Figure 13. To expand or contract the cylinders in the energy framework, a scale factor is applied to each cylinder's size based on the relative strength of molecular packing in the different directions ( Figure 13). Minor interactions below 5kJ/mol were ignored in order to avoid the crowding in the figures.    The best ligand-protein complex conformation was chosen by lower binding energy and hydrogen bond interactions for further analysis in Discovery studio visualizer. The best docked complex poses of M1, M2 and chloramphenicol with 4EJV protein are illustrated in Figure 14- Figure 14 and 15, respectively.
The docking results suggest that M1 and M2 are potential Staphylococcus epidermidis TcaR protein inhibitors and may be useful for the antibacterial drug development process.

Conclusions
The optimized structure, atomic Mulliken charges, molecular electrostatic potential, HOMO-LUMO energy gaps, and some other molecular characteristics of 5-Methoxy-1H-benzo[d] imidazole-2(3H)-thione (M1) and 4, 5-Dimethyl benzimidazolene-2-thione (M2) molecules, using DFT theory with B3LYP/6-311+G(d,p) level basic set, have been analyzed. The optimized parameters, by and large, agree well with the X-ray data. The HOMO-LUMO energy gap of M2 is slightly lower than that of M1, indicating that the molecular structure of M2 is more favorable for charge transfer than that of M1. The Hirshfeld surface analysis indicates that the critical interactions happening in the molecular structure results in π-π stacking (in M1). The proportion of volume occupied by voids in M1 and M2 is 0.36 % and 16.10 %, respectively, indicating that the physical strength of structure M1 is far greater than that of M2. The energy framework analysis reveals that electrostatic and dispersion components in both the structures have a major role to play in the total lattice energy. These findings suggest that packing density, packing manner, intermolecular interactions are inclusively expressed by the hydrogen bonds. The other non-hydrogen interactions, the amount of voids present in unit cell and interaction energies aresome of the governing factors that affect physical properties such as hardness and tablet-ability.
In silico molecular docking results of imidazole compounds (M1 and M2) against S. epidermidis protein 4EJV reveal higher binding scoring pose (-10.2 kcal/mol and -9.4 kcal/mol) compared to chloramphenicol (−9.1 kcal/mol). These outcomes enable us to consider M1 and M2 to act as lead molecules for potential S. epidermidis inhibitors and could be used in antibacterial drug design processes.