INTRODUCTION:

Antibiotic resistance, primarily driven by the overuse and misuse of antibiotics, has emerged as a global public health crisis¹. Multi-drug resistant (MDR) pathogens, which exhibited resistance to multiple antibiotics, have become increasingly prevalent, posing a significant challenge for healthcare systems worldwide². Consequently, the search for alternative strategies to combat MDR pathogens has intensified, with a particular focus on natural products as potential sources of novel therapeutic agents.

Endophytic microorganisms residing within plant tissues have garnered substantial attention as valuable reservoirs of bioactive compounds with diverse pharmacological properties³. Endophytic bacterium found in various plant species, has demonstrated significant promise as a producer of bioactive metabolites. These compounds have exhibited activity against a wide range of microorganisms, including MDR strains, making them intriguing candidates for further exploration in the quest to address antibiotic resistance⁴.

Molecular docking analysis is a powerful computational tool used to predict and analyze the binding interactions between small molecules and target proteins or genes^5,6. In the context of antibiotic resistance research, molecular docking provides valuable insights into the mechanisms by which compounds may inhibit MDR gene expression. Antibiotic resistance is an escalating global health concern, challenging the efficacy of antibiotics and necessitating innovative strategies to combat multidrug-resistant (MDR) pathogens^1,7. One crucial aspect of this resistance mechanism involves the interaction between antibiotics and their molecular targets specifically, the Penicillin-Binding Proteins (PBPs) found in bacterial cell walls which is essential for devising effective therapeutic interventions. Penicillin-binding proteins are a class of bacterial enzymes responsible for cell wall biosynthesis and remodeling⁷ andthey serve as the primary targets for β-lactam antibiotics, including penicillin, cephalosporin, and carbapenem. The antibacterial activity of these drugs hinges on their ability to bind to and inhibit PBPs, thereby disrupting the synthesis of the bacterial cell wall, leading to cell lysis and death.However, the emergence of MDR bacteria had revealed the adaptability of these microorganisms in circumventing the lethal effects of antibiotics. One well-documented mechanism of MDR involves the production of β-lactamases, enzymes capable of hydrolysing β-lactam antibiotics, rendering them ineffective⁸. This resistance strategy has prompted the development of β-lactamase inhibitors, such as clavulanic acid and sulbactam, which co-administered with β-lactam antibiotics, which can protect them from β-lactamase degradation⁹.

Moreover, MDR bacteria have evolved novel strategies to evade antibiotic action by altering their PBPs and in turn reduced affinity for antibiotics and making it more challenging for these drugs to bind and inhibit cell wall synthesis effectively¹⁰. This alteration in PBP structure and function is often attributed to mutations in the corresponding genes, leading to the development of antibiotic resistance. Understanding the interplay between PBPs and antibiotic resistance mechanisms is crucial for the design of new antibacterial agents and therefore research efforts have to be focused on developing antibiotics that exhibit increased affinity for PBPs or novel PBP inhibitors with distinct mechanism ofactions¹¹. Among the bioactive compounds isolated from Asaialannensis, 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro- and 3-Pyridinemethanol, 5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride showed noteworthy bioactivity profiles, which warrant investigation into their potential as inhibitors of MDR genes.

MATERIALS AND METHOD:

a) Compound Retrieval from Pub Chem:

In this present study, the chemical structures of two compounds, namely 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro- and 3-Pyridinemethanol,5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride, were procured from the PubChem database. To initiate this process, the compound names were employed as search queries within the PubChem database search engine. Subsequently, the respective chemical structures for both compounds were extracted from PubChem. These structures were obtained in standard file formats, including Structure-Data File (SDF), to ensure compatibility with subsequent computational analyses¹².

Following the successful retrieval and meticulous verification of these chemical structures, they were subjected for further subsequent analyses. This preparation phase encompassed converting the retrieved structures into appropriate file formats that are harmonious with molecular docking software and various other computational tools. This methodical approach ensures the accuracy and compatibility of the chemical structures for downstream computational investigations.

b) Protein Selection and retrieval from RCBS:

The RCSB PDB, a comprehensive repository of biological macromolecule structures, including proteins and nucleic acids, serves as a global research hub for structural data analysis. Protein retrieval was done by using specific identifiers (Penicillin Binding Protein 2X (PBP -2X) UNIPORT ID: 1QME) as search queries in the RCSB PDB database. Retrieved protein information underwent rigorous verification for accuracy, including PDB IDs, names, sources, and metadata. Three-dimensional structures were extracted in standard formats (PDB) for downstream analysis. Data quality was ensured by cross-referencing with experimental methods, resolution values, and relevant literature. Prepared structures underwent necessary adjustments, including solvent removal, hydrogen atom addition, and structural refinement using molecular modelling software.

Figure 1: 3D Structure of Penicillin Binding Protein 2X(PBP -2X) UNIPORT ID: 1QME

Penicillin-Binding Protein 2X (PBP-2X) was identified by its UNIPROT ID 1QME^[13]which is a pivotal bacterial protein with significant implications in antibiotic action and bacterial cell wall synthesis and it was retrieved through the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). PBP-2X is a member of the penicillin-binding protein family, a group of essential proteins in bacteria responsible for the synthesis and maintenance of the bacterial cell wall. This biological role makes PBP-2X a prime target for antibiotics and inhibition of PBP-2X by antibiotics, such as penicillin and cephalosporins, disrupts the crucial process of cell wall synthesis, leading to bacterial cell death. The 3D structure of PBP-2X,(Figure 1) represented by PDB ID 1QME, offered a detailed view of its binding sites, catalytic domains, and interactions with antibiotics. Researchers leverage this structural information for multiple purposes, including the design of novel antibiotics and a deeper understanding of antibiotic resistance mechanisms. Moreover, the structural data for PBP-2X is readily available for scientific exploration and computational analysis, contributing to our knowledge of bacterial physiology and the development of effective antimicrobial strategies.

c) Ligands for molecular Docking:

Ligands play a pivotal role in molecular docking studies, serving as potential candidates for interactions with target proteins.

i. 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro-

This compound, also known as 15-chloro-13-oxabicyclo[9.3.1]pentadecane, exhibits a unique bicyclic structure with chlorine substitution at position 15 which has hydrophobicity and potential for halogen bonding interactions with target proteins. Such interactions have been of interest in drug design due to their influence on ligand binding affinity¹⁴

Figure 2: 3D conformer of 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro-

ii. 3-Pyridinemethanol, 5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride

This ligand, often referred to as a pyridine derivative, features a pyridine ring substituted with a hydroxyl group and a methoxymethyl moiety. The presence of a hydroxyl group facilitate hydrogen bonding with amino acid residues in the active site of target proteins, potentially enhancing binding interactions¹⁵

Figure 3: 3D conformer of3-Pyridinemethanol, 5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride

d) Molecular Docking using Easy Dock Vina:

Molecular docking is a vital tool in drug discovery and molecular biology. Easy Dock Vina is a widely-used software for this purpose. The process involves meticulous preparation of molecular structures, including the target protein and ligand. This entails removing water molecules, trimming non-polar hydrogen atoms, and assigning atom charges. The protein's 3D structure is optimized, and the ligand is positioned near the binding site. AutoDock Vina explores various ligand orientations and conformations within the site, determining the energetically favoured binding pose. Crucial to this is the scoring function, assessing interaction energy based on Van der Waals forces, electrostatic interactions, and desolvation energy. It estimates binding affinity. This study perform multiple docking runs, exploring diverse ligand conformations and poses to refine outcomes. The software ranks poses by predicted binding energies, with lower values indicating stronger affinity. Top-ranked poses reveal binding modes and interactions, guiding experimental design and ligand optimization in drug development and structural biology. Molecular docking is indispensable in understanding ligand-protein interactions, facilitating crucial advancements in these fields.

RESULTS:

The molecular docking simulations have provided valuable insights into the binding interactions of two ligands, 13-Oxabicyclo[9.3.1]pentadecane, 15-chloro-, and 3-Pyridinemethanol, 5-hydroxy-4-(methoxymethyl)-6-methyl-, hydrochloride, with a target protein.

a) Binding Affinity:

13-Oxabicyclo[9.3.1]pentadecane, 15-chloro-, binding affinities ranged from -6.1 kcal/mol to -5.1 kcal/mol during the docking study. Lower binding affinity values indicated stronger interactions between the ligand and the target protein. Notably, the highest affinity score recorded was -5.1 kcal/mol. In contrast, the ligand 3-Pyridinemethanol, 5-hydroxy-4-(methoxymethyl)-6-methyl-, hydrochloride, exhibited binding affinities within the range of -5.4 kcal/mol to -4.6 kcal/mol. The ligand achieved its highest affinity score at -4.6 kcal/mol (Table 1). From the above results it is inferred suggested that both ligands established moderately to strongly favourable interactions with the target protein and the negative values signified energetically favourable binding, indicating effective ligand binding within the protein's active site.

b) Distance from rmsd Bounds:

When distance from rmsd bounds for 13-Oxabicyclo[9.3.1]pentadecane, 15-chloro-, was docked the distances from the rmsd lower bound (rmsdl.b.) varied from 0 Å to 45.145 Å, and upper bound (rmsdu.b.) ranged from 0 Å to 46.753 Å. These values provided insights into the spatial fit of the ligand within the binding site.Conversely, 3-Pyridinemethanol, 5-hydroxy-4-(methoxymethyl)-6-methyl-, hydrochloride, the distances from rmsdl.b. ranged from 0 Å to 30.563 Å, and from rmsdu.b. ranged from 0 Å to 31.186 Å, indicating different spatial arrangements within the binding site.

c) Amino Acid Interactions:

In addition to binding affinity and spatial compatibility, it's crucial to examine specific amino acid interactions within the ligand-binding site. 13-Oxabicyclo [9.3.1]pentadecane, 15-chloro-, key amino acid interactions included hydrogen bonding with GLN730, GLN728, LYS729, ASN501, and GLN590, contributing significantly to ligand stability (Figure4). Hydrophobic contacts with residues TYR700, F570, G597, W374, among others, enhance ligand binding. Aromatic stacking interactions with TYR700 and TRP374 further stabilize the complex. Comparatively,3-Pyridinemethanol, 5-hydroxy-4-(methoxymethyl)-6-methyl-, hydrochloride, exhibits similar interactions with amino acids, albeit with slight variations. Hydrogen bonds, hydrophobic interactions, and aromatic stacking contributed to the observed binding affinity, with specific residues involved.

Table 1: Binding affinity and distance from RMSD of two ligand against Penicillin-Binding Protein 2X (PBP-2X

Ligand	Affinity (kcal/mol)	Dist from rmsdl.b.	Dist from rmsdu.b.
13-Oxabicyclo [9.3.1] pentadecane, 15-chloro-	-6.1	0	0
	-5.9	35.035	36.141
	-5.5	27.131	28.783
	-5.4	19.696	21.251
	-5.4	25.481	27.253
	-5.4	29.696	31.03
	-5.2	16.301	17.701
	-5.1	45.145	46.753
	-5.1	32.335	34.383
3-Pyridinemethanol, 5-hydroxy-4-(methoxymethyl)-6-methyl-, hydrochloride	-5.4	0	0
	-5.1	10.212	11.874
	-4.9	1.661	2.304
	-4.9	3.041	5.513
	-4.8	28.6	30.446
	-4.7	2.167	3.074
	-4.7	30.563	31.186
	-4.6	27.786	30.254
	-4.6	7.349	9.506

Figure 4: Different poses of amino acid binding site of interaction 13-Oxabicyclo[9.3.1]pentadecane, 15-chloro-

DISCUSSION:

Molecular docking is pivotal in drug discovery and molecular biology. Easy Dock Vina, a widely-used software, aids in this process. It involves precise preparation of molecular structures, including the target protein and ligand. This includes eliminating water molecules, trimming non-polar hydrogen atoms, and assigning atom charges¹⁶. The protein's 3D structure is optimized, and the ligand is placed near the binding site. AutoDock Vina explores various ligand orientations and conformations within the site, determining the energetically preferred binding pose^17,18. Central to this is the scoring function, assessing interaction energy via Van der Waals forces, electrostatic interactions, and desolvation energy, estimating binding affinity. This study perform multiple docking runs, examining different ligand conformations and poses to refine results. The software ranks poses based on predicted binding energies, with lower values indicating stronger affinity¹⁹. Top-ranked poses reveal binding modes and interactions, guiding experimental design and ligand optimization in drug development and structural biology²⁰. Molecular docking plays a pivotal role in comprehending ligand-protein interactions, driving advancements in these fields.

CONCLUSION:

This research paper has explored the potential of two compounds, 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro-, and 3-Pyridinemethanol, 5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride, as inhibitors of Penicillin-Binding Protein 2X (PBP-2X) in the context of combating multidrug-resistant (MDR) pathogens. Molecular docking analysis was employed to investigate the binding interactions between these ligands and the target protein.The results indicated that both ligands exhibit favorable binding affinities with PBP-2X, with 13-Oxabicyclo[9.3.1]pentadecane, 15-Chloro- demonstrating slightly stronger binding than 3-Pyridinemethanol, 5-Hydroxy-4-(Methoxymethyl)-6-Methyl-, Hydrochloride. The spatial arrangements of the ligands within the binding site varied, suggesting potential differences in their specific interactions with the protein. Amino acid interactions analysis highlighted key interactions contributing to ligand stability, including hydrogen bonds, hydrophobic contacts, and aromatic stacking. These findings underscore the importance of molecular docking simulations in understanding ligand-protein interactions and provide valuable insights for rational drug design. Moreover, they contribute to the growing body of knowledge aimed at developing effective therapeutic agents against antibiotic-resistant pathogens. This study's findings lay a groundwork for future validation and optimization of these compounds as antibiotics or adjuvants against antibiotic resistance. It aids global efforts to combat this crisis via natural sources, emphasizing endophytic microorganisms and their bioactive metabolites as promising resources.Innovation and understanding antibiotic resistance are vital for safeguarding our ability to combat infectious diseases.

ACKNOWLEDGEMENT:

The authors acknowledge Vinayaka Mission’s Research Foundation, Salem, Tamil Nadu, India for providing facilities and funds to carry out our research.

CONFLICT INTEREST:

The authors declare that there is no conflict of interest.

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Received on 30.09.2023 Modified on 05.02.2024

Research J. Pharm. and Tech 2024; 17(9):4389-4393.

DOI: 10.52711/0974-360X.2024.00678