Investigation into the Interaction between Penicillin-Resistant and Penicillin-Susceptible Gonococcal Penicillin-Binding Protein 2 and Target Phenolic Ligands through Molecular Docking Studies and Structure-Activity Relationship Analysis

Gonococcal infections present a notable public health issue, and the major approach for treatment involves using β-lactam antibiotics that specifically target penicillin-binding protein 2 (PBP2) in Neisseria gonorrhoeae. This study examines the influence of flavonoids, namely, rutin, on the structural changes of PBP2 in both penicillin-resistant (FA6140) and penicillin-susceptible (FA19) strains. The research starts by clarifying the structural effects of certain mutations, such as the insertion of an aspartate residue at position 345 (Asp-345a), in the PBP2. The strain FA6140, which is resistant to penicillin, shows specific changes that lead to a decrease in penicillin binding. These mutations, namely, P551S and F504L, have a significant impact on the pace at which acylation occurs and the stability of the strain under high temperatures. Molecular docking analyses investigate the antibacterial activities of rutin and other phytocompounds, emphasising rutin's exceptional binding affinity and its potential as an inhibitor of PBP2. Quercetin and protocatechuic acid have encouraging antibacterial effectiveness, with quercetin displaying characteristics similar to those of drugs. Molecular dynamics simulations offer a detailed comprehension of the interactions between flavonoids and PBP2, highlighting rutin's exceptional antioxidant effects and strong affinity for the substrate binding site. The study's wider ramifications pertain to the pressing requirement for antiviral treatments, namely, in the context of the ongoing COVID-19 epidemic. Flavonoids have a strong affinity for binding to PBP2, indicating their potential as inhibitors to impair cell wall formation in N. gonorrhoeae. Ultimately, this study provides extensive knowledge on the interactions between proteins and ligands, the dynamics of the structure, and the ability of flavonoids to combat penicillin-resistant N. gonorrhoeae bacteria. The verified simulation outcomes establish a basis for the creation of potent inhibitors and medicinal therapies to combat infectious illnesses.


Introduction
Historically, the sexually transmitted illness gonorrhoeae, which is caused by the bacterium Neisseria gonorrhoeae (N.gonorrhoeae), was efectively treated by delivering a solitary dosage of penicillin [1][2][3].However, the emergence of penicillin-resistant bacterial species has led to the exploration of other antibiotics [4].Te increase in N. gonorrhoeae strains exhibiting intermediate resistance to routinely prescribed antigonococcal medication poses a signifcant obstacle to the efcacy of treatment [5,6].Recent research conducted by Vincent and Jerse has revealed alarming evidence of an escalating trend in the overall resistance, which may provide difculties in selecting appropriate treatment options [7].Te antibacterial activities of β-lactam antibiotics are attributed to their specifc binding to penicillin-binding proteins (PBPs) [8].Peptidoglycan biosynthesis proteins (PBPs), crucial enzymes responsible for the production of peptidoglycan in bacterial cells, may be classifed into three distinct classes (A, B, and C) based on their specifc structural and functional characteristics [3,9].Comprehending the transpeptidase function of PBPs in classes A and B is essential for promoting the creation of peptide cross-links between adjacent peptidoglycan strands.Class A PBPs possess a transglycosylase domain, which is accountable for the process of polymerisation and the formation of covalent bonds between glycan chains [8,9].Te study conducted by Straume et al. [10] emphasises the unique characteristics of class B PBPs. PBP1, PBP2, and PBPs 3 and 4 are categorised into classes A, B, and C, respectively [11].PBP2 enzymes have been identifed as the principal target of penicillin at the minimum inhibitory concentration (MIC) in susceptible bacteria [3,6,12].Te appearance of penicillin-resistant bacteria, caused by PBP2 variations with an insertion of aspartic acid at the amino acid junction 345-346 (Asp-345a or D-345a), emphasises the need for alternate treatment methods [13].Te variations exhibit 4-8 substitutions in close proximity to the protein's C-terminus [14,15].Our work has revealed the exceptional antibacterial activity of phenolic compounds present in Pleurotus ostreatus mushrooms.Tese compounds have demonstrated signifcant promise in fghting drug-resistant isolates of N. gonorrhoeae [6,16].Tis work highlights the need to discover new bioactive chemicals that can efectively fght against antibiotic-resistant bacteria, thereby tackling the growing problem of antimicrobial resistance.Molecular dynamics simulations enhance our understanding of the interactions between phenolic compounds and PBP2.Tese simulations ofer a comprehensive insight into the intricate molecular dynamics, highlighting the antioxidant efects and strong afnity of the compounds for the substrate binding site.Te integration of molecular simulations improves the accuracy and comprehensiveness of our research, enabling a detailed investigation of the complex interactions between proteins and ligands as well as the structural changes over time.Tis contributes to the progress of drug development methods.Moreover, this study expands the signifcance of its fndings to include more than just gonococcal infections.It also relates to the worldwide need for antiviral therapies during the current COVID-19 epidemic.Natural compounds' great afnity for target proteins positions them as prospective inhibitors capable of afecting cell wall formation in N. gonorrhoeae.Tis presents a fexible method in the larger context of infectious illnesses.Ultimately, this study provides a fresh viewpoint on the molecular foundations of antibiotic resistance in N. gonorrhoeae, presenting bioactive chemicals as promising inhibitors.By utilising the benefts of molecular simulations, our research establishes a strong basis for creating powerful inhibitors and medical treatments.Tis addresses the urgent issue of infectious illnesses and contributes to the worldwide fght against antibiotic resistance.

Method
2.1.Protein Identifcation.Te macromolecules studied in this research were penicillin-binding protein 2 (PBP2) of the clinical penicillin-resistant mutant N. gonorrhoea strain FA6140 (6HZJ) [17] and PBP2 from N. gonorrhoeae strain FA19 (3EQU) [18].Tese macromolecules were retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB), and the macromolecule studied in this research is the crystal structure of penicillin-binding protein 2.

Structure Validation.
Te structural integrity of both FA6140 and FA19 PBP2 was assessed using the use of Procheck Structure Verifcation Methodology.Te Procheck analysis generated a Ramachandran plot that depicted the bond angles (Phi and Psi angles) of each residue, thereby verifying the credibility of the anticipated secondary structure and its 3D conformations.Furthermore, a G-factor metric was computed to assess the likelihood of error or deviation in the anticipated confguration.

Protein Preparation.
Te protein complexes obtained from the RCSB PDB were processed using Discovery Studio 2021 to eliminate heteroatoms.Te protein structures were modelled and then optimised for docking purposes using AutoDockTools.Te resulting proteins were saved in the .pdbformat.oxychromen-4-one).Te ligands were retrieved in the .sdfformat and converted to the .pdbformat.

Ligand-Protein Docking.
Te PyRx software was utilised to execute the ligand-protein binding procedure by employing the AutoDock Vina plugin.Upon completion of a profcient docking procedure, the protein-ligand intermolecular associations were assessed in accordance with their corresponding binding energies.Te ligands' conformation was analysed through the use of Discovery Studio 2021.All molecular dynamics (MD) simulations were performed using the Desmond package.
Two independent replicates of the molecular dynamics simulations were performed for each investigated system, taking into account the intrinsic variety in molecular systems.Each replication started with unique random seed values for the initial velocities of atoms to introduce diversity in the trajectory sampling.Te decision to do several replicates is motivated by the understanding that a single trajectory may not fully capture the dynamic behaviour of the 2 Advances in Pharmacological and Pharmaceutical Sciences system and may yield results that are heavily impacted by the beginning conditions.To assess the statistical signifcance of our observations, we conducted separate analyses for each replicate.Tis approach allows us to determine and verify the coherence of patterns and the capacity to replicate the observed chemical interactions.By including several replicates, we greatly improve the trustworthiness of our results and get a more comprehensive understanding of the dynamics of the system.While examining the binding free energy landscape, we observed similar patterns throughout the replicates, indicating the presence of consistent binding events.Te RMSD profles from the two replicates exhibited similar patterns, confrming the reproducibility of the observed conformational changes in the protein-ligand complex.Furthermore, statistical calculations were performed independently for each duplicate to determine the mean binding afnities and residence lengths.Tis approach enables a more accurate computation of these parameters and also streamlines the assessment of the variability associated with the simulation outcomes.Te data presented in this article are the result of a comprehensive investigation of several duplicates, which improves the statistical significance and reliability of our molecular dynamics simulations.Te inconsistencies identifed in duplicate samples are thoroughly investigated in the subsequent sections, ensuring a comprehensive explanation of our fndings.

Results
Te structural integrity of the modelled FA6140 PBP2 was assessed using the Procheck software.Figure 1 depicts the Ramachandran plot analysis, which showcases the statistical distribution of the combinations of the backbone dihedral angles ϕ and ψ.Te Ramachandran plot delineates the permissible conformational space of the Phi/Psi torsion angles for an amino acid, X, situated within an ala-X-ala tripeptide, thus establishing the theoretically viable intervals.Te plot's narrative is dichotomised into two distinct domains predicated on the presence or absence of steric hindrances amidst atoms.Te region where steric collisions occur is conventionally designated as the disallowed region, while the region that lacks such collisions is commonly known as the allowed region.Te analysis of residue distribution within a protein molecule yielded signifcant insights pertaining to its structural and functional characteristics.Te study fndings indicate that a signifcant proportion of the protein, specifcally 91.7%, was situated in the most favourable regions, namely, A, B, and L. Additionally, a smaller proportion of 8.3% was observed in other allowed regions, including a, b, l, and p. Remarkably, residues were not detected within the permissive regions (∼a, ∼b, ∼l, and ∼p) or the impermissible regions.Te results indicate that the protein being studied exhibits a high degree of stability and proper folding, characterised by a compact core and limited conformational variability.Additional research is required to comprehensively comprehend the ramifcations of these fndings on the biological role of the protein.Te analysis encompassed a total of 869 residues.All of the 715 residues analysed were found to be nonglycine and nonproline residues.Furthermore, a total of 14 terminal amino acid residues (excluding glycine and proline) were detected.Te study revealed the identifcation of 90 glycine residues, which were denoted by triangles, and 50 proline residues.Te aforementioned fndings ofer signifcant insights into the composition of the scrutinised sample.Te identifcation of outliers in a dataset is a customary practice in statistical analysis.Outliers are data points that exhibit a substantial deviation from the remaining data.A common method for detecting outliers involves the implementation of a criterion based on a threshold of two standard deviations from the mean.Outliers in graphical data representations are commonly represented as black.Te small-molecule data are represented by the solid and dashed lines, denoting the mean and standard deviation values, respectively.Te Ramachandran plot analysis revealed a predominant 93.0% core region, accompanied by a minor 7.0% allowance region, while no residues were observed in the generously allowed or disallowed regions.Upon conducting an analysis of all Ramachandran, a total of three designated residues (out of 823) were detected.Additionally, on the chi1-chi2 plots, a total of seven designated residues (out of 451) were observed.Te side chain parameters exhibited 5 instances of improvement, 0 instances of being situated within the interior, and 0 instances of deterioration.Te analysis of residue properties revealed a maximum deviation of 11.8, 93 unfavourable contacts, 3.6 deviations in bond length and angle, and 4 cis-peptides.Te G-factors exhibited a dihedral value of −0.23, a covalent value of 0.46, and an overall value of 0.05.Te planar groups exhibited a 100.0%rate of conformity within the established parameters, with no instances of deviation or anomalies detected, as evidenced by the absence of any highlighted data points.Te congruence between a tridimensional atomic model and its corresponding unidimensional amino acid sequence was achieved by categorizing the model's structural class based on its specifc location and surrounding environment.Te outcomes were then juxtaposed against the established structural benchmarks.Te fndings indicate that a signifcant proportion of residues, specifcally 88.05%, exhibits an average 3D-1D score equal to or greater than 0.1.Figure 2 illustrates that a minimum of 80% of the amino acids have obtained a score of 0.1 or higher in the 3D/1D profle.Te structural integrity of the modelled FA19 PBP2 was assessed using the Procheck software.

Molecular Docking Analysis Using PyRx and Schrödinger.
Phytochemicals are natural molecules found in plants that have a crucial function in safeguarding them from both living and nonliving threats.Furthermore, these chemicals possess remarkable bioactive properties that might enhance human health and general well-being.Fruits and vegetables are the main sources of phenolic compounds in the human diet.Plant-based diets are known to include the main bioactive ingredients, which are these chemicals.Figure 3 displays a 2D chemical structure of the substance used in this experiment.Te current study utilised PyRx software to conduct molecular docking studies to assess the binding afnity between certain phytochemicals and the FA6140 PBP2 model.Te primary objective was to evaluate the binding energy of the mentioned chemicals.
In our investigation, we used computational techniques to replicate the process of binding and scrutinise the ensuing interaction.Te present study involved the performance of molecular docking analyses on all fve target compounds with the 3eqv [A] protein.Te experimental fndings suggest that the lowest binding energy was observed in the interaction between the 3eqv [A] FA6140 protein and rutin, followed by quercetin, ferulic acid, p-coumaric acid, and protocatechuic acid, as illustrated in Figure 4. Te binding energy is subject to notable infuence from the interactions that occur between ligands and the hydrophobic side chains present in proteins.It is widely recognised that hydrophobic amino acid residues demonstrate a tendency to repel water and other polar functional groups.Te depicted molecular interaction between rutin and FA6140 PBP2 results in a net attraction of the ligand's nonpolar groups.Tis phenomenon involves the association of nonpolar groups or molecules in an aqueous environment, as illustrated in Figure 5. Te results indicate that the compound rutin engages in noncovalent interactions with FA6140 PBP2, involving hydrogen bonding and hydrophobic interactions.Additionally, the research demonstrates that hydrophobic groups or molecules have a tendency to aggregate in an aqueous medium as a result of the hydrophobic efect.
In the context of the interaction between quercetin and FA6140 PBP2, the charge distribution within the molecule or complex system can be estimated or calculated.Te interpolated charge may be associated with the distribution of charges on the atoms within quercetin during the binding process.Having a clear grasp of the charge distribution is essential in order to fully grasp the electrostatic interactions between the ligand (quercetin) and the protein (FA6140 PBP2).We have extensive knowledge and expertise in the feld of bioinformatics.Te interaction between quercetin and FA6140 PBP2 is characterised by a robust binding affnity and specifc interactions with crucial amino acids.Te results obtained from the docking analysis confrm the molecular basis of the interaction and conformation of the specifed phytochemicals with the binding sites of FA6140 PBP2.Furthermore, the results indicate that the phytochemicals possess the capacity to establish numerous hydrogen bonds and noncovalent interactions with the crucial functional residues of the protein under investigation.Based on the empirical evidence, rutin demonstrates the most signifcant binding afnity towards the target protein when compared to the other compounds.Te molecule exhibits the capacity to form strong hydrogen bonds with essential functional amino acids, such as THR-485, SER-310, and a Pidonor hydrogen bond with THR-347, leading to a docking score of −8.1 kcal/mol.Te compound in question exhibited an interaction with the ALA-485 amino acid, which is classifed as an alkyl interaction.Furthermore, it demonstrates unfavourable donor-donor and acceptor-acceptor interactions with TYR-350 and SER-362, respectively, as illustrated in Figure 6.Quercetin displayed signifcant binding afnity to the target protein and formed strong interactions with the crucial functional amino acids, such as Pi-alkyl bonds at LER-165, ARG-75, PRO-72, and ALA-70; van der Waals bonds at GLY-186; Pi-anion bonds at GLU-164; and Pi-Pi stacked bonds and amine-Pi stacked bonds at TYR-201 and ASP-185, correspondingly.Te thermodynamic stability of the interaction was assessed by computing the docking score, which was found to be −7.6 kcal/mol.Furthermore, Figure 6 provides evidence of unfavourable interactions between the donor and acceptor moieties with TYR-350 and SER-362, respectively.
Te results indicate that hydrogen bonds were formed between ferulic acid and PRO-191 and ASN-299, while a Pialkyl bond was formed with HIS-177.An unfavourable acceptor-acceptor bond was observed between ferulic acid and SER-294, and a Pi-donor hydrogen bond was formed with ARG-297.Te binding afnity between ferulic acid and the aforementioned amino acids was calculated to be −6.0 kcal/mol.p-coumaric acid exhibited a binding afnity of −5.5 kcal/mol and engaged in hydrogen bonding interactions with the protein at SER-89 and ARG-75.Te compound exhibited interactions with LEU-165 through a Pi-alkyl bond, ASP-185 through an amine-Pi stacked bond, GLY-186 through a van der Waals bond, and TYR-201 Advances in Pharmacological and Pharmaceutical Sciences through a Pi-Pi stacked bond.Finally, the interaction between protocatechuic acid and PRO-397 was established through conventional hydrogen bonding and ASN-299 Pidonor hydrogen bonding, exhibiting a binding afnity of −5.3 kcal/mol.
Te outcomes derived from the docking analysis validate the molecular underpinnings of the interplay and confguration of the designated phytochemicals with the binding locales of FA19 PBP2.Te docking study's results are displayed in Table 1, which aimed to anticipate the binding  6 Advances in Pharmacological and Pharmaceutical Sciences and ASP-185, respectively; and Pi-alkyl bonds with ARG-75.Te thermodynamic stability of the interaction was evaluated through the calculation of the docking score, yielding a value of −7.8 kcal/mol, as presented in Table 1.Examining atom# 5, it possesses a "wrt_protein" value of 13.084 and a "wrt_ligand" value of 1.527.Tese fndings indicate that atom #5 in the protein exhibits a degree of fexibility, and there is a moderate level of variability in the ligand as well.Generally, the RMSF values ofer valuable information on the movement of atoms in both the protein and the ligand.Tis information is essential for comprehending the stability and interactions within the molecular system, as seen in Figure 7.

Molecular
Te torsion angles, also known as dihedral angles, in a molecular system are represented by data that are determined by the atomIDs in the system.Tese angles quantify the rotation between the planes of four successive atoms in the molecular structure.Te dihedral angle is determined by four atomIDs, namely, ai, aj, ak, and al.Every row in the data corresponds to a distinct frame or time point Advances in Pharmacological and Pharmaceutical Sciences  Te L_Properties are derived from a molecular dynamics simulation or similar computational research, and they represent distinct properties of a molecular system at diferent frames or time steps.Te simulation's frame or time step, which quantifes the average deviation of atomic locations between the current frame and a reference structure, provides insight into the extent of structural changes occurring during the simulation.Te radius of gyration provides a quantitative assessment of the molecular structure's compactness, while the intramolecular hydrogen bonds indicate the quantity of hydrogen bonds produced within the molecule.Te molecular surface area represents the overall surface area of the molecule, whereas the solventaccessible surface area specifcally indicates the portion of the molecule's surface that can be reached by solvent molecules.On the other hand, the polar surface area refers to the surface area of the molecule that is occupied by polar atoms.Our simulation revealed that the system experiences structural modifcations, characterised by an elevation in RMSD and variations in other characteristics.Notably, there is a large increase in RMSD, suggesting a more pronounced divergence from the initial structure.Some frames exhibit stabilisation, characterised by a generally constant rootmean-square deviation (RMSD) and other properties.Other frames, however, demonstrate oscillations in the system, but the properties do not display any major patterns.Tese interpretations are derived from overarching patterns, and a more comprehensive examination may be necessary to formulate precise conclusions on the behaviour of the molecular system during the simulation.
Figure 9 illustrates the progressive changes in the rootmean-square deviation (RMSD) of a protein, as indicated on the left Y-axis.Te protein frames are initially aligned with the reference frame backbone, and then, the rootmean-square deviation (RMSD) is computed using the selected atoms.Tracking the root-mean-square deviation (RMSD) of the protein can provide valuable information about its structural conformation during the simulation.RMSD analysis may determine if the simulation has reached equilibrium by examining the fuctuations near the conclusion of the simulation, which should be close to the average thermal structure.Small, globular proteins Advances in Pharmacological and Pharmaceutical Sciences may tolerate changes in the range of 1-3 Å without any issues.Signifcant alterations beyond the aforementioned magnitude suggest that the protein is experiencing a substantial structural transformation throughout the simulation.Additionally, it is crucial for your simulation to achieve convergence when the root-mean-square deviation (RMSD) values reach a stable and unchanging number.If the root-mean-square deviation (RMSD) of the protein exhibits a consistent increase or decrease during the simulation, it indicates that the system has not reached equilibrium.Consequently, the simulation duration may not be sufcient for rigorous analysis.Te ligand RMSD (shown on the right Y-axis) quantifes the stability of the ligand in relation to the protein and its binding pocket.Te graphic above displays the RMSD (root-mean-square deviation) of a ligand in the protein-ligand complex.Te alignment is performed by aligning the protein backbone of the complex to a reference structure, and then, the RMSD is calculated for the ligand's heavy atoms.If the measured values are considerably higher than the rootmean-square deviation (RMSD) of the protein, it is probable that the ligand has dispersed from its original binding site.Figure 10 illustrates the specifc interactions between ligand atoms and protein residues.Interactions that take place for more than 30.0% of the simulation duration in the chosen trajectory (0.00 through 100.00 nsec) are displayed.Interactions exceeding 100% can occur when certain residues form several interactions of the same kind with a single ligand atom.As an example, the ARG side chain possesses four H-bond donors, all of which may form hydrogen bonds with a single H-bond acceptor.Te fndings demonstrate Te interactions are standardised, enabling a precise comparison of their rates.For example, a number of 0.7 indicates that the particular contact was sustained for 70% of the duration of the experiment.It is worth noting that values greater than 1.0 can occur, suggesting that specifc protein residues may have numerous interactions of the same kind with the ligand.Tis extensive analysis enables researchers to comprehend the frequency and consistency of various interactions between the protein and ligand during the simulation, ofering vital insights into the dynamics of the molecular complex.Advances in Pharmacological and Pharmaceutical Sciences "wrt_Protein" and "wrt_Ligand."Te root-mean-square fuctuation (RMSF) is a valuable tool for quantifying localised variations along the protein chain.

Rutin-Penicillin
In Figure 12, the graphic demonstrates the dynamic behaviour of the protein during the experiment, with peaks denoting areas experiencing the most volatility.Generally, the N-and C-terminal tails display more signifcant changes compared to other areas.In contrast, secondary structural components such as alpha helices and beta strands exhibit more stifness in comparison with unstructured regions, leading to less fuctuation, particularly when compared to loop regions.Te ligand contacts section ofers signifcant insights into the protein residues that interact with the ligand.Tese interactions are graphically shown by vertical bars coloured in green.Understanding the particular residues involved in ligand interactions enhances our overall comprehension of the dynamics of protein-ligand binding during the simulation.Figure 13 depicts the essential simulation feature of monitoring protein interactions with the ligand.Te graphic presented above classifes and summarises these interactions based on their kind.Te interactions, also known as "contacts," are categorised into four primary types: hydrogen bonds, hydrophobic, ionic, and water bridges.Each type has further subtypes that are described in the "Simulation Interactions Diagram" panel.Hydrogen bonds have a crucial role in the binding of ligands, afecting the specifcity of drugs, their metabolism, and their adsorption.Tere are four subtypes under this category: backbone acceptor, backbone donor, side chain acceptor, and side chain donor.Te protein-ligand H-bond is evaluated based on precise distance and angle measurements, which provide a thorough insight into the dynamics of the interaction.Tis category has three subtypes: π-cation, π-π, and other nonspecifc interactions.Te geometric criteria for each subtype are defned by the proximity and orientation requirements, usually requiring a hydrophobic amino acid and an aromatic or aliphatic group on the ligand.Ionic interactions, also known as polar interactions, take place between atoms having opposing charges that are located within a specifc distance from each other.Tere are two distinct kinds, which may be diferentiated based on whether the contact is facilitated by the protein backbone or the side chains.Furthermore, the monitoring of protein-metalligand interactions includes the observation of a metal ion  that is coordinated within a particular distance of protein and ligand heavy atoms.Water bridges refer to the hydrogen-bonded connections facilitated by water molecules between the protein and ligand.Te geometric criteria for these interactions involve more lenient parameters in comparison with conventional H-bond defnitions, ensuring a subtle and detailed evaluation.Te stacked bar charts in the graphic are normalised based on the trajectory, representing the proportion of simulation time that each given interaction type is sustained.Values above 1.0 can occur, indicating cases when a protein residue establishes several interactions of the same subtype with the ligand.Tis research provides a thorough knowledge of the complex network of interactions that control the protein-ligand complex.It ofers useful insights for drug design and understanding structural dynamics.
Te graphic depicts the progression of the root-meansquare deviation (RMSD) of the protein and ligand during the simulation, illustrated in Figure 14.Te Y-axis on the left side depicts the root-mean-square deviation (RMSD) values of the protein, which indicate the degree of structural differences from the reference frame backbone.Tracking the root-mean-square deviation (RMSD) of proteins allows for the examination of the changes in structural conformation during the course of the simulation.Te presence of fuctuations around the thermal average structure at the end of the simulation indicates that equilibration is occurring.Small, globular proteins are deemed acceptable if they exhibit changes within the range of 1-3 Å.Nevertheless, more substantial modifcations may suggest noteworthy conformational shifts.
Te Y-axis on the right side of the graph represents ligand RMSD, which indicates the stability of the ligand in relation to the protein and its binding pocket.Te "Lig ft Prot" values represent the root-mean-square deviation (RMSD) of the ligand when it is aligned with the protein backbone of the reference frame.If the ligand RMSD values are signifcantly greater than the protein RMSD, it indicates the possible difusion of the ligand from its original binding site.Tis observation is essential for evaluating the stability of the ligand within the binding pocket and comprehending its behaviour with respect to the protein during the simulation.

Discussion
When dealing with gonococcal infections, the main objective of β-lactam antibiotics is to target penicillin-binding protein 2 (PBP2) in N. gonorrhoeae with precision.Certain strains of N. gonorrhoeae, such as FA6140, exhibit the addition of an aspartate residue following the 345th position in the PBP2.According to a source, there are 4-8 additional changes that occur alongside this insertion, referred to as Asp-345a.Nevertheless, the impact of these mutations on the protein's structural integrity remains uncertain.I used the crystal structure of PBP2 from the penicillin-resistant strain FA6140 [15] to perform the analysis on the target protein.Tis strain shows four mutations in the C-terminal region of the protein, resulting in a notable fvefold reduction in the rate of penicillin binding compared to the standard type.Trough extensive kinetic investigations, it has been discovered that the acylation rate experiences a signifcant decrease due to two specifc mutations, namely, P551S and F504L [13].Tese mutations also lead to a reduction in the enzyme's thermal stability, as evident from the melting curves.Furthermore, previous studies have highlighted the impressive antibacterial and antifungal properties of rutin, enabling it to efectively combat a wide range of harmful microorganisms.By exploring molecular interactions and binding afnities, our aim is to uncover promising inhibitors that can efectively hinder the activity of penicillin-binding protein 2. Tis research aligns with the ongoing eforts to develop bioactive inhibitors that are both safe and efcient for targeting viral proteins associated with COVID-19 [19].Additional investigation has unveiled the remarkable ability of rutin to efciently attach to the PBP2 of FA19 Neisseria gonorrhoeae, a vital catalyst in the creation of the microbial cellular barrier.Prior research has shown the efcacy of rutin in fghting gram-negative bacteria, indicating a possible mechanism that involves its interaction with PBP2 [20,21].Advances in Pharmacological and Pharmaceutical Sciences By studying the FA6140 and FA19 PBP2, one can gain a deep understanding of how rutin afects important proteins in both penicillin-resistant and penicillin-susceptible strains of N. gonorrhoeae.Tis study uncovers the complex interactions between these proteins and rutin.Having a deep understanding of the intricate molecular mechanisms involved in antibiotic resistance is of utmost importance.Tese specifc mutations have been discovered to interfere with the active site, resulting in challenges for antibiotics to bind or for essential conformational changes to take place during β-lactam antibiotic acylation.Furthermore, certain phenolic compounds have demonstrated exceptional antibacterial activity, in addition to their widely recognised antioxidant properties [15].A selection of fve compounds was made to investigate the interactions between the penicillin-resistant FA6140 PBP2 and certain phytocompounds with antibacterial properties.Te selection of these compounds was based on their prior in vitro antibacterial activity.Te study aimed to gain insights into the antibacterial mechanisms of these compounds, which have been observed to afect the permeability of cell membranes, interact with enzymes through hydrogen bonding to infuence intracellular functions, and modify the rigidity of cell walls.Tese interactions with the cell membrane can result in damage to its integrity, as shown in previous studies [6,[21][22][23].Advanced analytical techniques were employed to simulate the binding process and evaluate interactions.Extensive molecular docking analyses were conducted on all fve target compounds against the FA6140 PBP2 enzyme's 3eqv [A].According to the experimental fndings, it was noticed that rutin exhibited the lowest energy consumption during the binding process.After that, quercetin, ferulic acid, p-coumaric acid, and protocatechuic acid were observed in that specifc sequence.Tese fndings provide further support for previous in vitro research, suggesting that specifc favonoids, such as quercetin and rutin, demonstrate superior antibacterial efcacy in comparison with other compounds.Rutin, with its molecular formula C 27 H 30 O 16 , exhibits remarkable antioxidant properties, effectively diminishing various oxidising species such as superoxide, peroxyl, and hydroxyl radicals.In addition, it shows noteworthy interactions with the FA6140 PBP2 enzyme.Tis comprehensive analysis deepens our comprehension of the antibacterial properties of these compounds, particularly with regard to the penicillin-resistant FA6140 PBP2 enzyme.Moreover, these compounds exhibit pharmacological properties, including anticancer, antibacterial, and anti-infammatory efects.Our research indicates that both quercetin and rutin have the ability to bind to the substrate binding sites of both FA19 and FA6140 PBP2 enzymes.Te FA6140 PBP2 binding site shows a signifcant afnity for rutin, as indicated by its remarkable binding energy of −8.1 kcal/mol.Despite its strong binding afnity, rutin does not possess drug-like properties, according to Lipinski's rule.However, quercetin, with a log Po/w of 1.63 and no drug-likeness violations, seems to be a more encouraging prospect.In previous studies, it was noted that caulerpin adheres to Lipinski's rule and has a satisfactory ADMET profle, indicating its potential as a safe drug against the SARS-CoV-2-3CL main protease.Tere is potential for this natural compound to be utilised in drug development in the future [24].In line with this investigation, our research showcases the successful blocking of the FA6140 PBP2's main function by these plant compounds, leading to the prevention of the formation of penicillin-resistant N. gonorrhoeae bacteria.Quercetin exhibits a signifcant afnity of −7.6 kcal/mol for FA6140 PBP2, suggesting a strong attraction.Te primary factors that govern this interaction are the energies associated with van der Waals and electrostatic forces.Acquiring three-dimensional structures through crystallography can be quite a complex process due to various factors such as crystal packing and static disorder.Building upon our previous laboratory research, we discovered that favonoids such as quercetin and rutin have shown improved efectiveness.In our latest study, we found that protocatechuic acid exhibited a binding energy of −6.4 kcal/mol, indicating a strong afnity for the FA19 PBP2 binding site.It seems that protocatechuic acid has the ability to disrupt the main function of the FA19 PBP2, which could hinder the growth of penicillin-resistant gonorrhoea in cells.Studies have shown that rutin, with its remarkable antioxidant properties, has the ability to signifcantly mitigate the efects of diferent oxidising agents.In addition, it demonstrates a range of pharmacological activities, such as antineoplastic, antibacterial, and antiinfammatory properties.Upon analysing the interaction with FA19 PBP2, it was observed that both rutin and quercetin exhibited a signifcant afnity towards the substrate binding site.However, rutin displayed a higher binding energy of −8.1 kcal/mol.Stable complexes were observed in molecular dynamics simulations with FA6140 PBP2.After conducting a thorough analysis, it was noticed that the presence of rutin resulted in a reduction in the solvent-accessible surface area.Tere appears to be a favourable alignment between the ligand and the binding pocket.Flavonoids possess remarkable binding capabilities that surpass those of other plant compounds, making them highly intriguing substances for optimising leads and driving pharmaceutical development forward.As part of a broader  Advances in Pharmacological and Pharmaceutical Sciences context, this study emphasises the signifcance of creating medications for infectious diseases, with a particular focus on the urgent need for antiviral therapies that can combat the SARS-CoV-2 virus amidst the current pandemic [25,26].
Flavonoids have a strong binding afnity to the central functional units of FA6140 PBP2 and FA19 PBP2.Blind docking is a computational method that enables ligands to autonomously navigate the whole surface of a target protein without any prior knowledge of the binding location.Tis approach enhances our comprehension of the binding location and pharmacophoric interactions through many means.Blind docking is a method that may anticipate probable binding sites on a protein, uncovering specifc areas where ligands exhibit a strong attraction.Tis is particularly advantageous in cases where the specifc binding site is unidentifed or when there are several putative binding pockets.Ligands have the ability to interact with many parts of the protein surface, rather than being limited to specifc binding sites.Blind docking allows for a comprehensive examination, whereas focused docking studies provide specifc and detailed information about known binding areas.Te simultaneous use of both methodologies enhances our comprehensive comprehension of ligand-protein interactions.Te analysis of the interaction between rutin-and penicillin-suppressed PBP2, as well as the simulation of rutin-and penicillin-resistant PBP2, produced promising results.Tese results indicate a successful and accurate simulation.Te RMSD plot showed fuctuations within the range of 1-3 Å, suggesting that there were acceptable variations for small, globular proteins.Te simulation successfully reached convergence, displaying consistent RMSD values towards the end, indicating equilibration.Te RMSD fuctuations remained within 1.5 Å throughout the simulation, indicating a well-equilibrated system.Peaks of ligand RMSD were observed, which corresponded to areas of signifcant protein fuctuation, such as the N-and Cterminal tails.Te ligand RMSD displayed periodic peaks that coincided with protein fuctuations, yet it consistently stayed below 2.0 Å. Tis suggests that the ligand remains stable within the binding pocket.Te plot clearly highlighted the protein residues that interacted with the ligand, and we observed that structural changes were gained through the distribution of secondary structure elements (SSEs).Prominent ligand contacts were observed, with signifcant interactions in important secondary structure elements, demonstrating a strong binding pattern.Te ligand RMSF exhibited localised fuctuations, highlighting specifc atomlevel interactions that contribute to stable binding events.Te maintenance of hydrogen bonds and hydrophobic interactions was consistent throughout, as indicated by the normalised frequencies of binding.Te timeline representation provided a concise overview of the specifc contacts, while the schematic diagrams emphasised the interactions that occurred for a signifcant portion of the simulation time.
Te timeline representation revealed sustained contacts, while the schematic diagrams highlighted important interactions, giving a clear picture of the stable binding interface.Te ligand torsions displayed smooth transitions, and the radial plots indicated minimal conformational strain, thus confrming the stability of the ligand in the protein-bound state.Te simulation showcased promising attributes, including smooth RMSD, stable ligand behaviour, consistent interactions, and minimal conformational strain.
Tese fndings collectively suggest a dependable portrayal of the dynamics of the protein-ligand complex.In summary, these analyses ofer a thorough grasp of the dynamic behaviour of the protein-ligand complex, revealing insights into structural changes, stability, and interactions during the simulation.

Conclusion
We are investigating the antibacterial efects of rutin and other phytocompounds on two strains of N. gonorrhoeae in our research.Te binding afnity of rutin is highlighted through molecular docking analyses, suggesting its potential as an inhibitor of PBP2.Additional research on quercetin and protocatechuic acid highlights their potential as efective antibacterial agents, particularly quercetin, which exhibits properties similar to those of pharmaceutical drugs.Te molecular dynamics simulations ofer a thorough comprehension of the interactions between favonoids and PBP2.Te afnity of rutin for the substrate binding site indicates its potential to inhibit penicillinresistant N. gonorrhoeae, thanks to its impressive antioxidant properties.

Figure 1 :
Figure 1: Te identifcation of the allowed and disallowed regions of protein backbone conformation.

FA19Figure 2 :
Figure 2: Te congruence between a three-dimensional atomic model and the corresponding one-dimensional amino acid sequence of the FA19 PBP2 chain A.

Figure 6 :
Figure 6: A graphical representation of the interactions between the target protein and the selected ligands in two dimensions.(a) Protocatechuic acid.(b) Ferulic acid.(c) p-Coumaric acid.(d) Quercetin.(e) Rutin.
, SER-89, TYR-201 GLY-186; GLU-164; TYR-201, ASP-185, ARG-75 Rutin 5280805 −8.1 THR-485, SER-310, GLY-481, GLY-482 THR-347, ALA-496 8 Advances in Pharmacological and Pharmaceutical Sciences in a molecular dynamics simulation.Te columns display the numerical values of the dihedral angles (measured in degrees) for each specifed torsion (dihedral) in the system.Te numbers in each cell of the table represent the precise angle (measured in degrees) for the matching dihedral at a certain frame.Analysing a particular entry (e.g., entry at Frame 0, dihed1), the dihedral angle formed by atoms with the IDs 27, 24, 10, and 64 measures 24.876 degrees at Frame 0. Te dihedral angles exhibit variability across successive frames, suggesting the dynamic character of the molecular system, as seen in Figure 8. Te system investigates various torsional conformations, as indicated by the varying values of the dihedral angles.Positive and negative values signify the direction of rotation at these angles.Te ligand torsion graphic provides a concise representation of the changes in conformation of each rotatable bond (RB) in the ligand during the whole simulation track (0.00 to 100.00 nsec).Te upper panel displays a two-dimensional diagram of a ligand, with rotatable bonds highlighted using diferent colours.A dial plot and bar plots of the same colour are provided for each rotatable bond torsion.Dial (or radial) graphs depict the confguration of the torsion angle across the duration of the simulation.Te simulation starts at the central point of the radial plot, and the temporal progression is depicted in a radial outward manner.Te bar plots provide a concise representation of the data from the dial plots, displaying the probability density of the torsion.If data on torsional potential are provided, the fgure will further display the potential of the rotatable bond by aggregating the potential of the corresponding torsions.Te potential values are displayed on the Y-axis of the chart, positioned to the left, and are denoted in units of kcal/mol.Examining the correlation between the histogram and torsion potential might provide valuable information about the structural stress experienced by the ligand in order to retain its shape while attached to a protein.

Figure 7 :
Figure 7: Atomic fuctuations in protein and ligand: RMSF analysis of molecular dynamics simulation.

igure 8 :
Torsion angle profles in molecular dynamics simulation.10Advances in Pharmacological and Pharmaceutical Sciences that the ligand's interactions with proteins were consistently observed and categorised into certain kinds throughout the simulation.Te classifcation comprises hydrogen bonds, hydrophobic interactions, ionic interactions, and water bridges.Each of these interaction types possesses more precise subtypes, ofering intricate understanding of the characteristics of the interactions.Te graph displayed in Figure11provides a concise summary of the distribution of protein-ligand interactions across the simulation trajectory.

Figure 10 :
Figure 10: Exploring ligand-protein interactions: contact analysis between rutin and penicillin-binding protein 2 in Neisseria gonorrhoeae.

Figure 13 :
Figure 13: Protein-ligand contacts: categorisation and dynamics of interactions throughout the simulation.

Figure 14 :
Figure 14: Structural dynamics analysis: RMSD evolution of the protein and ligand in the simulation.

Table 1 :
Te molecular docking of various target compounds with the 3equ [A] protein.
Tis enables a thorough examination of the whole protein surface.Tis aids in comprehending the presence of alternate binding areas outside established locations.Blind docking can reveal allosteric binding sites, which are locations that are far from the active site and can infuence the activity of proteins.Tese websites are essential for comprehending regulatory systems.Ligands have the ability to assume diverse conformations during blind docking, which allows for a better understanding of their fexibility and the manner in which they interact with different areas of the protein.
Quercetin shows great potential as a promising candidate with strong attraction and drug-like properties.Te study's importance reaches beyond the Advances in Pharmacological and Pharmaceutical Sciences scope of infectious disease medications, highlighting the crucial role of antiviral therapies, particularly in the midst of the ongoing COVID-19 pandemic.Flavonoids have strong binding afnities to PBP2, indicating their potential as inhibitors to disrupt cell wall synthesis in N. gonorrhoeae.Our research ofers a thorough and insightful analysis of the protein-ligand interactions, revealing the antibacterial properties of favonoids against penicillin-resistant N. gonorrhoeae.Te simulation results indicating stability and accuracy provide valuable insights for the development of efective inhibitors and the advancement of pharmaceutical interventions against infectious diseases.