Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 1, April 2025 | Pages: 541-552 | DOI: 10.14421/biomedich.2025.141.541-552 ISSN 2540-9328 (online) Antimicrobial Potential of Phytochemicals from Coccinia grandis Leaves: A Molecular Docking Study Against Penicillin-Binding Protein 5 of Escherichia coli and DNA Topoisomerase IV Subunit B (ParE 24kDa) of Staphylococcus aureus and Escherichia coli Malshan Isuranga*, Dulki Nihinsa Danthanarayana Faculty of Science, University of Kelaniya, Sri Lanka. Corresponding author* malshanisuranga98@gmail.com Manuscript received: 06 February, 2025. Revision accepted: 20 May, 2025. Published: 04 August, 2025. Abstract Coccinia grandis (C. grandis) leaves, traditionally used in Sri Lanka for diabetes management, also have a potential antimicrobial activity. In this study, site-specific molecular docking was performed to investigate the antimicrobial activity of phytochemicals of Coccinia grandis leaves against Penicillin-binding protein 5 (PBP 5) and DNA topoisomerase IV subunit B (ParE 24kDa) of Escherichia coli (E. coli) and DNA topoisomerase IV subunit B (ParE 24kDa) of Staphylococcus aureus (S. aureus). Penicillin was selected as the reference molecule for Penicillin-binding protein 5 and for DNA topoisomerase IV subunit B (ParE 24kDa), Novobiocin was selected as the reference molecule. The results identified Lupeol (-7.72 kcal/mol) and Beta-Sitosterol (-8.21kcal/mol) have a higher binding affinity to PBP5 of E. coli than Penicillin (-7.20 kcal/mol). Quercetin (-6.70 kcal/mol), Kaempferol (-6.95 kcal/mol), Naringenin (-7.07 kcal/mol), Isoquercetin (-6.15 kcal/mol), Lupeol (-7.87 kcal/mol), Beta-Sitosterol (-9.42 kcal/mol) and Sanguinarine (-9.07 kcal/mol) show higher binding affinity to DNA topoisomerase IV subunit B (ParE 24kDa) of S. aureus than novobiocin (-6.04 kcal/mol). As well Quercetin (- 6.85 kcal/mol), Kaempferol (-6.82 kcal/mol), Naringenin (-7.23 kcal/mol), Isoquercetin (-6.20 kcal/mol), Lupeol (-7.67 kcal/mol), Beta- Sitosterol (-9.08 kcal/mol) and Sanguinarine (-9.03 kcal/mol) show higher binding affinity to DNA topoisomerase IV subunit B (ParE 24kDa) of E. coli than novobiocin (-5.76 kcal/mol). In silico pharmacokinetic and physicochemical parameter predictions were also conducted to study drug-likeness of above molecules using specialized web servers. Keywords: Coccinia grandis; Escherichia coli; Staphylococcus aureus; Penicillin-Binding Protein 5; DNA topoisomerase IV subunit B (ParE 24kDa); Molecular Docking. INTRODUCTION Antibiotics are the most wonderful innovation in the history of medicine. From the first antibiotic, Penicillin, discovered by Alexander Fleming in 1928, antibiotics have a long development history. These drugs have saved numerous numbers of lives worldwide. Some antibiotics kill the microorganisms while others reduce their growth rate. Antibiotic Resistance (ABR) is the ability of microorganisms to withstand the activity of the antibiotic. Mutations, Horizontal Gene Transfer, and Human Actions such as misuse of antibiotics contribute to the ABR (Larsson & Flach, 2022). According to the World Health Organization (WHO), ABR is one of the most significant global health threats (Global Antimicrobial Resistance and Use Surveillance System (GLASS) Report 2022, 2022). Coccinia grandis belongs to the family Cucurbitaceae, predominantly distributed in tropical Asia and Africa including Pakistan, India, and Sri Lanka (Farrukh et al., 2008). C. grandis is commonly known as Ivy Gourd or Scarlet Gourd in English; Kowakka in Sinhala. In Sri Lanka, this plant is predominantly distributed in the North Central, Southern, and Western regions. From centuries local population of Sri Lanka used leaves of this plant for diabetes management (Attanayake et al.,2016). Researchers suggest that leaf extract of C. grandis exhibits Anti-hyperglycemic, Xanthine Oxidase inhibitory, Analgesic, Anti- inflammatory, Antipyretic, Antioxidant, Anti- hyperlipidemic, Antimicrobial, and Anti-hepatotoxic activities (Ramachandran et al., 2014). Penicillin-binding protein 5 (PBP 5) of E. coli engages in cell wall synthesis. The main constituent of the bacterial cell wall is Peptidoglycan. PBP 5 enzyme performs a DD-carboxypeptidase reaction on the bacterial Peptidoglycan. The active site of PBP 5 contains a specific serine residue, which acts like a hook to grab a unit of the Peptidoglycan chain during the https://doi.org/10.14421/biomedich.2025.141.541-552 542 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 541-552 enzymatic reaction. Near the active site, two lysine residues (Lys47 and Lys213) play a critical role in proton-transfer events during acylation and deacylation events (Zhang et al., 2007). PBP 5 can be inactivated by β-lactam antibiotics such as penicillin. Inactivation occurs when the antibiotic forms a covalent bond with a serine residue in PBP 5, creating a stable complex that inhibits the protein’s enzymatic activity and disrupts its normal function. When PBP 5 binds to the antibiotic, it is dormant in an acylation state, thus, disrupting the cell wall synthesis, and resulting in cell death (Nicholas et al., 2003). DNA topoisomerase IV subunit B (ParE 24kDa) of Escherichia coli (E. coli) is an important enzyme that regulates DNA topology during replication and transcription. Its primary role is to separate the intertwined daughter chromosomes after DNA replication, ensuring that genetic material is correctly distributed to daughter cells. ParE functions with another subunit, ParC, to form a heterotetrameric complex (C2E2) that utilizes the energy derived from ATP hydrolysis to introduce and relax supercoils in DNA (Bellon et al., 2004). MATERIALS AND METHODS Ligand Preparation: Phytochemicals of Coccinia grandis leaves identified through a literature survey. The structure of the active site of E. coli PBP 5 was also obtained through literature survey. The structures of selected molecules obtained from PubChem database (PubChem, n.d.) in .sdf format. The energy-minimization of ligands was performed using Avogadro (version 1.2.0) software and saved in .pdb format. Figure 1. (A) Crystal structure of Escherichia coli PBP 5 in complex with a peptide-mimetic penicillin PDB DOI: https://doi.org/10.2210/pdb3BEB/pdb, (B) Crystal structure of E. coli Topoisomerase IV ParE 24kDa subunit PDB DOI: https://doi.org/10.2210/pdb1S14/pdb, (C) Crystal Structure of S. aureus ParE 24kDa in complex with Novobiocin PDB DOI: https://doi.org/10.2210/pdb4URN/pdb, (D) Structure of active site of E. coli PBP 5 (Sauvage et al., 2008), (E) Structure of active site of E. coli ParE 24kDa (Bellon et al., 2004) (F) Structure of active site of S. aureus ParE 24kDa (Lu et al., 2014b). Protein Preparation: The crystal structures of proteins were obtained from protein data bank in .pdb format (PDB ID – 3BEB: Crystal structure of E. coli penicillin-binding protein 5 in complex with a peptide-mimetic penicillin - PDB DOI: https://doi.org/10.2210/pdb3BEB/pdb (Sauvage et al., 2008), PDB ID – 1S14: Crystal structure of Escherichia coli Topoisomerase IV ParE 24kDa subunit - PDB DOI: https://doi.org/10.2210/pdb1S14/pdb (Bellon et al., 2004), PDB ID – 4URN: Crystal Structure of Staph ParE 24kDa in complex with Novobiocin – PDB DOI: https://doi.org/10.2210/pdb4URN/pdb (Lu et al., 2014). The protein was prepared using AutodockTools (version 1.5.7). Heteroatoms and water molecules were removed, and Polar hydrogen and Kollman chargers were added to the protein. AD4 type atoms were assigned to the protein. Molecular Docking: Autodock version 4.2.6 was used to perform site-specific molecular docking and results were generated in .dlg format (output – LamarckianGA-4.2). The grid parameters were set to cover the active site of each protein complex. A B C D E F Isuranga & Danthanarayana – Antimicrobial Potential of Phytochemicals from Coccinia grandis … 543 Table 1. Grid map dimensions (A0) and Grid-center coordinates (A0). Protein Complex Grid map dimensions (A0) Grid-center coordinates (A0) x-axis y-axis z-axis x-axis y-axis z-axis E. coli PBP 5 15.8 26.3 16.5 42.732 4.638 26.112 E. coli ParE 24kDa subunit 18.0 18.0 17.3 19.236 26.093 48.526 Staph ParE 24kDa subunit 17.3 21.0 19.5 -27.52 5.92 0.958 The genetic algorithm (GA) parameters were set to 50 runs with 300 population size. The maximum number of evaluations: 25,000,000 (medium) and maximum number of generations 27 000 (Lawan & Tharakee, 2023). Molecular dynamics simulation: The site-specific molecular docking results were analyzed using Autodocktools (Version 1.5.7). Binding energies and Inhibition constants were examined,and the interactions were visualized using Discovery studio visualizer (v24.1.0.23298). Drug-likeness and Pharmacokinetics of Selected Molecules: The potential of selected molecules as drugs was evaluated by assessing their drug-likeness and pharmacokinetic properties. Pharmacokinetics parameters of the selected molecules were predicted using the PkCSM server (https://biosig.lab.uq.edu.au/pkcsm/prediction) and SwissADME (http://www.swissadme.ch/index.php). These parameters describe how a drug behaves in the body including absorption, distribution, metabolism, excretion, and toxicity (Table 9). The drug-likeness of the molecules was analyzed using SwissADME (http://www.swissadme.ch/index.php), which evaluates molecules against Lipinski’s rule of five (Lipinski et al., 2001) and Verber’s rules (Veber et al., 2002) (Table 8). Docking Validation (Quality control): The docking procedure was validated by removing the inhibitor from each obtained protein complex and re- docking the inhibitor. The re-docked complex was aligned with the reference crystalized complex using PyMOL (version 2.5.8), and the difference in their positions was measured using the root mean square deviation (RMSD) (Shivanika et al., 2022). RESULTS Table 2. Calculated Binding Energies and Inhibition Constants of Selected Molecules against PBP 5 of E. coli. Molecule Reference RMSD (A0) Binding Energy (kcal/mol) Inhibition Constant-Ki (µM) at 298.15 K Penicillin 51.41 -7.20 5.32 Quercetin 49.21 -6.19 28.88 Kaempferol 49.70 -5.90 47.19 Naringenin 51.17 -6.27 25.47 Isoquercetin 51.46 -6.08 34.69 Lupeol 49.67 -7.72 2.19 Beta-Sitosterol 47.94 -8.21 0.953 Rutin 50.44 -5.24 143.35 Sanguinarine 48.95 -6.53 16.23 p-Coumaric Acid Unsuccessful Table 3. Calculated Binding Energies and Inhibition Constants of Selected Molecules against Topoisomerase IV ParE 24kDa subunit of E. coli. Molecule Reference RMSD (A0) Binding Energy (kcal/mol) Inhibition Constant-Ki (µM) at 298.15 K Novobiocin 57.21 -5.76 60.25 Quercetin 60.87 -6.85 9.45 Kaempferol 60.68 -6.82 10.08 Naringenin 60.92 -7.23 5.01 Isoquercetin 60.71 -6.20 28.57 Lupeol 59.13 -7.67 2.39 Beta-Sitosterol 58.98 -9.08 222.53 Rutin 58.62 -4.38 611.52 Sanguinarine 60.40 -9.03 0.241 p-Coumaric Acid Unsuccessful 544 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 541-552 Table 4. Calculated Binding Energies and Inhibition Constants of Selected Molecules against Topoisomerase IV ParE 24kDa subunit of S. aureus. Molecule Reference RMSD (A0) Binding Energy (kcal/mol) Inhibition Constant-Ki (µM) at 298.15 K Novobiocin 25.79 -6.04 37.19 Quercetin 24.15 -6.70 12.27 Kaempferol 25.23 -6.95 7.99 Naringenin 23.41 -7.07 6.63 Isoquercetin 25.88 -6.15 31.28 Lupeol 26.02 -7.87 1.70 Beta-Sitosterol 23.93 -9.42 0.125 Rutin 26.99 -5.42 106.89 Sanguinarine 23.39 -9.07 0.224 p-Coumaric Acid Unsuccessful Figure 2. (A) Superimposition of re-docked penicillin (yellow) on active site of crystalized structure (Green) of Escherichia coli PBP 5 using PyMOL (RMSD = 0.000 A0), (B) Superimposition of re-docked novobiocin (yellow) on active site of crystalized structure (Green) of E. coli Topoisomerase IV ParE 24kDa subunit using PyMOL (RMSD = 0.000 A0), (C) Superimposition of re-docked novobiocin (yellow) on active site of crystalized structure (Green) of S. aureus ParE 24kDa using PyMOL (RMSD = 0.000 A0). Figure 3. Interactions between selected molecules and Escherichia coli PBP 5 (A) Penicillin, (B) Quercetin, (C) Kaempferol, (D) Naringenin, (E) Isoquercetin, (F) Lupeol, (G) Beta-Sitosterol, (H) Rutin, (I) Sanguinarine A B C E A B C D F G H I Isuranga & Danthanarayana – Antimicrobial Potential of Phytochemicals from Coccinia grandis … 545 Figure 4. Interactions between selected molecules and Topoisomerase IV ParE 24kDa subunit of E. coli (A) Novobiocin, (B) Quercetin, (C) Kaempferol, (D) Naringenin, (E) Isoquercetin, (F) Lupeol, (G) Beta-Sitosterol, (H) Rutin, (I) Sanguinarine. Figure 5. Interactions between selected molecules and Topoisomerase IV ParE 24kDa subunit of S. aureus (A) Novobiocin, (B) Quercetin, (C) Kaempferol, (D) Naringenin, (E) Isoquercetin, (F) Lupeol, (G) Beta-Sitosterol, (H) Rutin, (I) Sanguinarine. A B C D E F G H I A B C 546 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 541-552 Figure 5. Cont. Table 5. Hydrogen bonding interactions (Intermolecular Conventional Hydrogen Bond) between ligands and PBP 5 of E. coli. Molecule Name Distance (A0) H-Donor H-Acceptor DHA Angle (0) Penicillin A:SER44:HG - :UNL1:N 1.70075 A:SER44:HG :UNL1:N 153.622 A:SER87:HN - :UNL1:OXT 2.15591 A:SER87:HN :UNL1:OXT 163.07 A:ASN112:HD21 - :UNL1:OXT 2.10638 A:ASN112:HD21 :UNL1:OXT 159.893 Lupeol :UNL1:H - A:SER44:OG 1.90396 :UNL1:H A:SER44:OG 147.908 Beta-Sitosterol :UNL1:H - A:ASN112:OD1 2.42915 :UNL1:H A:ASN112:OD1 137.911 Table 6. Hydrogen bonding interactions (Intermolecular Conventional Hydrogen Bond) between ligands and Topoisomerase IV ParE 24kDa subunit of E. coli. Molecule Name Distance (A0) H-Donor H-Acceptor DHA Angle (0) Novobiocin A:HIS1079:HE2 - :UNL1:O 2.89395 A:HIS1079:HE2 :UNL1:O 112.624 :UNL1:H - A:ILE1078:O 2.44228 :UNL1:H A:ILE1078:O 176.688 Quercetin A:ASN1042:HD21 - :UNL1:O 2.88793 A:ASN1042:HD21 :UNL1:O 101.638 :UNL1:H - A:GLU1046:OE1 2.19212 :UNL1:H A:GLU1046:OE1 114.214 :UNL1:H - A:GLU1046:OE1 2.12888 :UNL1:H A:GLU1046:OE1 137.021 :UNL1:H - A:VAL1039:O 2.9988 :UNL1:H A:VAL1039:O 91.088 Kaempferol A:ARG1072:HN - :UNL1:O 2.62735 A:ARG1072:HN :UNL1:O 101.889 A:GLY1073:HN - :UNL1:O 1.74046 A:GLY1073:HN :UNL1:O 144.668 :UNL1:H - A:VAL1039:O 2.02003 :UNL1:H A:VAL1039:O 176.714 :UNL1:H - A:ASP1069:OD1 2.30913 :UNL1:H A:ASP1069:OD1 114.577 :UNL1:H - A:GLU1046:OE1 2.04473 :UNL1:H A:GLU1046:OE1 167.188 Naringenin A:ARG1072:HN - :UNL1:O 2.89481 A:ARG1072:HN :UNL1:O 97.009 A:GLY1073:HN - :UNL1:O 1.85791 A:GLY1073:HN :UNL1:O 158.693 :UNL1:H - A:VAL1039:O 2.24921 :UNL1:H A:VAL1039:O 159.885 :UNL1:H - A:GLU1046:OE1 1.87455 :UNL1:H A:GLU1046:OE1 135.297 D E F H IG Isuranga & Danthanarayana – Antimicrobial Potential of Phytochemicals from Coccinia grandis … 547 Table 6. Cont. Molecule Name Distance (A0) H-Donor H-Acceptor DHA Angle (0) Isoquercetin A:ASN1042:HD21 - :UNL1:O 2.7382 A:ASN1042:HD21 :UNL1:O 100.17 A:ASN1042:HD21 - :UNL1:O 1.92763 A:ASN1042:HD21 :UNL1:O 164.831 A:GLY1073:HN - :UNL1:O 1.67752 A:GLY1073:HN :UNL1:O 141.974 :UNL1:H - A:ASN1042:O 2.23832 :UNL1:H A:ASN1042:O 102.23 :UNL1:H - A:GLU1046:OE1 1.86696 :UNL1:H A:GLU1046:OE1 133.412 :UNL1:H - A:GLU1046:OE1 2.33954 :UNL1:H A:GLU1046:OE1 128.18 :UNL1:H - A:VAL1039:O 2.95873 :UNL1:H A:VAL1039:O 103.791 Table 7. Hydrogen bonding interactions (Intermolecular Conventional Hydrogen Bond) between ligands and Topoisomerase IV ParE 24kDa subunit of S. aureus. Molecule Name Distance (A0) H-Donor H-Acceptor DHA Angle (0) Novobiocin A:ARG138:HH11 - :UNL1:O 2.07271 A:ARG138:HH11 :UNL1:O 116.16 :UNL1:H - A:THR92:OG1 2.30294 :UNL1:H A:THR92:OG1 142.972 :UNL1:H - A:GLY80:O 2.37333 :UNL1:H A:GLY80:O 114.849 :UNL1:H - A:GLU53:OE1 2.28073 :UNL1:H A:GLU53:OE1 153.157 :UNL1:H - A:ASP76:OD2 1.84227 :UNL1:H A:ASP76:OD2 129.527 Quercetin A:ARG79:HN - :UNL1:O 2.74275 A:ARG79:HN :UNL1:O 97.988 A:GLY80:HN - :UNL1:O 1.68791 A:GLY80:HN :UNL1:O 145.046 :UNL1:H - A:ILE46:O 2.13321 :UNL1:H A:ILE46:O 152.776 :UNL1:H - A:ASP76:OD1 2.27165 :UNL1:H A:ASP76:OD1 112.554 :UNL1:H - A:GLU53:OE2 1.92621 :UNL1:H A:GLU53:OE2 122.031 Kaempferol :UNL1:H - A:SER50:OG 2.19725 :UNL1:H A:SER50:OG 150.997 :UNL1:H - A:ASN49:O 1.75646 :UNL1:H A:ASN49:O 133.732 :UNL1:H - A:ASN56:OD1 1.98098 :UNL1:H A:ASN56:OD1 144.601 :UNL1:H - A:GLY80:O 2.00352 :UNL1:H A:GLY80:O 143.187 Naringenin :UNL1:H - A:GLY80:O 2.08123 :UNL1:H A:GLY80:O 138.264 :UNL1:H - A:ASP76:OD2 2.21723 :UNL1:H A:ASP76:OD2 145.165 :UNL1:H - A:THR168:O 2.91224 :UNL1:H A:THR168:O 111.039 Isoquercetin A:ASN49:HD21 - :UNL1:O 2.8621 A:ASN49:HD21 :UNL1:O 119.408 :UNL1:H - A:ASN56:OD1 2.05503 :UNL1:H A:ASN56:OD1 155.619 :UNL1:H - A:GLU53:OE1 1.71719 :UNL1:H A:GLU53:OE1 133.802 :UNL1:H - A:ASN56:OD1 2.06169 :UNL1:H A:ASN56:OD1 97.614 :UNL1:H - A:GLU53:OE1 2.16274 :UNL1:H A:GLU53:OE1 107.551 :UNL1:H - A:ASP76:OD2 2.11531 :UNL1:H A:ASP76:OD2 141.204 Sanguinarine A:ARG79:HE - :UNL1:O 2.65739 A:ARG79:HE :UNL1:O 90.653 Table 8. Predicted physiochemical properties of selected molecules (MW = Molecular Weight, NHA = Number of Heavy Atoms, NRB = Number of Rotatable Bonds, NHBA = Number of H-Bond Acceptors, NHBD = Number of H-Bond Donors, TPSA = Topological Polar Surface Area, O/W-PC = Octanol/Water Partition Coefficient, Lip = Lipinski’s rule of five. Veb = Veber’s rule, BAS = Bio-Availability Score. Molecule MW (g/mol) NHA NRB NHBA NHBD TPSA (Ų) O/W-PC (Mlogp) Lip Veb BAS Penicillin 334.39 23 5 4 2 112.01 1.55 Yes Yes 0.56 Novobiocin 612.62 44 10 11 5 200.01 0.65 No No 0.17 Quercetin 302.24 22 1 7 5 131.36 -0.56 Yes Yes 0.55 Kaempferol 286.24 21 1 6 4 111.13 -0.03 Yes Yes 0.55 Naringenin 272.25 20 1 5 3 86.99 0.71 Yes Yes 0.55 Isoquercetin 464.38 33 4 12 8 210.51 -2.59 No No 0.17 Lupeol 426.72 31 1 1 1 20.23 6.92 Yes Yes 0.55 Beta-Sitosterol 414.71 30 6 1 1 20.23 6.73 Yes Yes 0.55 Rutin 610.52 43 6 16 10 269.43 -3.89 No No 0.17 Sanguinarine 332.33 25 0 4 0 40.80 2.72 Yes Yes 0.55 548 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 541-552 Table 9. Predicted pharmacokinetic parameters of the selected molecules. Penicillin Novobiocin Quercetin Kaempferol Naringenin Absorption CaCo2 permeability (log Papp in 10-6 cm/s) 0.114 -4.836 -0.229 0.032 1.029 Water solubility (log mol/L) -2.47 -0.427 -2.925 -3.04 -3.224 Intestinal absorption (human) (% Absorbed) 59.901 66.418 77.207 74.29 91.31 Skin Permeability (log Kp) -2.735 -2.787 -2.735 -2.735 -2.742 P-glycoprotein substrate Yes Yes Yes Yes Yes P-glycoprotein I inhibitor No Yes No No No P-glycoprotein II inhibitor No Yes No No No Distribution VDss (human) (log L/kg) -1.905 -1.336 1.559 1.274 -0.015 Fraction unbound (human) (Fu) 0.328 0.127 0.206 0.178 0.064 BBB permeability (log BB) -0.864 (No) -1.914 (No) -1.098 (No) -0.939 (No) -0.578 (No) CNS permeability (log PS) -2.943 -3.492 -3.065 -2.228 -2.215 Metabolism CYP2D6 substrate No No No No No CYP3A4 substrate No Yes No No No CYP1A2 inhibitor No No Yes Yes Yes CYP2C19 inhibitor No No No No No CYP2C9 inhibitor No No No No No CYP2D6 inhibitor No No No No No CYP3A4 inhibitor No Yes No No No Excretion Total Clearance (log ml/min/kg) 0.197 -0.228 0.407 0.477 0.06 Renal OCT2 substrate No No No No No Toxicity AMES toxicity No No No No No Max. tolerated dose (human) (log mg/kg/day) 0.692 0.125 0.499 0.531 -0.176 hERG I inhibitor No No No No No hERG II inhibitor No Yes No No No Oral Rat Acute Toxicity (LD50) (mol/kg) 1.716 2.085 2.471 2.449 1.791 Oral Rat Chronic Toxicity (LOAEL) (log mg/kg_bw/day) 2.542 2.095 2.612 2.505 1.944 Hepatotoxicity Yes No No No No Skin Sensitization No No No No No T. Pyriformis toxicity (log ug/L) 0.285 0.288 0.288 0.312 0.369 Minnow toxicity (log mM) 3.698 1.146 3.721 2.885 2.136 Pharmacokinetic parameters Ligand Isuranga & Danthanarayana – Antimicrobial Potential of Phytochemicals from Coccinia grandis … 549 Isoquercetin Lupeol Beta- Sitosterol Rutin Sanguinarin e Absorption CaCo2 permeability (log Papp in 10-6 cm/s) 0.242 1.226 1.201 -0.949 2.107 Water solubility (log mol/L) -2.925 -5.861 -6.773 -2.892 -5.56 Intestinal absorption (human) (% Absorbed) 47.999 95.782 94.464 23.446 100 Skin Permeability (log Kp) -2.735 -2.744 -2.783 -2.735 -2.707 P-glycoprotein substrate Yes No No Yes Yes P-glycoprotein I inhibitor No Yes Yes No Yes P-glycoprotein II inhibitor No Yes Yes No Yes Distribution VDss (human) (log L/kg) 1.846 0 0.193 1.663 0.298 Fraction unbound (human) (Fu) 0.228 0 0 0.187 0.265 BBB permeability (log BB) -1.688 (No) 0.726 (No) 0.781 (No) -1.899 (No) -0.105 (Yes) CNS permeability (log PS) -4.093 -1.714 -1.705 -5.178 -1.419 Metabolism CYP2D6 substrate No No No No No CYP3A4 substrate No Yes Yes No Yes CYP1A2 inhibitor No No No No Yes CYP2C19 inhibitor No No No No Yes CYP2C9 inhibitor No No No No No CYP2D6 inhibitor No No No No Yes CYP3A4 inhibitor No No No No Yes Excretion Total Clearance (log ml/min/kg) 0.394 0.153 0.628 -0.369 1.051 Renal OCT2 substrate No No No No No Toxicity AMES toxicity No No No No Yes Max. tolerated dose (human) (log mg/kg/day) 0.569 -0.502 -0.621 0.452 0.172 hERG I inhibitor No No No No No hERG II inhibitor Yes Yes Yes Yes Yes Oral Rat Acute Toxicity (LD50) (mol/kg) 2.541 2.563 2.552 2.491 2.588 Oral Rat Chronic Toxicity (LOAEL) (log mg/kg_bw/day) 4.417 0.89 0.855 3.673 1.729 Hepatotoxicity No No No No No Skin Sensitization No No No No No T. Pyriformis toxicity (log ug/L) 0.285 0.316 0.43 0.285 0.308 Minnow toxicity (log mM) 8.061 -1.696 -1.802 7.677 -0.718 DISCUSSION Quercetin, Kaempferol, Naringenin, Isoquercetin, Rutin are considered as flavonoids. Lupeol is a pentacyclic triterpenoid, while Beta-Sitosterol is a Plant sterol (phytosterol). Sanguinarine belongs to Alkaloids. p- Coumaric Acid is a Hydroxycinnamic acid. The inhibitory constant, denoted as Ki (µM), is a measurement used in pharmacology to understand how tightly a drug binds (binding affinity) to a specific molecule (target molecule). It reflects the drug concentration required to occupy half of the available binding sites. A Lower Ki value indicates stronger binding. This means a lower drug concentration sufficient to occupy half (50%) of the available binding sites. Molecules with Ki values less than 100 µM are considered potent inhibitors while molecules with higher Ki values than 100 µM are considered as non-potent inhibitors (Zheng & Polli, 2010). Lupeol (-7.72 kcal/mol), Beta-sitosterol (-8.21 kcal/mol) show higher binding affinity to PBP 5 of E. coli than the Penicillin (-7.20 kcal/mol) which is the reference molecule. Both Beta-sitosterol (0.953 µM) and Lupeol (2.19 µM) show lower inhibition constant than Penicillin (5.32 µM), indicating higher potency than Penicillin (Table 2). Quercetin (-6.85 kcal/mol), Kaempferol (-6.82 kcal/mol), Naringenin (-7.23 kcal/mol), Isoquercetin (- 6.20 kcal/mol), Lupeol (-7.67 kcal/mol), Beta-Sitosterol (-9.08 kcal/mol) and Sanguinarine (-9.03 kcal/mol) show higher binding affinity to Topoisomerase IV ParE 24kDa subunit of E. coli than Novobiocin (-5.76 kcal/mol). Except for Beta-sitosterol, all above mentioned Pharmacokinetic parameters Ligand 550 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 541-552 molecules exhibit a lower inhibition constant than novobiocin (Table 3). Quercetin (-6.70 kcal/mol), Kaempferol (-6.95 kcal/mol), Naringenin (-7.07 kcal/mol), Isoquercetin (- 6.15 kcal/mol), Lupeol (-7.87 kcal/mol), Beta-Sitosterol (-9.42 kcal/mol) and Sanguinarine (-9.07 kcal/mol) show higher binding affinity to DNA topoisomerase IV subunit B (ParE 24kd) of S. aureus than novobiocin (-6.04 kcal/mol). All these molecules exhibit lower inhibition constant than novobiocin (Table 4). Among these molecules Beta-Sitosterol shows the highest binding affinity and the lowest inhibition constant towards Topoisomerase IV ParE 24kDa subunit of S. aureus. Interactions between a ligand and a protein are crucial for understanding biochemical processes, particularly in protein function and stability. Two major types of interactions that can be observed are hydrogen bonds (H- bonds) and hydrophobic interactions. Hydrogen bonds significantly contribute to the stability of the protein structure. Furthermore, H-bonds can provide specificity in ligand binding. The precise arrangement of H-bond donors and acceptors facilitates selective binding of ligands. Higher the number of H-bonds, greater the binding efficiency and the inhibition (Kumar et al., 2015). Hydrophobic interactions drive protein folding and significantly contribute to the stability of the protein. In H-bond interactions analysis, Penicillin forms three H-bonds with three amino acids (SER44, SER87, and ASN112) located in the active site of the PBP5. In the case of PBP5 and Lupeol one H-bonding interaction was observed (SER44), while PBP5 and Beta-sitosterol exhibit one H-bond (ASN112) (Figure 3 and Table 5). Two H-bonds were observed between the Topoisomerase IV ParE 24kDa subunit of E. coli and novobiocin (HIS1079 and ILE1078). Additionally, quercetin forms four H-bonds with three amino acids (ASN1042, GLU1046 and VAL1039) in the active site of this protein. Five H-bonds with five amino acids in the active site, were observed between kaempferol and the protein (ARG1072, GLY1073, VAL1039, ASO1069 and GLU1046). Similarly, four H-bonds with four amino acids in the active site, were observed between naringenin and the protein (ARG1072, GLY1073, VAL1039 and GLU1046). The highest number of H- bonds was observed between Isoquercetin and the protein, seven H-bonds with four amino acids in the active site (ASN1042, GLY1073, GLU1046 and VAL1039). Lupeol, beta-sitosterol and sanguinarine do not form H-bonds with the Topoisomerase IV ParE 24kDa subunit of E. coli, but they exhibit hydrophobic interactions with the protein (Figure 4 and Table 6). Considering the H-bonds between Topoisomerase IV ParE 24kDa subunit of S. aureus and novobiocin, five H- bonds were observed with five amino acids present in the active site (ARG138, THR92, GLY80, GLU53 and ASP76). Between quercetin and the protein, five H- bonds with five amino acids present on the active site, were observed (ARG79, GLY80, ILE46, ASP76, and GLU53). Kaempferol shows four H-bonds with the protein (SER50, ASN49, ASN56, and GLY80). Three H- bonds were observed between the protein and naringenin (GLY80, ASP76, and THR168). The highest number of H-bonds observed is six between Isoquercetin and the protein (ASN49, ASN56, GLU53, and ASP76). Sanguinarine exhibits one H-bond with the protein (ARG79). Both lupeol and beta-sitosterol show hydrophilic interactions with the protein but do not form any H-bonds (Figure 5 and Table 7). Considering the predicted physiochemical properties of selected molecules, Isoquercetin and Rutin do not fulfil Lipinski’s and Veber’s rules. Even novobiocin, a commercially available antibiotic, does not fulfil Lipinski’s and Veber’s rules. Except for Sanguinarine, none penetrates the Blood brain barrier (BBB). Oral rat acute toxicity (LD50) is defined as the amount of substance that is required to kill 50% of a tested population within a specific time frame. Among the selected molecules, all except Naringenin exhibit higher LD50 values compared to commercially available antibiotics which are Penicillin and Novobiocin. A higher LD50 value suggests that these molecules may be less toxic than Penicillin and Novobiocin. Hepatotoxicity which is also known as liver toxicity of the selected molecules was also evaluated. All the molecules, except Penicillin, were found to be non-hepatotoxic. When considering the number of selected molecules that exhibit higher binding affinity to the protein than the reference molecule, only two molecules (Lupeol and Beta-sitosterol) exhibit higher binding affinity than penicillin against PBP 5 of E. coli. In the case of Topoisomerase IV ParE 24kDa subunit, seven molecules exhibit higher binding affinity compared to the reference molecule which is novobiocin in both E. coli and S. aureus. Further studies should be conducted to investigate the combined effect of the molecules against each protein. These results reveal that selected phytochemicals show a higher inhibitory effect on the DNA Topoisomerase IV ParE 24kDa subunit compared to their effect on PBP5. This suggests that selected molecules may inhibit enzymes crucial to bacterial structural component synthesis and DNA replication. These molecules may exhibit a dual mechanism of action. This dual action mechanism may potentially kill or reduce the growth of microorganisms. The re-docking was done to validate the docking procedure. Penicillin bound to the active site of the Penicillin-binding protein 5 of E. coli with binding energy of -7.20 kcal/mol. Penicillin formed three H- bonds with three amino acids (SER44, SER87, and ASN112) located in the active site of the PBP 5 of E. coli with a distance ranging from 1.7 – 2.1 A0. These bonds are well within the optimal range for H-bonding, indicating a strong interaction. The re-docked complex Isuranga & Danthanarayana – Antimicrobial Potential of Phytochemicals from Coccinia grandis … 551 was then superimposed on to the native crystalized structure of E. coli PBP 5 from PDB using PyMOL observed 0.000 A0 RMSD value. The same methodology was followed for both Topoisomerase IV ParE 24kDa subunit of E. coli and S. aureus with novobiocin. Novobiocin formed two H-bonds with two amino acids (HIS1079 and ILE1078) located in the active site of the Topoisomerase IV ParE 24kDa subunit of E. coli with a distance ranging from 2.4 – 2.9 A0. Novobiocin formed five H-bonds with five amino acids (ARG138, THR92, GLY80, GLU53, and ASP76) located in the active site of the Topoisomerase IV ParE 24kDa subunit of S. aureus with a distance ranging from 1.8 – 2.4 A0. These bonds are well within the optimal range for H-bonding, indicating a strong interaction. The re-docked complexes were then superimposed on to their native crystalized structures using PyMOL and observed 0.000 A0 RMSD value (Figure 2). Obtaining 0.000 A0 for RMSD indicates no difference between the re-docked complex and the referenced crystallized structure. Overall, this study provides a strong foundation for further investigations to study anti-microbial activity of selected molecules derived from phytochemicals of Coccinia grandis leaves. These findings may pave the way for developing novel antibiotic agents with enhanced inhibitory efficacy. CONCLUSION Selected phytochemicals of Coccinia grandis leaves were evaluated in silico to determine their antimicrobial activity against Penicillin-binding protein 5 (PBP 5) and Topoisomerase IV ParE 24kDa subunit of Escherichia coli and Topoisomerase IV ParE 24kDa subunit of Staphylococcus aureus. Physiochemical and pharmacokinetic properties of selected molecules were also evaluated. Molecular docking, physiochemical and pharmacokinetic results were compared with reference molecules which are Penicillin and Novobiocin which are commercially available antibiotics. Out of the tested molecules, Lupeol and Beta- sitosterol exhibited better inhibition capabilities against PBP5 of Escherichia coli than Penicillin. Quercetin, Kaempferol, Naringenin, Isoquercetin, Lupeol, Beta- Sitosterol and Sanguinarine exhibit higher binding affinity to Topoisomerase IV ParE 24kDa subunit of both Escherichia coli and Staphylococcus aureus than Novobiocin. Selected natural molecules exhibit higher binding affinity towards Topoisomerase IV ParE 24kDa subunit than PBP5 of E. coli. Results in this study suggest that these selected phytochemicals of Coccinia grandis Leaves may serve as promising candidates for development of antimicrobial agents against resistant strains of Escherichia coli and Staphylococcus aureus. Further studies should focus on optimizing the structure of these selected molecules to enhance their binding affinity and the inhibitory potency. Acknowledgements: I would like to express my sincere gratitude to Dr. M.G.A.N. Perera from the faculty of Applied Science at the University of Sabaragamuwa for his invaluable guidance and support throughout this research. Authors’ Contributions: M. Isuranga designed the study. Computational analysis and data interpretation were conducted by M. Isuranga and D.N. Danthanarayana. The manuscript was written by both M. Isuranga and D.N. Danthanarayana. 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