Chromatographic Analyses, Virtual Screening and Pharmacokinetics of Yellow Malaysian Rambutan (Nephelium lappaceum L.) Fruit Epicarp Extracts Reveal Potential Antibacterial Compounds

The emergence and spread of antimicrobial resistance have been of serious concern on human health and the management of bacterial infectious diseases. Effective treatment of these diseases requires the development of novel therapeutics, preferably free of side effects. In this regard, natural products are frequently conceived to be potential alternative sources for novel antibacterial compounds. Herein, we have evaluated the antibacterial activity of the epicarp extracts of the Malaysian cultivar of yellow rambutan fruit (Nephelium lappaceum L.) against six pathogens namely, Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella enterica. Among a series of solvent extracts, fractions of ethyl acetate and acetone have revealed signicant activity towards all tested strains. Chemical proling of these fractions, via HPLC, LC-MS and GC-MS, has generated a library of potential bioactive compounds. Downstream virtual screening, pharmacological prediction, and receptor-ligand molecular dynamics simulation have eventually unveiled novel potential antibacterial compounds, which can be extracted for medicinal use. To this end, we report novel mechanistic aspects of these compounds in competitively inhibiting the ATP-binding domain of the DnaK chaperone of P. aeruginosa and MRSA. Our work takes a step forward to discover antimicrobials capable of perforating the barrier of resistance posed by both the gram positives and the negatives. Ethyl acetate and acetone extracts were used as samples for qualitative phytochemical screening via HPLC using Agilent-1260 innity system, according to the reported method of Zeb 76 . Briey, one-gram sample extract was mixed in methanol and water (1:1; 20 mL; v/v) and heated at 70˚C for 1 hour in water bath. This was centrifuged at 4000 rpm for 10 minutes and 2 mL of the supernatant was ltered into HPLC vials through Whatman lter paper. The separation was performed via Agilent-Zorbax-Eclipse column (XDB-C18). Column gradients system comprised solvent B and C. Solvent B consisted of deionized water: methanol: acetic acid having a ratio of 180: 100: 20; v/v while solvent C had deionized water: methanol: acetic acid in the ratio of 80: 900: 20; v/v. Gradient system was started by solvent B for 100%, 85%, 50% and 30% at 0, 5, 20 and 25 minutes followed by solvent C (100%) from 30-40 minutes. Elution occurred after 25 minutes. The ultraviolet array detector (UVAD) was set at 280 nm for the antioxidants analysis and chromatogram were documented using retention times. UV spectra of compounds and accessible standards along with quantication was carried out by taking the percent peak area. Quantication of the antioxidants was measured by formula:


Introduction
Antimicrobial resistance (AMR) has been projected as one of the serious global concerns with issues in the management of infectious diseases caused by Multidrug Resistant (MDR) bacterial pathogens 1 .
These pathogens have been reported to cause 700,000 deaths each year which is estimated to cross over 10 million by 2050 2 . Such alarming rise can be attributed mostly to the prevalent misappropriation of antibiotics in human healthcare systems 3 delineated by the overuse and poor appropriate prescriptions coupled with the lack of new drug development, which have reduced the e ciency of antibiotics as the resistant strains rapidly increases 4 . This necessitates the urgency for unearthing alternate natural therapeutics focused on natural products to be exploited as repertoires of natural bioactive compounds, preferably devoid of side effects and hence, potential for future drug development.
Natural products, frequently discovered with potent antimicrobial potential, have been used as alternatives with the hope of eliminating the use of synthetic antibiotics, since a long time 5 . Besides serving a widespread range of secondary plant metabolites, for instance, alkaloids, tannins, avonoids and phenolic compounds, with remarkable antimicrobial effects 6 , many plant products have been reported for their antimicrobial potentials, namely, citrus peel 7 , grape seed 8 , cranberry pomace 9 , pomegranate peel 10 and passion fruit seed 11 . In this regard, the fruit rambutan (Nephelium lappaceum L.) has gained attention due to its vast range of bioactive constituents including, but not limited to, vitamin C, vitamin E, carotenes, xanthophylls, tannins and phenolic compounds like, geraniin, ellagic acid, quercetin, corilagin and rutin 12 .
While the broad range of biological activities of rambutan include anticancer, antiproliferative and anti hypercholesterolemic properties 13,14 , few researchers have even reported its in vitro antibacterial activities. For instance, Malini & Maheshkumar 15 have disclosed signi cant antibacterial activity of rambutan fruit sap extracts towards Pseudomonas aeruginosa while Bhat and Al-daihan 16 revealed antibacterial activities of its seeds extracts against Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Escherichia coli and P. aeruginosa. Moreover, antibacterial potential of rambutan peel extracts have also been reported against Vibrio cholerae, Enterococcus faecalis, S. aureus and Staphylococcus epidermidis 17 . Furthermore, Sekar et al. 18 comparatively evaluated the antibacterial potency of red and yellow rambutan fruit peels against S. aureus and S. pyogenes, to reveal better e cacy of the latter extracts against the tested pathogens.
In this study, we have delineated a stepwise approach of determining the e cacy of the crude extracts of the epicarp of yellow Malaysian rambutan against clinically important MDR bacterial pathogens e.g. B. subtilis, methicillin-resistant S. aureus (MRSA), S. pyogenes, P. aeruginosa, K. pneumoniae and S. enterica. Further chemical pro ling of the extracts through HPLC, LC-MS and GC-MS revealed their potential chemical determinants which were screened virtually to pharmacologically unveil novel bioactive compounds through molecular dynamics simulation. To this end, a competitive inhibition of the ATPbinding domain of the DnaK chaperone of P. aeruginosa and MRSA, by our shortlisted compounds, portrayed novel antibacterial mechanism, representative of targeting both the gram-positive and thenegative pathogens.

Results
Variable antibacterial activity of crude extracts by disc diffusion A preliminary screening for the antibacterial activities of the yellow-variety Malaysian Rambutan epicarp crude extracts was assessed through disc diffusion assay. Only freshly prepared solutions were used for all the tested pathogens (TP) for the crude extracts. Throughout such trials, the solvent control (SC), DMSO, showing no inhibtion zone, did not exercise antibacterial activity against the TP. Moreover, none of the sequential extracts exhibited activity against the TP (Table 1, Fig. S1), while in case of direct extracts, the picture was quite different. Extracts of ethyl acetate (EA) displayed visible activity against MRSA, B. subtilis and S. enterica but did not show activity against the rest of the TP (RTP). Again, acetone (AC) extracts exhibited markedly prominent activity against B. subtilis along with visible acitivty against S. pyogenes and P. aeruginosa, which was not observed among the RTP (Table 1, Fig. S2).   Revelation of antioxidants from crude extracts using HPLC-UV For a preliminary identi cation of the basic antioxidants present in the Malaysian yellow-rambutan epicarp extracts, HPLC-UV was conducted. All compounds with known antioxidant capacities were identi ed in comparison with standard phenolic compounds. The identi ed compounds and their quanti cation, along with their speci c peak position and retention time (Rt) in chromatogram, are shown in Table 3 and Fig. S4. Only three compounds were identi ed in the EA extract namely, malic acid, vitamin C and chlorogenic acid along with three more in the AC extract. These are epigallocatechin gallate, catechin hydrate and quercetin. Identi cation of volatile constituents by Gas chromatography-mass spectrometry (GC-MS) To identify any volatile organic compounds, present in the EA and AC extracts of Malaysian yellow-variety N. lappaceum epicarp, they were exposed to GC-MS analysis (Fig. S7 a-d). Most of the compounds from EA and AC fractions, extracted directly, have been reported with antibacterial activities (Table 4). On the contrary, the compounds of these fractions from sequential extraction have not been reported for any such activity. The chromatogram of these compounds showed mentionable area % scores (above 0.5%)   (Table 4). Of these, compound 2 is known as Eplerenone ( Fig. 1) and was found to be an important one in the upcoming analyses. Ensuing virtual screening of 91 chemical compounds obtained through the chromatographic analyses, 41 of them, having binding energy less than − 7 kcal/mol, were considered as good binders (Table S9; Fig. 1, 2A). Among these, potential drug candidates were chosen based on the predicted pharmacokinetic properties. For example, good gastrointestinal (GI) absorption, bad BBB permeability, and non-Pglycoprotein (PGP) substrates were preferred for absorption properties. For metabolism, non-cytochrome P450 inhibitors were preferred. Violations on drug-likeness rules were, of course, not favored. To cater to the need, ve rules have been considered, namely, the Lipinski, Ghose, Veber, Egan, and Muegge rules [25][26][27][28][29] . Lastly, higher bioavailability scores were preferred. The Abbot Bioavailability Score utilized herein was to predict chances of drug bioavailability to be more than 10% upon oral intake 30 .
Among the virtually screened compounds, catechin (C), eplerenone (E) and oritin-4-beta-ol (O) stood out to be good binders with their average binding energies being − 8.205, -7.980 and − 7.190 kcal/mol, respectively for S. aureus (Sa) and P. aeruginosa (Pa) DnaK proteins. C, E, O also exhibited good predicted pharmacological properties except that C is a PGP substrate ( Fig. 1, Table S9). The binding conformations of C ( Validation of inhibitory effects of selected compounds by Molecular Dynamics Simulation Molecular Dynamics (MD) simulations for 10 nanoseconds were carried out for C, E, and O Ligand-SaD/PaD complexes to observe ligand-receptor interactions. Over the course of MD simulations, the ligands were retained in the docking pocket of respective DnaK receptors, except for C in SaD system (CSaD) of which the ligand seemed to be escaping from the initial binding pocket ( Fig. 3A; B). Moreover, the upper part of the DnaK NBD domain was completely disintegrated in CSaD. Besides, the total number of receptor-ligand intermolecular hydrogen bonds were maintained stably at around 4 and 5 in P. aeruginosa DnaK complexed with C (CPaD) and O (OPaD) respectively, and 4 in S. aureus DnaK complexed with O (OSaD) (Fig. 3C). Moreover, both the E complexes of SaD (ESaD) and PaD (EPaD) have maintained the total number of hydrogen bonds at around 1. However, in CSaD, a sharp decline in the number of intermolecular hydrogen bonds can be observed at 5 ns time point from around 4 to between 0 and 1, which can explain the escape of ligand from its initial docking pocket. Stable active residues have been observed in CPaD (LYS 70, GLU 171, GLU 267), EPaD (ARG 345), and OPaD (THR 11, ASP 194) complexes, as well as in OSaD (GLY 312) complex (Fig. 3G).
In all MD simulation systems, the root-mean-square uctuations of DnaK receptor maintained at around 0.5 nm (5 Å) except for the C-terminal end where the disordered regions were localized (Fig. 3E). Besides, the RMSD of C and E maintained at 0.5 nm in PaD receptor, while higher RMSD values of around 0.8 nm have been observed in CSaD and ESaD (Fig. 3D). The ligand RMSD were relatively lower in O, compared to others, for which RMSD of 0.4 nm was reported in both SaD and PaD cases. The interaction energies of all systems maintained stably throughout the simulation, except for CSaD complex of which a sharp decrease of Coulomb potential can be observed at around 6 ns time point (Fig. 3F). In general, E maintained the lowest total interaction energies, followed by C and O (Fig. 3H).

Discussion
Over the years, the commendable development in the eld of virtual screening has enabled time-and cost-e cient drug discovery along with repurposing 31 . Herein, we have carried out a scaffolded approach to antimicrobial drug discovery from yellow variety of Malaysian N. lappaceum L. fruit epicarp crude extracts. The rst upstream set of experimental work comprised the extraction of the plant product, followed by characterization of their antimicrobial property and chromatographic identi cation of chemical compounds from therein. This was coupled with downstream set of computational analyses comprising virtual screening and pharmacological predictions of extracted chemical compounds against potential drug targets. To this end, molecular dynamic simulation has taken a step forward to uncover new potent bioactive compounds which can target both gram negative and positive bacteria at the same time. Our study delineates a method to uncover potent chemicals which might have contributed in the antibacterial activities of plant products like Nephelium lappaceum epicarp, to be further utilized for drug discovery, repurposing, or other ab initio synthetic enhancements.
Extraction is the key stage to obtain the diverse bioactive chemical compounds from plant products.
These chemical determinants display different solubility with different organic solvents such that a screening with different solvents helps to bring forth the best one for further exploration 32 . Thus, in order to explore the extraction of biologically active constituents, several organic solvents were utilized in our study. This was initiated with a sequential extraction process of utilizing solvents like chloroform (CF), ethyl acetate (EA), acetone (AC), ethanol (ET), methanol (MT) and water (WT), in order of their increasing polarity. Our study revealed that the yellow variety of Malaysian N. lappaceum epicarp crude extracts exhibited varied inhibitory activities against the six tested MDR pathogens, namely, B. subtilis, methicillinresistant S. aureus (MRSA), S. pyogenes, P. aeruginosa, K. pneumoniae and S. enterica. In fact, the EA and AC fractions from the sequential mode remarkably inhibited about all the Gram-positive and Gramnegative tested pathogens (TP) while the remaining solvent fractions responded moderately or poorly, thereby providing a strong clue to proceed for further direct extraction from these solvents. To this end, an extensive spectrum of chemical classes was revealed after LC-MS analysis and included, terpenes, alkaloids, polyunsaturated and monounsaturated fatty acids among others, that were present in both the extracts. Among these, only 21 of the 54 compounds (with above 86% MFG scores) of the EA fractions have been reported to possess antibacterial activities (Table S3) With a set of 91 compounds obtained through chromatographic analyses, we have conducted a computational analysis for virtual screening through molecular docking to short list a selective set of chemicals via pharmacokinetics consideration (Table S9). Pharmacokinetics is an important criterion when it comes to drug discovery and drug design, especially pertaining to bioavailability and toxicity. Herein, we have considered several parameters for absorption, metabolism, drug-likeness, and bioavailability for selecting the ideal drug for potential pharmacological application in the future. For instance, good GI absorption can allow absorption into bloodstream during oral consumption, while bad blood-brain barrier (BBB) permeability can avoid interruption to the central nervous system 59  crucial proteins indispensable for the cellular integrity of the bacteria 70 . Also, the ATP-binding pocket of DnaK chaperone has been indicated to be druggable and shown promise to cope with MDR in both gram negatives and positives (Tan & Lahiri, unpublished data). Hence, DnaK protein has been selected for the in-silico study, herein, as a promising drug target for MDR bacteria by inhibiting its ATP binding pocket, which can result in its impairment of chaperone function.
Through our computational screening of the chemical libraries of the N. lappaceum L. fruit epicarp extractions, Catechin (C), Eplerenone (E), and Oritin-4-beta-ol (O) were shortlisted as the promising antimicrobials in combating the MDR pathogens by dint of their capacity in targeting the DnaK protein and having good pharmacological pro les. Despite being PGP substrate, C has manifested strong binding a nity to DnaK and therefore, can result in effective DnaK functional inhibition with a small amount. Otherwise, PGP inhibitors like C can be co-prescribed easily as it has a good metabolic pro le.
Moreover, C has been well-characterized for its antibacterial activities and known for its ability to cause leakage of bacterial cellular contents along with increased intracellular reactive oxygen species production in both gram negatives and positives 71,72 . However, the biological targets of C have not been described. As DnaK protein is crucial in bacterial stress response, by inhibiting the DnaK chaperone function, the bacterial cellular and biomolecular integrity can be affected upon receiving environmental oxidative stress. Herein, we showed that in P. aeruginosa, C could bind stably to the ATP-binding pocket of DnaK throughout the MD simulation with 3 stable active residues (LYS 70, GLU 171, and GLU 267), while maintaining the ATP-bound conformation of the DnaK protein without the necessity for ATP binding (Fig. 2B, 3A). This re ected the inability of the ATP molecules to bind the CPaD (Catechin-bound DnaK protein of P. aeruginosa) as also a complete halting of the normal DnaK chaperone function via conformational changes ensuing ATP hydrolysis. However, C could not inhibit SaD (DnaK of S. aureus) the same way, due to its inability to maintain the integrity of NBD domain and thereby escaping from the binding pocket. It is this binding pocket which allows subsequent binding of ATP molecules on DnaK to continue the chaperone function. On the contrary, herein, we present the discovery of two novel potential compounds, E and O, whose antibacterial activities have not been reported and/or described earlier.
Notably, E has been widely utilized in cardiovascular implications and as diuretics 73,48 . O, however, has not been explored to confer any biological signi cance. Despite that, it is notable that the chemical structure of O is analogous to C (Fig. 1), with the sites of hydroxylation being slightly different.
Throughout the molecular dynamics simulation (MDS) processes, only 1 or 2 hydrogen bonds can be observed in EPaD and ESaD, which suggested weak protein-ligand electrostatic interactions. This can be explained by the chemical structure of E, being crowded with carbonyls and ethers which are weak bases, and hydroxyl groups are lacking. The ligand, however, has been retained in the docking pocket over the course of MDS. This probably suggests that hydrophobic (van der Waals) interactions were dominant in this case. This was re ected through the intermolecular interaction energies (Fig. 3F), of which the Lennard-Jones potentials were much higher than Coulomb potentials in Eplerenone-DnaK (ED) complexes, while the reverse was observed in for C and O. Moreover, the binding conformation of E in PaD did not "cover up" completely at the binding site of phosphate groups of the ATP for which further wet-lab con rmation is required. Furthermore, among the three ligands simulated, O manifested the best binding capabilities to both PaD and SaD with rich intermolecular electrostatic interactions and highest total interaction energies. After MDS, the active residues THR 11 and ASP 194 were retained in OPaD, while GLY 312 was retained in OSaD. Again, despite being structurally analogous to C, O manifested good predicted pharmacological properties in all the aspects considered. Therefore, with better binding capabilities to DnaK receptor and pharmacological properties, herein we report O to be a more potent antibacterial compound compared to the well-known C, which is active against both the gram positive and negative bacteria.

Conclusion
Our ndings reinstate the promising antibacterial effects, of the yellow variety of Malaysian Rambutan (N. lappaceum L.) fruit epicarp crude extracts, against selected Gram-positive and Gram-negative MDR pathogens. In this context, particularly ethyl acetate and acetone (sequential and direct) extracts demonstrated remarkable antibacterial effects toward all tasted pathogens, while remaining fractions including, chloroform, ethanol, methanol and water did not exhibit such potential. Nevertheless, N. lappaceum presents itself to be a novel source for antibacterial compounds with high potential for the development of pharmaceutically valuable drugs. Further studies are mandatory to separate the speci c compound(s) responsible for the desired effects, and to develop our knowledge on the other unseen potentials in N. lappaceum.

Materials And Methods
Solvents.
All solvents, used for preparation of crude extractions, were of HPLC grades In order of increasing polarities, these were Chloroform (99.9%, Sigma-Aldrich, LiChrosolv, Malaysia), Ethyl acetate, Acetone Preparation of crude extracts.
Epicarp crude extracts were prepared following the method of Do et al. 75 using the solvents mentioned earlier for the direct extracts. For the sequential method of extraction, the mentioned solvents were used in order of increasing polarity viz chloroform<ethyl acetate<acetone<ethanol<methanol<water. In both the cases, essentially, the peels of N. lappaceum were removed from the fruit and washed thoroughly with running, followed by, distilled water to remove contaminants and thereafter dried using freeze-dryer. Dried peels were ground into ne powder using an electric grinder. To produce different fractions of crude extracts, 10 g of powder was extracted in 100 mL of selected solvents. The solution was mixed thoroughly by using incubator shaker (Yihder LM-530D Incubator Shaker, Taiwan) for 24 h. To separate supernatant, the solution was centrifuged (Eppendorf 5810 R Centrifuge, Germany) at 4000 rpm for 10 min at 4 °C to completely eliminate the leftover ne sediments. The solvent extracts were concentrated using Rotary evaporator, and further with vacuum concentrator until a viscous extract was obtained. All extracts were stored at 4°C for future experiments.
Potential in-vitro antibacterial activities of yellow rambutan fruit epicarp extracts.

Disc Diffusion assay.
Seed culture of the tested pathogen was consistently swabbed on agar plate. Sterilized blank paper discs were separately impregnated by different concentration of extracts (250 to 2000µg/ml) and placed on agar plate. The plates were incubated at 37ºC for 16h. The antibacterial activity was noted by measuring the diameter of inhibition zone. Gentamicin (10µg/disc) was used as positive control while DMSO (<1%) was kept as negative control. All the experiments had technical triplicates and were performed twice to render two biological replicates.
Broth Dilution Assay. Exploration of chemical constituents through chromatographic analyses.
Ethyl acetate and acetone extracts were used as samples for qualitative phytochemical screening via HPLC using Agilent-1260 in nity system, according to the reported method of Zeb 76 . Brie y, one-gram sample extract was mixed in methanol and water (1:1; 20 mL; v/v) and heated at 70˚C for 1 hour in water bath. This was centrifuged at 4000 rpm for 10 minutes and 2 mL of the supernatant was ltered into HPLC vials through Whatman lter paper. The separation was performed via Agilent-Zorbax-Eclipse column (XDB-C18). Column gradients system comprised solvent B and C. Solvent B consisted of deionized water: methanol: acetic acid having a ratio of 180: 100: 20; v/v while solvent C had deionized water: methanol: acetic acid in the ratio of 80: 900: 20; v/v. Gradient system was started by solvent B for 100%, 85%, 50% and 30% at 0, 5, 20 and 25 minutes followed by solvent C (100%) from 30-40 minutes. Elution occurred after 25 minutes. The ultraviolet array detector (UVAD) was set at 280 nm for the antioxidants analysis and chromatogram were documented using retention times. UV spectra of compounds and accessible standards along with quanti cation was carried out by taking the percent peak area. Quanti cation of the antioxidants was measured by formula: Cx= Sample concentration; As= Standard peak area; Ax= Sample peak area; Cs= Standard concentration (0.09 µg/ml).

Liquid Chromatography and Mass Spectrometry (LC-MS).
A mixture of standards and new metabolites found in the ethyl acetate and acetone fractions were analyzed via LC-MS exactly as per the method reported by Yap et al. 77 . In order to eradicate systematic errors, reference solution was used with the two ions, having m/z of 121.0508 and 92266.0097, being selected for mass calibration. Gas chromatography-mass spectrometry (GC-MS).
Ethyl acetate and Acetone fractions were subjected to gas chromatography-mass spectrometry (GC-MS) analysis, using Agilent technologies model 7890B GC System coupled with Pegasus HT High Throughput TOFMS (Leco Corp., MI, USA). An aliquot of an extract of 1ml was injected to the GC-MS apparatus. Next, Agilent J&W HP-5MS (phenyl methyl siloxane, length 30 m, Dia. 0.32 mm, Film, 0.25µm) analytic column was used to separate components under an inert atmosphere of helium (1.5 mL/min).
Other standardized parameters utilized during the process: oven temperature of 80°C (2 min) was increased to a temperature of 300°C at the rate of 3°C/min, solvent delay time was 5 min, inlet line temperature was 225°C, and ion source temperature was 250°C. Mass spectra were taken at 70 eV and acquisition mode-scan was 20-1000 amu while sixty-four (64)  For Sa DnaK (SaD), besides the aforementioned Ec DnaK (EcD) models, one additional template, from Geobacillus kaustophilus DnaK protein (PDB ID: 2V7Y, Chain: A), was selected due to the high percentage of sequence identity expected as per the gram positive character of S. aureus and G. kaustophilus. As this template structure was in closed conformation and we were only interested in the open conformation, only the Nucleotide Binding Domain (NBD, residues 1 to 350 in template model) which does not differ much in both conformations, were taken into consideration for homology modelling, and the remaining Cterminal residues modelling were guided by the Ec models to shape an open conformation. Therefore, the templates for SaD were (PDB ID: 2V7Y, Chain: A, Template Residues: 1-350, QC: 57%, PI: 83.19%, R: 2.37 Å; PDB ID: 5NRO, Chain: A, QC: 93%, PI: 56.19%, R: 3.25 Å; PDB ID: 4JNE, Chain: A, QC: 92%, PI: 55.54%, R: 1.96 Å; and PDB ID: 4B9Q, Chain: A, QC: 94%, PI: 55.43%, R: 2.40 Å). 5 homology models were generated for each protein of SaD and PaD, and the models with lowest DOPE (discrete optimized protein energy) scores were selected for downstream virtual screening for both. The SaD and PaD homology models were validated via Swiss-Model Structure Assessment and SAVES v5.0 servers 80 (Table S7).

Druggable Pocket Validation.
To validate the druggability of the ATP docking pocket, we have conducted ligand binding site prediction using P2Rank from PrankWeb server 81 . P2Rank predicts the chemical druggability on protein solventaccessible surface via a non-templated machine learning approach. The ATP binding pocket was predicted to be druggable and ranked rst in both cases of SaD and PaD (Table S8; Fig. S8). These pockets from SaD and PaD were further considered to be targeted for virtual screening.
Molecular Docking with Chemical Determinants. POAP pipeline, Samdani & Vetrivel 82 was followed for an in silico virtual screening of the chemical compounds obtained through different chromatographic separation. SMILES notations of these compounds were obtained and their 3D models (in mol2 format) were generated through POAP Ligand Preparation pipeline. To this end, Chimera was utilized to generate physiological protonation states of ligands, and PDBQT les were prepared 83 . Ligand optimizations were carried out via POAP Ligand Preparation pipeline utilizing MMFF94 force eld, being optimized for drug-like organic molecules and molecular docking 84 . Out of the 50 conformers, generated for each ligand through Weighted Rotor Search approach, only the best conformers were retained. Finally, the ligands were subjected to energy minimization for 5000 steps by the conjugate algorithm.
The macromolecule receptors, pertaining to the SaD and PaD proteins, were prepared using AutoDockTools. AutoDock 4.2, aided by POAP pipeline, was utilized for the virtual screening process 85 . For AutoDock parameters, 100 generations of Lamarckian Genetic Algorithm were set for each proteinligand complex. To t in the previously predicted pocket, docking grids were adjusted into squares of 24 Å with x, y, z coordinates of 17  Chemical Structure of Catechin, Oritin-4-beta-ol, and Eplerenone.

Figure 2
Results of virtual screening targeting P. aeruginosa and S. aureus DnaK proteins. A. Distribution of binding energies were plotted against screened compounds. Top binders with good pharmacological properties, which are B. Catechin, C. Eplerenone, and D. Oritin-4-beta-ol were selected for analyses. P2Rank predicted binding pockets were coloured in green for visualization of ligands (magenta) binding to the receptor DnaK proteins (brown), with active sites (yellow) labelled. E. Intermolecular hydrogen bonds were tabulated with active residues listed. Figure 3