Naturally Occurring Quorum Sensing Inhibitors for Pseudomonas aeruginosa by Molecular Modeling

: The quorum-sensing (QS) system allows organisms to communicate and form virulence factors such as biofilm. Pseudomonas aeruginosa is one of the Gram-negative bacteria leaving many casualties every year. In the past years, the bacteria's QS system has gained attention as a valuable target to design and develop new efficient antibiotics. Nature has always been an inspiration source for designing many new medicines, and using natural compounds is always the first approach to discover and design new drugs. Here, we in-silico studied 24,000 natural compounds, including first and secondary metabolites, on five known QS-related receptors of Pseudomonas aeruginosa . After several levels of screening, compounds NP-1 (3,6′-di-O-sinapoylsucrose), NP-2 (ZINC000257412737), NP-3 (3-O-β-D-rutinosin) showed the best inhibition activity on AHL synthase LasI with the docking scores of -8.988, -8.690, and -8.925 kcal/mol, respectively. Also, NP-101 (ZINC000077264779), NP-102 (ZINC000225518732), and NP-103 (ZINC000096269362) were selected for LasR-LBD with the scores of -13.355, -13.038, and -12.917 kcal/mol, respectively. In the case of LasA, docking scores of -13.357, -10.796, and -10.564 kcal/mol were obtained for NP-201 (Parishin), NP-202 (7-O-galloyl-D-sedoheptulose), and NP-203 (1,2-di-O-galloyl-6-O-p-coumaroyl-β-D-glucose), respectively. Furthermore, when the LasR-TP4 was the target, the best results were observed from NP-301 (ZINC000514288841), NP-302 (ZINC000077264754), and again NP-103 (ZINC000096269362), in which they showed docking scores of -13.175, -13.020, and -12.998 kcal/mol, respectively. Finally, the highest docking scores belonged to NP-401 (ZINC000150351649), NP-402 (ZINC000150351636), NP-403 (ZINC000150349056), which interacted with the ATPase Type IV pilus with the docking scores of -16.274, -15.773, and -15.405 kcal/mol, respectively.

and biofilm formation [28]. Hence, disrupting the mechanisms that power pilus assembly and disassembly can significantly debilitate the bacteria to use pili for virulence.
In this study, the virtual screening of the 24,000 compounds against the five targets was carried out using high throughput virtual screening (HTVS). The top 100 structures for each receptor entered the next phase and were evaluated by the extra precision (XP) docking method. Binding free energies, as well as pharmacokinetic characteristics (ADMET), were calculated for hit candidates. Finally, the top three promising in silico inhibitors for each of the five QS receptors in P. aeruginosa were identified and reported.

3D-ligand preparation & protein preparation.
The structures of 24,000 bioactive natural compounds were obtained from the ZINC15 database (https://zinc15.docking.org/). The entire compounds were optimized using the OPLS3 force field, and by applying Epik in the pH range of 7.0 ± 2, conformations were generated. The compounds were desalted and tautomerized. Sixteen stereoisomers and four law energy ring conformation were made per ligand for computation using the Ligprep module of Maestro.
The crystallography structures of the five target receptor including AHL synthase LasI (PDB ID: 1RO5), LasR-LBD (PDB ID: 2UV0), LasA (PDB ID: 3IT7), LasR-TP4 (PDB ID: 3JPU), and ATPase Type IV pilus (PDB ID: 3JVV) were downloaded from the Protein Data Bank (https://www.rcsb.org/). Proteins were prepared using the Protein Preparation Wizard Tool of Maestro (2015). To this end, hydrogen atoms were added to the receptors, disulfide and zeroorder metal bonds were created in the protein's chains. The water molecules that do not participate in interactions, the native ligand, and the heteroatom(s) were removed. The PROPKA was employed to predict the pKa of ionizable groups in proteins at pH 7.00. Ultimately, the OPLS3 force field was optimized and minimized at RMSD of 0.3 Å the proteins.

Molecular docking & docking validation.
The 24,000 natural compounds were docked into the previously mentioned receptors active site using Glide high-throughput virtual screening (HTVS) followed by extra precision (XP) for the top 100 structures obtained in the HTVS method for each receptor. For optimizing the docking setting, the Ligand sampling option was set on Flexible, and the "Sample nitrogen inversion" and "Sample ring conformation" were checked, and Epik state penalties were added to the docking score. In the end, RMSD to input ligand geometries was computed. Obtain a more detailed assessment of the in silico activity of the three candidates for each target, and their in-silico results were compared with those of the positive controls: Tobramycin ( Figure  1a), a well-known antibiotic that is especially effective against species of Pseudomonas, N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) (Figure 1b), a reported quorumsensing signaling molecule [25], and the specified native co-crystallized ligands where existed (Figure 1c, 1d).

Calculating of ΔGBinding.
For calculating the Gipps Free Energy of bindings, the molecular mechanicsgeneralized Born surface area (MM-GBSA) tool in the Prime program was used. The docking results were imported into the program, and the "Solvation model" option was set on 'VSGB' mode, and the calculation was performed using the OPLS3 force field. Free binding energy results were expressed in kcal/mol [38]. ΔGbind = Gcomplex -Gligand -Greceptor 2.5. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction.
Lipinski's rule of five for the top 100 structures was calculated using Qikprop v4.4, updated by February 10, 2014, tool of Maestro. Pharmacokinetic aspects, namely molecular weight (MW), logPo/w, H-bond acceptor and donor, central nervous system (CNS), percentage of oral absorption, and polar surface area (PSA) were estimated for the top hit compounds.

Results and Discussion
The virtual screening protocol was applied to discover new inhibitors against different enzymes involved in the quorum-sensing process of P. aeruginosa (PAO1). To this end, 24,000 natural compounds, including primary and secondary metabolites, were virtually screened against five known QS-involved receptors in P. aeruginosa (PAO1). The target receptors were chosen carefully based on their significant roles in PAO1's QS. At first, the entire 24,000 compounds were screened against each receptor using high throughput virtual screening (HTVS). Then, the top 100 metabolites for each protein structure were again examined by the extra precision (XP) method followed by MM-GBSA and Pharmacokinetic features calculation. The most promising three in-silico inhibitors for each of the targets in PAO1 were identified and presented. The obtained data of docking and free binding energy are reported in Table 2, and the 2D structure of these hit compounds is illustrated in Figure 2.  As it is represented in Figure 3a, all three compounds showed a high docking score in comparison with the positive controls. The first compound (NP-1), 3,6′-di-O-sinapoylsucrose, has been previously reported for its anticancer properties and is extracted from Polygala Flavescens DC, a sub-specie of Polygala family plants [39]. Hydroxyl groups in the furanose ring interact with amino acid Ile107. Also, the hydroxyl group in the pyranose ring forms hydrogen bonds with Thr145 and Glu171 (Figure 4). Amino acid Arg154 also establishes a hydrogen bond with the carboxyl group. The NP-1 with a docking score of -8.988 kcal/mol showed the most effective inhibition against AHL synthase LasI. The free binding energy for this compound was reported to be -25.016 kcal/mol. The other structure (NP-3), 3-O-β-Drutinosin, is the well-known main compound of Alhagi sparsifolia and A. pseudalhagi. It has been reported to have some anti-neuroinflammatory activities [40]. According to our search, there were no previous biological activity reports related to the top three structures for this target. The native co-crystallized ligand in this protein is the same as one of the positive controls, 3-oxo-C12-HSL. This ligand showed a docking score of -7.289 kcal/mol in the interaction with its native receptor ( Figure 5). In contrast, the other positive control, Tobramycin, did not show any affinity and interaction with the receptor. Meanwhile, the NP-101, NP-102, and NP-103 revealed a high attraction to LasR-LBD ( Figure  3b). The compound of NP-101 with a docking score of -13.355 kcal/mol and ΔGbind of -51.516 kcal/mol was recognized as the best structure in inhibiting LasR-LBD protein. The benzene ring of the benzenesulfonamide group makes a π-π stacking bond with Tyr47. The hydroxyl group of the phenolic ring forms a hydrogen bond with Trp60, and the secondary amine gives hydrogen and salt bridge bonds with Asp73.  The first member of this group is NP-201, one of the Parish family and is extractable from rhizomes of Gastrodia elata. They have a history of headache, dizziness, vertigo, and convulsions epilepsy therapy [41]. Hydroxyl groups in pyranose sugar form hydrogen interactions with Gln18, Asp40, Trp41, His81, Thr117, and Tyr151. Also, Tyr151 creates a ππ stacking bond with the benzene ring ( Figure 6). These interactions lead to a docking score of -13.357 kcal/mol and binding free energy of -35.891 kcal/mol. The other structure with a docking score of -10.796 kcal/mol is 7-O-galloyl-D-sedoheptulose (NP-202), and it is one of the principal compounds of Corni Fructus and Cornus officinalis. It has shown some promising biological activity against Alzheimer's, diabetes, and diabetic complications. Besides, it has received attention as a preventive agent against oxidative stress-related diseases [42][43][44][45][46][47][48][49][50]. The last structure of the group, 1,2-di-O-galloyl-6-O-p-coumaroyl-β-D-glucose (NP-203) with the binding energy of -59.458 kcal/mol and docking score of -10.564 kcal/mol, is one of the main constituents of Rhubarb, an important crude drug in Asiatic regions which is produced from different Rheum species [51]. As this receptor does not have any native co-crystallized ligand, the natural compounds were compared to Tobramycin and 3-oxo-C12-HSL as positive controls. Figure 3c shows the docking score differentiation between the selected structures and positive controls. All three compounds showed a significant increase in the docking score in comparison with the positive controls. No preceding reported activity for NP-301, NP-302 were found in the literature. Among positive controls, both Tobramycin and the native co-crystallized ligand of the receptor, 4-Bromo-2-((2-chlorobenzamido) methyl)-6-methylphenyl-2,4-dichlorobenzene, displayed a high docking score and affinity for LasR-TP4. However, the selected structures showed more affinity for the mentioned receptor (Figure 3d). Compound NP-301 with a docking score of -13.175 kcal/mol and free binding energy of -87.824 kcal/mol became the best LasR-TP4 inhibitor. In this compound, the benzene ring forms a stacking bond with amino acid Tyr47, a salt bridge bond between amino groups and Asp73. In addition, a hydrogen bond was established between the hydroxyl group of the tetrahydrofuran ring and Thr75. Secondary amine interacts with Asp73 ( Figure 7).

ATPase Type IV pilus.
Similar to the LasR-TP4, there is no record of the biological activity of NP-401 to NP-403. Within the positive controls of this enzyme, the enzyme's native ligand (Phosphomethyl phosphonic acid adenylate ester) has the highest affinity for ATPase Type IV pilus with the docking score of -7.988 kcal/mol. On the other hand, structures NP-401, NP-402, and NP-403 showed the best in-silico inhibition activities (-16.274, -15.773, and -15.405 kcal/mol, respectively) for the ATPase Type IV pilus in comparison with the positive controls as well as the other promising candidates for the other four targets (Figure 3e). In compound NP-401, the carboxylate group establishes a hydrogen bond with Gly135, Lys136, Ser137. Other amino acids involved include Thr98, Thr139, and Glu204 ( Figure 8).

Pharmacokinetic characteristics.
Bioavailability properties for the 15 final compounds are summarized in Table 3. Compounds with a molecular weight of 130 to 725 g/mol, hydrogen bond donors of 0 to 6, hydrogen bond acceptors of 2 to 20, octanol/water partition coefficient of -2 to 6.5, CNS of -2 (inactive) to +2 (active) and PSA of 7 to 200 Å are in the acceptable range. 3,6′-di-Osinapoylsucrose (NP-1) with a molecular weight of 586.54 g/mol and CNS of -2 was not able to cross the blood-brain barrier and was therefore considered inactive. Compounds of NP-103, NP-102, and NP-101 showed 100, 76.89, and 65.91% oral absorption, respectively. All listed properties for these compounds except CNS for NP-103 were acceptable. NP-201 showed a CNS of -2, logP of -1.878, and molecular weight of 728.65 g/mol. Compounds NP-103, NP-301, and NP-302 displayed 100, 86.95, and 73.69% oral absorption, respectively. The NP-301 and NP-103 with CNS of +2 are active and can cross the blood-brain barrier. The rest of the predicted characteristics for these three compounds were appropriate. Finally, compounds NP-401, NP-402, and NP-403 do not cross the blood-brain barrier, but their oral absorption is zero.

Conclusions
Fast-growing bacterial resistance is a vital concern, hence discovering and developing novel and efficient antibiotics that can defeat bacteria resistance. P. aeruginosa is a Gramnegative bacteria responsible for many yearly deaths worldwide. Its advanced quorum-sensing system protects the organism from the human defense system and a broad spectrum of known antibiotics. So, targeting the protective QS system can attenuate the bacterium following its destruction. We used in-silico methods to screen 24,000 natural metabolites against five critical PAO1's QS receptors. After screening entire compounds, the top 100 effective structures for each target were assessed by the extra precision (XP) approach followed by calculating binding energies (MM-GBSA) and ADMET properties. Finally, the best three potent in-silico inhibitors for each of the five QS-involved receptors were identified and reported to be used in further in-vitro and in-vivo studies for antimicrobial activity against P. aeruginosa.

Funding
This research received no external funding.