Evaluation of the Antibacterial and Investigation of the Molecular Docking of New Derivatives of 1, 3, 4-Oxadiazole as Inhibitors of Quorum Sensing System in the Human Pathogen Pseudomonas Aeruginosa

Background: Oxadiazoles are a group of anti-inflammatory compounds that have a wide range of activity due to their higher efficacy. Pseudomonas aeruginosa is an opportunistic pathogen and a major pathogen of nosocomial infections. This study aimed to evaluate the antibacterial and investigation of the molecular docking of new derivatives of 1, 3, 4-oxadiazole against P. aeruginosa, in vitro & in silico. Materials and Methods: Four new derivatives were synthesized and added to our previous synthetic derivatives of 1, 3, 4-oxadiazole. The antibacterial activity of all derivatives was measured based on three standard species of P. aeruginosa using inhibition zone (IZ) and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Then, employing the computational design of the drug by the molecular docking method, the inhibitory effect of synthetic compounds on the LasR regulatory protein of P. aeruginosa quorum sensing system was investigated, which plays an important role in regulating the expression of pathogenic genes in bacteria. Results: The chemical structures of new compounds were characterized by IR spectra and 1H-NMR. A variety of inhibitory effects were observed by the synthesized compounds – compound 4d and 4g, in particular. Also, the inhibitory effect of these two compounds on the LasR regulatory protein under the control of the quorum sensing system in P. aeruginosa was demonstrated by molecular docking. Conclusions: The results of this study showed that the two compounds containing the functional group of naphthalene and fluorophenyl have a significant effect on the inhibition of P. aeruginosa, as well as on the LasR protein of this bacterium.

isomers of this 5-membered heterocycle, including 1, 2, 4-oxadiazole 1, 2, 3-oxadiazole 1, 2, 5-oxadiazole, and 1, 3, 4-oxadiazole (8). 1, 3, 4-oxadiazoles are of special importance due to their prominent biological properties such as antibacterial (7), antifungal (9), anti-tuberculosis (10) and anti-cancer (11) properties. The introduction of 1, 3, 4-oxadiazole ring to the inhibitors can change their polarity and flexibility, as well as their metabolic stability (12). As the acceptor of hydrogen bonds formation, meanwhile, the 1, 3, 4-oxadiazole scaffold has the potential to be an isosteric substituent for amide or ester groups, so as to interfere protein or lipid biosynthesis in pathogens (13). Given the fact that bioinformatics and information technology are advancing today at an unexpected pace and their importance cannot be denied in the fields of medicine, biotechnology, and pharmacology, now along with traditional methods of drug synthesis, computersynthesized structures are also approved in terms of antibacterial power through computer calculations with the least cost, which, if appropriate, lead to the therapeutic use (14). In the present study, the synthesis of new 1, 3, 4-oxadiazole compounds along with our previous derivatives, and the inhibitory effect of these compounds on three different strains of P. aeruginosa and LasR protein (chain H) as the most important regulatory protein in the quorum sensing system of this bacterium were investigated experimentally and computationally.
All beginning and lawful materials were prepared from Merck Company (Germany) and used moving forward without any more filtration. Infrared spectrum was measured by a Shimadzu IR-460 spectrometer. Nuclear magnetic resonance spectrum was obtained by a Bruker DRX-300 AVANCE spectrometer (1H NMR at 300 Hz) in CDCl 3 . Chromatography columns were prepared using silica gel powder (Merck, Germany).

One-step Process for the Synthesis of 1, 3, 4-Oxadiazole Derivatives (4j-o)
First 0.01 mL of 2-pyridine-carbaldehyde was dissolved with 0.07 mL of di-ethyl-amine in acetonitrile, which was used as a solvent. After stirring vigorously for 5 minutes, 0.03 g of N-iso-cyano-imino-triphenyl-phosphorane was slowly added. TLC showed that after 2 hours the reaction was complete and the product was formed and was separated by a plate with a ratio of 10:6 (petroleum ether to ethyl acetate). After evaporation of the solvent, the product was obtained (15).  (14). The results were reported as the ±mean of three independent experiments.

Inhibition Zone
After preparing the suspension of each strain of bacteria in the tube with distilled water and matching the turbidity of the suspensions with the turbidity equivalent to 0.5 McFarland standards (1.5 × 10 8 CFU/mL), some of each suspension were removed with sterile cotton and were pure culture culture in Müller Hinton agar medium. Then, wells were made in the plate by Pasteur pipette. Then, 10 μL of solutions prepared from 1, 3, 4-oxadiazole derivatives (at concentrations of 0.5 mg/mL) were injected into the wells. Finally, the plates were closed and they were incubated for 24 hours at 37°C. After the mentioned time, the plates were examined for the presence of growth inhibition zone. It should be noted that ciprofloxacin (at concentrations of 0.5 mg/mL) was considered as a control sample (15).

Minimum Inhibitory Concentration Experiment
Minimum inhibitory concentration of the compounds was determined using dilution agar method. Concentrations of 1000, 500, 250, 125, 62.50, 31.25, 15.62 µg/mL were prepared in sterile molten Muller Hinton Broth from the stock solution. The microplates were inoculated with 1.5×10 6 cfu/mL of bacterial suspension at 37°C for 24 hours. The MIC was defined as the lowest concentration of the compounds that prevented visible growth of microorganisms after 24h incubation. The results were compared with ciprofloxacin (at concentrations of 0.5 mg/ mL) as standard antibacterial agents. Stock solutions of 1, 3, 4-oxadiazole derivatives were prepared at concentration of 0.5 mg/mL in dimethyl sulfoxide (0.001 g of each derivative in 2 cc of DMSO). The lowest concentration that inhibited growth of bacteria was defined as the MIC (16) . All experiments were performed in duplicate and results were reported as mean ± standard deviation.

MBC Experiment
MBC refers to the lowest concentration of antibiotic that can reduce the bacterial population by 9.99% after 24 hours; in other words, it can reduce the initial population a thousand times more. After determining the MIC, the tubes are cultured on a sterile Müller Hinton agar culture medium with a swap, which is placed in a 37°C incubator for 24 hours (17).

Molecular Docking Protein Preparation
The crystallographic structure of the LasR protein complex of P. aeruginosa with a resolution of 1.83 angstroms was downloaded from the protein database (PDB ID: 2UV0). By removing all ligand molecules, waters, excess molecules, as well as the chains of protein that were not required for docking and then by adding polar hydrogen and charge to it, the desired protein was prepared for the docking process. Chain (H) was used for this docking ( Figure 1).

Ligand Preparation
The fourteen compounds of 1, 3, 4-oxadiazole, that are called ligands in this program, were plotted by professional ChemDraw software (19.1) and then the energy optimization of structures using molecular mechanical (MM +) and quasi-experimental methods ( AM1) was applied by Chem3D (19.1).

Docking
Using the AutoDockTools (1.5.6) program, all hydrogen atoms were added to the protein structure, and the partial loads of the ligand were calculated and added with Discovery Studio 4.5 Client program; Grid box being 40×40×40 dimensional and Dimensions were also considered as X-center = 26.668, Y-center = 32.892 and Z-center = 41.388. Then using AutoDock Vina (18), the best format with the least amount of connection energy was selected as the stable format and the best combination in terms of (-ΔG) was examined by Discovery Studio (4.5 Client) software (2D and 3D).

Chemicals
The structures (4a-4o), infrared and NMR of all compounds (4j-4o) were obtained (Table 1). Our study is novel in chemistry since it investigated the synthesis of 4j-4o structures for the first time in the form of singlestage synthesis. The results of the study showed that the synthetic products could be obtained with a purity of over 95%. To continue the experiments, all products were used as liquid solutions at a concentration of 0.5 mg/mL. Chemical formula, exact mass and molecular weight of all compounds were calculated with chem3D ( Table 2).
In Vitro According to Figure 2, which deals with Inhibition zone of compounds on the target bacterium, structures containing         naphthalene (N) and difluorophenyl (Di-Fl-Ph) showed the best performance. These results are reported in Table 2.
As Table 2 and Figure 2 show, it seems that the compound 4d, which has an aromatic hydrocarbon and is widely used for disinfection and insecticide (mostly dissolved in methanol), had a very favorable effect on the bacteria along with the oxadiazoles ring. Compound 4g, which has one of the aromatic compounds and by pairing the two aromatic compounds of fluoro, created a favorable property against the studied bacteria. Following Table 2, the effect of compound 4d was against P. aeruginosa with IZ= ± (35.00 ± 0.50) mm, MIC=125 mg/mL, and MBC=250 mg/mL. This result can be due to the presence of naphthalene group in the main compound (17). The effect of compound 4g was against P. aeruginosa with IZ= ± (35.00 ± 0.33) mm, MIC=125 mg/mL, and MBC = 250 mg/mL. This result may be due to the presence of Fluorophenyl group in the main compound with tow site (18).

In Silico
As shown in Table 2, all affinities of the compounds were calculated, and the best compounds with low ΔG (-ΔG) were selected to continue experiments and to investigate chemical interactions. According to Figure  3, the highest amount of hydrogen bonds was related to the 4d compound with the amino acid alanine by 127 hydrogen bonds, valine:76, leucine:110, tyrosine:64, asparagine:73, tryptophan:88 and tyrosine:115, among which the compound bonds were the case . The opinion can be considered by hydrogen bonding the site of alanine, leucine and tyrosine . With 4g compound, the highest amount of hydrogen bonds was related to alanine: 127, valine: 76, tryptophan: 64, asparagine: 73, leucine: 110, Tyrosine: 115. The opinion can be considered by hydrogen bonding the site of alanine, leucine and tyrosine.

Discussion
Generally, 1, 3, 4-oxadiazoles are multi-functionalized compounds with an assortment of organic properties and, therefore, their unused compound blend is vital. This study evaluated the antibacterial and in silico effects of 15 synthesized 1, 3, 4-oxadiazole derivatives (4j-4o new structures) against the Human Pathogen P. aeruginosa. These compounds, mainly Di-Fluorophenyl (4g) and Naphthalene (4d), showed relatively acceptable antibacterial effects against Gram-Negative P. aeruginosa. In a study conducted by Rayam et al, it was reported that the antibacterial activity of new synthesis compounds revealed 2-(1-(4-isobutylphenyl)ethyl)-5-(((1-phenyl-1H-1,2,3triazol-4-yl)methyl)thio)-1,3,4-oxadiazole (8b) and demonstrated more potent antibacterial activity against E. coli and P. aeruginosa (19). In another study, Chortani et al showed how synthesis of novel 1, 3, 4-oxadiazole linked benzopyrimidinones conjugates. Biological results of their study revealed that some of these compounds demonstrated excellent to moderate antimicrobial activities with minimum inhibitory concentration (MIC) values ranging from 10.8 to 140.7 μM. Notably, compounds 5g(1,4-dimethylbenzene) and 5h(4chlorophenyl) showed the highest inhibitory activity against all bacterial strains with MIC values ranging from 111.3 to 10.8 μM against S. aureus, E. coli and P. aeruginosa (20). The results of these studies are consistent with the results of ours since the main role of the 1, 3, 4-oxadiazole ring has been mentioned in all given studies. In addition, in silico results indicated that Fluorophenyl ring and piridine-methanol ring in compound 4g play an important role in the formation of hydrogen bonds. Also, pirydine-methanol ring and oxadiazole ring with naphthalene in compound 4d are important factors in the formation of hydrogen bonds. Moreover, the simple workup, tall abdicates, and brief response times make the strategy a valuable expansion for planning advanced was related to alanine: 127, valine: 76, tryptophan: 64, asparagine: 73, leucine: 110, Tyrosine: 115. The opinion can be considered by hydrogen binding the site of alanine, leucine and tyrosine. Evaluation of the Antibacterial of New Derivatives of 1, 3, 4-Oxadiazole pharmaceutical synthetics. It is noteworthy that these derivatives (4j-4o) were synthesized for the first time and the relevant tests were performed to ensure the biological properties of the compounds. This research was done in order to provide new structures and confirm the existence of antibacterial activity and drug design (4a-4o) of these compounds.

Conclusions
It seems that 1, 3, 4-oxadiazole subordinates would be a supportive structure for conceivable improvement of unused drugs but this result must be affirmed by other broad clinical trials that would be a portion of our future plans.

Ethical Approval
The disclaimer implies that ethical principles have been considered in relation to the proposed work and no ethical issues have been found to be applied to this research proposal.