Antibacterial, Anticandidal, and Antibiofilm Potential of Fenchone: In Vitro, Molecular Docking and In Silico/ADMET Study

The aim of the present study is to investigate the effective antimicrobial and antibiofilm properties of fenchone, a biologically active bicyclic monoterpene, against infections caused by bacteria and Candida spp. The interactions between fenchone and three distinct proteins from Escherichia coli (β-ketoacyl acyl carrier protein synthase), Candida albicans (1, 3-β–D-glucan synthase), and Pseudomonas aeruginosa (Anthranilate-CoA ligase) were predicted using molecular docking and in silico/ADMET methods. Further, to validate the in-silico prediction, the antibacterial and antifungal potential of fenchone was evaluated against E. coli, P. aeruginosa, and C. albicans by determining minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and minimum fungicidal concentration (MFC). The lowest MIC/MBC values of fenchone against E. coli and P. aeruginosa obtained was 8.3 ± 3.6/25 ± 0.0 and 266.6 ± 115.4/533.3 ± 230.9 mg/mL, respectively, whereas the MIC/MFC value for C. albicans was found to be 41.6 ± 14.4/83.3 ± 28.8 mg/mL. It was observed that fenchone has a significant effect on antimicrobial activity (p < 0.05). Our findings demonstrated that fenchone at 1 mg/mL significantly reduced the production of biofilm (p < 0.001) in E. coli, P. aeruginosa, and C. albicans by 70.03, 64.72, and 61.71%, respectively, in a dose-dependent manner when compared to control. Based on these results, it has been suggested that the essential oil from plants can be a great source of pharmaceutical ingredients for developing new antimicrobial drugs.


Results and Discussion
The current research mainly focuses on predicting the physicochemical parameters, molecular modeling, and docking analysis of fenchone molecules against three different proteins involved in antimicrobial infections.

Pharmacokinetic/ADME Properties of Fenchone
Several physicochemical traits and TPSA (Topological Polar Surface Area), a basic physiochemical parameter used to evaluate drug delivery characteristics, along with the pharmacokinetic property of fenchone, are predicted using Swiss ADME and are listed in Table 1. The compounds showed high gastrointestinal (GI) absorption and are P-gp (p-glycoprotein) non-inhibitors that can also penetrate the blood-brain barrier (BBB). Fenchone inhibits the Cytochrome P450 isomers that possess a low on skin permeability of −4.73 cm/s. Drug likeness property was examined, which provides the molecules with a powerful drug based on molecular mass, Log P, hydrogen bond acceptors, and donor's ratio. The compound violated any rules and disclosed a bioavailability score of around 0.55 (Table 1).

Physicochemical Properties of 1, 3-β-D-Glucan Synthase and Anthranilate-CoA Ligase
The physicochemical parameters and primary structure of 1, 3-β-D-glucan synthase and the Anthranilate-CoA ligase were computed using ExPasy ProtParam tools ( Table 2). The presence of more non-polar amino acid residues suggests that the proteins are more hydrophobic [35]. The theoretical Pi values of 1, 3-D-glucan synthase and Anthranilate-CoA ligase sequences are 9.74 and 5.81, respectively, indicating the acidic nature of the sequence, which is useful in developing recombinant protein purification buffers [36]. The extinction coefficient (EC) values of 57,090 (1, 3-β-D-glucan synthase) and 57,130 (Anthranilate-CoA ligase) determine the quantitative investigation of protein-protein and protein-ligand interactions [37]. The sequence's instability index indicates the protein's stability; a score of less than 40 indicates that the protein is stable, whereas a score greater than 40 indicates it is unstable [38]. In the case of 1, 3-β-D-glucan synthase, the predicted structure is stable with an instability index value of 37.99, whereas the Anthranilate-CoA ligase had a value of 41.80. The addition of aliphatic amino acids to the side chain of a protein increases its thermal stability. The aliphatic index value for anti-freeze protein ranges from 57.83 to 125.23 [35], which is almost similar in both the proteins, indicating the protein could be stable for huge temperature variations. Pymol was used to visualize the modeled 3D structure using I-TASSER and protein structures retrieved from the PDB (Figure 1). The I-Tasser theoretical tool that integrates ab initio modeling, threading, and atomic level structure modification technologies [39] is used to predict the three-dimensional protein structure of 1, 3-β-D-glucan synthase and the Anthranilate-CoA ligase.
I-Tasser was used to generate five models, each with a C-Score of −2.50, −3.01, −4.19, −5, and −5.00, and TM-Score and RMSD values of 0.42 ± 0.14 and 11.5 ± 4.5 Å for 1, 3-D-glucan synthase. In the case of the Anthranilate-CoA ligase, the C-Scores were 0.35, 0.07 and −0.49, whereas the TM-Score and RMSD values were 0.76 ± 0.10 and 6.6 ± 4.0 Å. These values are generated by the modeled structure's threading pattern alignment and resolution parameter. The higher the C-Score, the more certain model 1 is, and it was selected for further study [40]. I-Tasser was used to generate five models, each with a C-Score of −2.50, −3.01, −4.19, −5, and −5.00, and TM-Score and RMSD values of 0.42 ± 0.14 and 11.5 ± 4.5 Å for 1, 3-D-glucan synthase. In the case of the Anthranilate-CoA ligase, the C-Scores were 0.35, 0.07 and −0.49, whereas the TM-Score and RMSD values were 0.76 ± 0.10 and 6.6 ± 4.0 Å. These values are generated by the modeled structure's threading pattern alignment and resolution parameter. The higher the C-Score, the more certain model 1 is, and it was selected for further study [40].

Structure Validation
The predicted structure of the Anthranilate-CoA ligase and 1, 3-β-D-glucan synthase were validated by ProSA, which compares the Z-Score of the predicted model against structures refined from empirical techniques including X-ray and NMR ( Figure 2). The total energy sandwiched in between the initial fold and misfolds determines the Z-Score of a protein in the ProSA web tool. The Z-Score primarily calculates the variation from the energy distribution produced by random conformation and determines the reliability of the modeled protein structure [41]. The Z-Score of the Anthranilate-CoA ligase is −8.94 and 1, 3-β-D-glucan synthase is −3.02, which had no significant variation from native structures of a similar size obtained from X-ray and NMR. Based on the ϕ and ψ angles between C∞-C and N-Cα, the position of amino acid residue in a segment is graphed on the Ramachandran plot [42]. The Ramachandran plot was employed in this study to investigate the predicted protein structure utilizing the SAVES Server. The statistical analysis of the modeling revealed that the majority of the residues fall in the most favored and

Structure Validation
The predicted structure of the Anthranilate-CoA ligase and 1, 3-β-D-glucan synthase were validated by ProSA, which compares the Z-Score of the predicted model against structures refined from empirical techniques including X-ray and NMR ( Figure 2). The total energy sandwiched in between the initial fold and misfolds determines the Z-Score of a protein in the ProSA web tool. The Z-Score primarily calculates the variation from the energy distribution produced by random conformation and determines the reliability of the modeled protein structure [41]. The Z-Score of the Anthranilate-CoA ligase is −8.94 and 1, 3-β-D-glucan synthase is −3.02, which had no significant variation from native structures of a similar size obtained from X-ray and NMR. Based on the φ and ψ angles between C ∞ -C and N-Cα, the position of amino acid residue in a segment is graphed on the Ramachandran plot [42]. The Ramachandran plot was employed in this study to investigate the predicted protein structure utilizing the SAVES Server. The statistical analysis of the modeling revealed that the majority of the residues fall in the most favored and additionally allowed region, including non-glycine and non-proline residues, which ensures good stereochemistry quality of the modeled structure [43]. additionally allowed region, including non-glycine and non-proline residues, which ensures good stereochemistry quality of the modeled structure [43]. Based on the position of amino acids present in the Anthranilate-CoA ligase, the geometry of the structure is shown in Figure 3a, which has 77.8% residue in the most superior sections, 18.9% in further acceptable sections, 2.5% in freely allowed sections, and 1.6% in prohibited sections. Similarly, the predicted 1, 3-β-D-glucan synthase had 76.7% residues in the most superior sections, 17.9% in further acceptable sections, 4.5% in freely allowed sections, and 0.9% in prohibited sections, which shows the geometry of the structure (Figure 3b). Based on the position of amino acids present in the Anthranilate-CoA ligase, the geometry of the structure is shown in Figure 3a, which has 77.8% residue in the most superior sections, 18.9% in further acceptable sections, 2.5% in freely allowed sections, and 1.6% in prohibited sections. Similarly, the predicted 1, 3-β-D-glucan synthase had 76.7% residues in the most superior sections, 17.9% in further acceptable sections, 4.5% in freely allowed sections, and 0.9% in prohibited sections, which shows the geometry of the structure (Figure 3b).

Molecular Docking
The docking study was performed using PatchDock for three different proteins, namely β-ketoacyl acyl carrier protein synthase I, the Anthranilate-CoA ligase, and 1, 3β-D-glucan synthase with fenchone. The docking score, atomic content energy, hydrogen bonds, and amino acid residues involved in binding are listed in Table 3 along with the

Molecular Docking
The docking study was performed using PatchDock for three different proteins, namely β-ketoacyl acyl carrier protein synthase I, the Anthranilate-CoA ligase, and 1, 3-β-D-glucan synthase with fenchone. The docking score, atomic content energy, hydrogen bonds, and amino acid residues involved in binding are listed in Table 3 along with the interactions in Figures 4-6. The obtained results showed that the compound fenchone exhibited low ACE against the three used proteins. The possible reason for this could be the presence of functional side chains in the fenchone structure. The docking results of the molecules with the protein β-ketoacyl acyl carrier protein synthase I showed interactions with essential amino acids in the binding pocket with Pro272, as well as Van der Waals interactions with Phe390, Phe392, Phe229, Gly391, and, finally, a Pi-cation interaction with His298. The compound showed better interaction with the amino acid residues PRO283, ALA259, and LEU494, forming four hydrogen bond interactions with the Anthranilate-CoA ligase protein with an ACE value of −113.15 kcal/mol. FKS1 expression is cell-cycle controlled and associated with cell wall reorganization, and catalytic subunits of glucan synthase (GS) are upregulated in cell wall construction [15]. Because FKS transcription factors are linked with the catalytic domain of GS, interrupting FKS1 lowers glucan synthase activity [44]. FKS and RHO1 proteins are known to be the preserved among fungi and are essential for cell survival [45]. As a result, inhibiting 1,3-β-D-glucan synthase disrupts cell wall construction and inhibits fungal growth. The docking results showed five interactions with TYR197, TYR228, LEU232, PRO193, and ILE235 with ACE −130.89 (kcal/mol).       FKS1 expression is cell-cycle controlled and associated with cell wall reorganization, and catalytic subunits of glucan synthase (GS) are upregulated in cell wall construction [15]. Because FKS transcription factors are linked with the catalytic domain of GS, interrupting FKS1 lowers glucan synthase activity [44]. FKS and RHO1 proteins are known to be the preserved among fungi and are essential for cell survival [45]. As a result, inhibiting 1,3-β-D-glucan synthase disrupts cell wall construction and inhibits fungal growth. The docking results showed five interactions with TYR197, TYR228, LEU232, PRO193, and ILE235 with ACE −130.89 (kcal/mol).

Antimicrobial Activity of Fenchone
The rising development of antibiotic resistance, which leads to antimicrobial treatment insufficiency, is the fundamental challenge in antimicrobial chemotherapy. Overuse of antibiotics and the resulting antibiotic selection pressure are thought to be the most critical attributes significant to the emergence of different forms of antibiotic resistant bacteria [46], therefore, there is an immediate requirement to find new antimicrobial agents with various chemical structures and unique modes of action. Essential oils and their active components are broadly utilized in medicine as ingredients in a variety of medical products, as flavoring additives in food, and as perfumes in cosmetics [46]. The antimicrobial potential of fennel essential oil has been investigated against a variety of bacterial and fungal strains [46]. In the present study, the antibacterial and antifungal efficacy of commercial fenchone (a constituent of absinthe and the essential oil of fennel) against E. coli, MDR-PA, and C. albicans has been investigated by using the microbroth dilution method. The MIC/MBC/MFC values of fenchone against E. coli, MDR-PA, and C. albicans were shown in Table 4. The MIC and MBC results for Gram-negative bacterial, i.e., E. coli, and MDR-PA were found to be 8.3 ± 3.6/25 ± 0.0, and 266.6 ± 115.4/533.3 ± 230.9 mg/mL, respectively (Table 4; Figure 7A,B). Compared to Gram-negative MDR-PA, the MIC/MFC values of fenchone for C. albicans were found to be significantly lower, i.e., 41.6 ± 14.4/83.3 ± 28.7 mg/mL ( Figure 7C). It was observed that fenchone has a significant effect on antimicrobial activity (p < 0.05). In a previous study, the lowest MIC values of fennel essential oil have been reported for E. coli and C. albicans [46]. Bassyouni et al. (2019) reported an MIC value of 0.78 to 6.25% against clinical isolates of C. albicans [47]. Kawther (2007) reported that the essential oil from fennel seeds exhibits prominent anticandidal activity against different Candida species [48]. It was reported that fennel essential oil and seed extracts have varying degrees of antimicrobial potential depending on the doses used [47,49]. Fennel essential oil has a higher sensitivity to Gram-negative and Gram-positive bacteria due to the occurrence of volatile compounds such as polyphenols in greater quantities [50]. It also enhances plasma membrane fluidity, which leads to greater fluid loss from bacterial species and inhibits microbial respiration [51,52]. As a result, the post-diffusion effect of essential oils on bacterial and fungal growth and metabolism appears to be the most important antibacterial activity [53,54]. Without a doubt, the essential oil of fenchone includes a plethora of interesting compounds that can be employed for medicinal purposes, primarily pharmacological potential [55]. Natural essential oils, similar to other plant products, provide broad-spectrum antibacterial action against pathogenic microbial strains. The present findings suggested that fenchone could be used as a natural antimicrobial treatment to treat various infections caused by pathogenic microorganisms and may provide pharmaceutical ingredients for the development of novel therapeutic and antimicrobial medicines.

Inhibition of Biofilm Formation
Biofilm production by Candida and bacterial spp is one of the most critical elements in wound infections, and it can lead to biofilm-related sepsis, which is the leading cause of wound-related death. The rate of antibiotic resistance is increasing due to the formation of organized bacterial and candida biofilm communities, which complicates the treatment therapy and leads to the development of chronic infection [56]. In recent decades, there has been a growing interest in exploring the diverse biological potential of plant metabolites. In this regard, plant-based essential oils have been suggested as a potential substitute for most commonly used antibiotics as well as an addition to conventional therapy. Essential oils derived from plants have been broadly used as antimicrobial, antioxidants, and flavoring agents due to the presence of various secondary metabolites such as terpenoids, alkaloids, and polyphenolic compounds [57]. The antibacterial, antifungal, and antibiofilm properties of a large number essential oils have been demonstrated against a diverse variety of pathogenic bacteria [57]. Despite numerous studies evaluating a variety of biological activities, none have looked at fenchone's antibiofilm potential against bacteria and Candida biofilms. Therefore, the aim of this study was to assess the potency of fenchone against E. coli, MDR-PA, and C. albicans. The result in Figure 8 demonstrates the inhibition of biofilm production of E. coli, MDR-PA, and C. albicans by fenchone at varying concentrations. It has been observed that E. coli, MDR-PA, and C. albicans biofilms treated with a lower concentration of fenchone, i.e., 0.25 mg/mL, inhibit biofilm formation by 51.7, 40.85, and 44.27%, respectively. Whereas, under similar conditions, fenchone at 1.0 mg/mL inhibits biofilm production of E. coli, MDR-PA, and C. albicans by 71.03, 59.15, and 61.71%, respectively. The data is presented as the average of three independent tests in triplicates with standard deviation. The asterisks (**) represent the significance as p < 0.001 against their control experiments by One-Way ANOVA-based Pairwise Multiple Comparison Procedures (Holm-Sidak method), whereas the overall significance level = 0.05, was conducted on SigmaPlot 11.05 statistical analysis software (Figure 8). Previously, it has been reported that fennel essential oil significantly decreased the formation of S. aureus biofilm and reduced the metabolic activity of attached cells as well [56]. In another study, it has been investigated that fennel oil at concentrations ranging from 6.25 to 25% reduces the biofilm formation in C. albicans by 50% [47]. Whereas, in the present study, it has been found that fenchone inhibits biofilm formation in C. albicans by 61.71%, which is much lower than the previous report [47].
other plant products, provide broad-spectrum antibacterial action against pathogenic microbial strains. The present findings suggested that fenchone could be used as a natural antimicrobial treatment to treat various infections caused by pathogenic microorganisms and may provide pharmaceutical ingredients for the development of novel therapeutic and antimicrobial medicines. The statistically significant difference between MICs a and b , b and c , and MBCs d and e and, e and f , were found to be as p < 0.010 and p < 0.013, and p < 0.011 and p < 0.013, respectively, whereas, overall significance level = 0.05.   Note: The statistically significant difference between MICs a and b , b and c , and MBCs d and e and, e and f , were found to be as p < 0.010 and p < 0.013, and p < 0.011 and p < 0.013, respectively, whereas, overall significance level = 0.05.

Retrieval of the Ligand Molecule and Protein Structure for ADME Studies
The 3D structure of the fenchone compound was retrieved from the Pub Chem dat base using the Swiss ADME web server (Swiss Institute of Bioinformatics, Lausanne, Sw zerland) [58], and the Simplified Molecular Input Line Entry System (SMILES) format the compounds was used to develop their ADME/pharmacokinetic profile and drug-lik ness characteristics. The Protein Data Bank (PDB) is a collection of 3-D structural data f essential biological substances [59]. A 3D structure for β-ketoacyl acyl carrier protein sy thase I (KAS I), which is involved in cell wall formation, was discovered using the Prote Data Bank.

Molecular Docking Analysis
The sequences of 1, 3-β-D-glucan synthase and Anthranilate-CoA ligase protein we retrieved from the Swiss-Prot database. The ProtParam application was used to weigh t physicochemical characteristics of the obtained protein sequence by using the Expasy w browser. The 3D structures of the protein sequences were developed using the I-Tass server. Based on the C-Score (−5 to +2), TM-score, and RMDS, the accuracy of the gene ated protein structure was estimated. The quality of the model was validated using t structural validation algorithm ProSA, and the overall structure geometry was calculat using the Ramachandran plot and the SAVES Server (https://saves.mbi.ucla.edu/; a cessed on 11 November 2021).
Molecular modeling investigation was conducted for the retrieved 3D structure an modeled protein structure using the PatchDock online server and the interactions are v

Retrieval of the Ligand Molecule and Protein Structure for ADME Studies
The 3D structure of the fenchone compound was retrieved from the Pub Chem database using the Swiss ADME web server (Swiss Institute of Bioinformatics, Lausanne, Switzerland) [58], and the Simplified Molecular Input Line Entry System (SMILES) format of the compounds was used to develop their ADME/pharmacokinetic profile and druglikeness characteristics. The Protein Data Bank (PDB) is a collection of 3-D structural data for essential biological substances [59]. A 3D structure for β-ketoacyl acyl carrier protein synthase I (KAS I), which is involved in cell wall formation, was discovered using the Protein Data Bank.

Molecular Docking Analysis
The sequences of 1, 3-β-D-glucan synthase and Anthranilate-CoA ligase protein were retrieved from the Swiss-Prot database. The ProtParam application was used to weigh the physicochemical characteristics of the obtained protein sequence by using the Expasy web browser. The 3D structures of the protein sequences were developed using the I-Tasser server. Based on the C-Score (−5 to +2), TM-score, and RMDS, the accuracy of the generated protein structure was estimated. The quality of the model was validated using the structural validation algorithm ProSA, and the overall structure geometry was calculated using the Ramachandran plot and the SAVES Server (https://saves.mbi.ucla.edu/; accessed on 11 November 2021). Molecular modeling investigation was conducted for the retrieved 3D structure and modeled protein structure using the PatchDock online server and the interactions are visualized using Discovery Studio.

Evaluation of Antibacterial and Antifungal Activity
The antimicrobial activity of fenchone was investigated against E. coli, C. albicans, and multidrug resistant Pseudomonas aeruginosa (MDR-PA). Fenchone [(+)-fenchone] was obtained from Alfa Aesar with 98% purity. Bacterial cultures were grown for 24 h in a shaker incubator in Luria Bertani (LB) broth at 37 • C, whereas C. albicans was grown in Sabouraud Dextrose Broth (SD) broth at 28 • C. In the following step, the bacterial culture was rinsed with phosphate buffer saline, and the pellet was collected and re-suspended in fresh LB broth.

Minimal Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) value of fenchone was investigated by the standard microbroth dilution method in a 96-well microtiter plate. Briefly, 20 µL of freshly grown cultures of each tested organism was inoculated in 180 µL of Brain Heart Infusion (BHI) broth containing a two-fold concentration (800-1.56 mg/mL) of fenchone for 24 h at 37 • C. MICs are defined as the low concentration of a tested compound at which no obvious growth was observed [60]. Penicillin and fluconazole were used as reference drugs, and it was found that multidrug resistant P. aeruginosa was resistant to penicillin. For E. coli, the MIC value for Penicillin was 64 ug/mL, whereas fluconazole showed a 64 ug/mL MIC for C. albicans.

Minimal Bacterial and Fungicidal Concentration (MBC and MFC)
To determine the MBCs and MFCs, a 10µL suspension of bacteria and Candida was taken from microtiter plate wells that had no bacterial and fungal growth and spread on Mueller Hinton Agar (MHA) and Sabouraud Dextrose Agar (SDA) plates, respectively, for 24 h. MBCs/MFCs values of the tested compound could be defined as the minimum concentration of tested fenchone at which no bacterial or fungal growth or less than 3 CFUs were detected [60].

Evaluation of Anti-Biofilm Activity
Polystyrene flat bottom microtiter tissue culture was used to determine the anti-biofilm efficacy of fenchone against E. coli, MDR-PA, and C. albicans biofilms [61]. Briefly, 20 µL of fresh culture was added to 180 µL of LB broth containing different concentrations of fenchone and incubated for 24 h at 37 • C. After that, the content of the plate was discarded and washed three times with PBS before being stained with crystal violet dye (0.1% w/v) for 15 min. The excess dye was washed off and followed by rinsing in PBS and drying. The ethanol (95%) was then used to solubilize the stain, and the biofilm production was then measured at OD595 using an ELISA reader.

Statistical Analysis
All the obtained results were analyzed statistically using SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA).

Conclusions
In silico analysis revealed that the peptide-ketoacyl acyl carrier protein synthase I from E. coli and the anthranilate-CoA ligase from P. aeruginosa have favorable stereochemical, physiochemical, and functional properties. The fenchone molecule showed four H-bond interactions with the amino acid residues PRO283, ALA259, and LEU494 of anthranilate-CoA ligase protein with an ACE value of −113.15 kcal/mol. The docking results of fenchone with 1, 3-β-D-glucan synthase of C. albicans had five H-bond interactions with amino acid residues TYR197, TYR228, LEU232, PRO193, and ILE235 that have an ACE value of −130.89 (kcal/mol). The preliminary antibacterial, antifungal, and antibiofilm screening results demonstrated that the antimicrobial and antibiofilm potency of fenchone molecules was found to be moderate to high and were effective against E. coli, P. aeruginosa, and C. albicans. Among the tested pathogens, E. coli was the most sensitive to fenchone, showing the lowest MIC/MBC (i.e., 6.25 and 25 mg/mL). In addition, it has also been observed that fenchone had a significant effect on the biofilm forming abilities of tested pathogens. These findings suggested that the fenchone molecule has biological significance and might be explored as a new antimicrobial agent. However, additional electron microscopic and molecular research is still required to completely understand the mode of action of fenchone against bacteria and candida in order to justify the real-world applications of fenchone as a natural antimicrobial agent.

Data Availability Statement:
The authors confirm that the data supporting the study's findings are included in the article.