Molecular Docking Study of the C-10 Massoia Lactone Compound as an Antimicrobial and Antibiofilm Agent against Candida tropicalis

Antimicrobial resistance is now considered a global health problem because it reduces the effectiveness of antimicrobial drugs. According to the World Health Organization (WHO), the highest mortality rate is associated with infections caused by multidrug-resistant microorganisms, with approximately 700,000 deaths worldwide each year. The aim of this study was to determine the potential of C-10 massoia lactone to inhibit the growth of fungi and C. tropicalis biofilm, and molecular docking studies were performed to determine the nature of the inhibition. The study was conducted using the microdilution method for antifungal and antibiofilm testing and designed with a molecular docking approach. Furthermore, an analysis using the scanning electron microscope (SEM) was performed to evaluate the mechanism of effect. The results obtained showed that C-10 massoia lactone can inhibit the growth of fungi by 84.21% w/v. Meanwhile, the growth of C. tropicalis biofilm in the intermediate phase was 80.23% w/v and in the mature phase was 74.23% w/v. SEM results showed that C-10 massoia lactone damaged the EPS matrix of C. tropicalis so that hyphal formation was hindered due to damage to fungal cells, resulting in a decrease in attachment, density, and lysis of C. tropicalis fungal cells. Based on molecular docking tests, C-10 massoia lactone was able to inhibit biofilm formation without affecting microbial growth, while docking C-10 massoia lactone showed a significant binding and has the potential as an antifungal agent. In conclusion, the C-10 massoia lactone compound has the potential as an antibiofilm against C. tropicalis, so it can become a new antibiofilm agent.


Introduction
Bioflm is one of the problems in the treatment of human infections, making it a health problem throughout the world, especially for patients sufering from immune system disorders such as cancer, organ transplants, urinary tract disorders, and malnutrition [1].On the surface of hosts and inanimate objects such as tissues, blood vessels/heart valves, and wounds on the body or medical devices, bioflms can grow simply by adhering to the surface to form a group of microorganisms to form bioflms [2][3][4].
Bioflm bacteria are 1000 times more resistant to antibiotics than planktonic cells, which do not form bioflms [5].Tere are few available antibiotics that can efectively combat bioflm infections, resulting in very high levels of drug resistance.To date, there has been an increase in bioflmassociated infections [6].By forming bioflms, bacteria are able to protect themselves from antibiotics and disinfectants; therefore, antibiotic resistance often occurs in bioflms [2][3][4].Tis is because bioflms contain extracellular polymeric substances (EPS) that envelop or wrap a collection of microorganisms that gather into colonies, making it difcult for antibiotics to penetrate the EPS to attack these microorganisms [7].In addition, phenotypic and genotypic changes in gene expression are another cause of antibiotic resistance in bioflms [8].
Tis makes bioflm bacteria very resistant to antibiotic treatment and immune responses.Although it is known that antibiotic treatment is currently the most important and efective way to control microbial infections, it is almost impossible for antibiotic treatment to eradicate bioflm infection.Bioflm development via bacterial adhesion to human tissues and medical devices is an important virulence factor associated with increased antibiotic resistance, decreased phagocytosis, and overall survival of microorganisms [9][10][11].
Te management of infections caused by bioflms is very important because infections caused by bioflms can afect the morbidity and mortality of a person and can cause death if not properly treated [8,12].It is known that bioflm infections cause the death of 7500 people per year [13].Bioflms also cause economic losses of up to $11 billion in the United States [5,14].According to the World Health Organization (WHO), infections are the second leading cause of death in the world, and according to the US National Institutes of Health, infections caused by bioflms account for over 60% of all infections [15].Furthermore, 80% of infections are caused by bioflms [16].In several countries, the prevalence of bioflm infection in UTI cases is 46% in Tailand, 72% in India, and 95% in Iran [17].
In vitro and in vivo experiments show that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for bioflm bacterial cells are usually much higher (about 10-1000 times) than those for planktonic bacterial cells [18][19][20][21].
Terefore, due to the increasing antibiotic resistance of clinically relevant bacteria, there is an urgent need to develop new classes of antibiotics that are not afected by preexisting resistance mechanisms in the bacterial population [22,23].Tere is also a need for new anti-infectives that work through new mechanisms of action [24].Plants are a good source of natural materials to fnd bioactive compounds [25,26].Te study of alternative mechanisms of antibiotic resistance can open new avenues for the development of antibiotic resistance [24], and natural products can become an important reservoir of antibiotics to overcome mechanisms of antibiotic resistance [27,28].C-10 massoia lactone is a compound from the masoyi plant (Cryptocarya massoy) which has many health benefts such as antibioflm [29], antibacterial [30], antifungal [29][30][31], antioxidant, antidiabetic, and anticytotoxic [32], and immolator [33].Other microbes inhibited by the C-10 massoia lactone compound included Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus [34], Actinomyces viscosus, Streptococcus mutans, Streptococcus sanguinis, and Lactobacillus acidophilus [35], and Escherichia coli and Pseudomonas aeruginosa [29].
Terefore, the aim of this study was to investigate the inhibition of the antibioflm activity of the C-10 massoia lactone and to identify the targets of its inhibitory efect on bioflms using molecular docking.

Fungal Preparation for Testing.
Te test isolate used in this study was C. tropicalis JFM 1541.First, several fungal colonies on SDA media were inoculated into 15 mL of YPD medium, which was then incubated at room temperature in a shaker for 24 hours.Te fungal culture was then centrifuged at 3000 rpm for 15 minutes and washed with PBS.Te pellets obtained were then resuspended in RPMI medium, and a C. tropicalis 1 × 10 8 CFU/mL suspension was prepared using 5 mL of RPMI medium (equivalent to McFarland standard 0.5).Ten, 1 mL of the suspension was added to 9 mL of RPMI medium to obtain a 1 × 10 7 CFU/mL C. tropicalis colony suspension as a stock solution.To obtain a 1 × 10 6 CFU/mL C. tropicalis suspension, the working solution was prepared at a 1 : 10 dilution [36].

Antifungal Testing Using the Microdilution Method.
Antifungal testing was performed using the microdilution method, where samples were pure isolates with several concentration series (0.125%-1% w/v).Nystatin 1% w/v was 2 Te Scientifc World Journal used as a positive control for comparison with the study results.Tis test was performed on 96-well fat-bottomed polystyrene microtiter plates, with the sample placed in each microtiter plate well containing RPMI media.Te samples were then incubated at 37 °C for 72 hours, after which the absorbance was read using a microplate reader at a wavelength of 595 nm.

Determination of Minimum Bioflm Inhibitory Concentration (MBIC) Using the Microbroth Dilution Method.
Te novel C-10 massoia lactone was tested on the bioflm strain C. tropicalis JFM 1541.Te bioflm was inoculated into 96-well fat-bottomed polystyrene microplates.Each well received 200 μL of C. tropicalis suspension (10 6 CFU/mL) and was then incubated at ± 37 °C for 90 minutes [37].After incubation, the plates were washed with PBS.Media containing pure isolate at a concentration (0.125%-1% w/v) of 200 μL was then added to each well.Media containing 1% DMSO was used as a solvent control, and microbial suspension was used as a negative control.Te antifungal drug nystatin was added to the microbial suspension at 1% w/v as a positive control and media with no added microbes as a control medium [38].Te plates were then incubated at 37 °C for 24 hours to form bioflms in the intermediate phase and for 48 hours in the maturation phase [39,40].Te plates were then washed with PBS.125 μL of 1% crystal violet solution was added to each well and incubated for 15 minutes at room temperature.After incubation, the microtiter plate was washed with PBS, and 200 μL of 96% ethanol was added to each well to dissolve the bioflm.Optical density (OD) readings were performed using a microplate reader at a wavelength of 595 nm [39][40][41][42].

Scanning Electron Microscopy (SEM) Testing.
For the scanning electron microscopy (SEM) test, 24-well fatbottomed polystyrene microplates containing the test suspension were treated in the same way as for the bioflm test.Te microplates were then incubated at 37 °C for 24-48 hours.After incubation, the wells containing the test sample on the microplate were carefully rinsed three times with sterile distilled water.Ten, the wells containing the sample were fxed with cacodylate internal glutaraldehyde 0.5% (v/v) for ±24 hours at the cell passing point without changing the cell structure to be observed.Te microplate was then hydrated with methanol for 30 minutes to reduce the water content in the wells so that the results could be clearly seen.Scanning electron microscopy was used to examine the samples at a voltage of 10 kV [42,43].

Molecular Docking.
Te possible pathway for the binding of phytochemicals found in the massoia lactone compound to the protein 3HEM (mycolic acid cyclopropane synthase CmaA2 in complex with dioctylamine) is a protein target from the bacteria/EPS bioflm matrix, which was modelled using molecular docking.Te three-dimensional structure of the 3HEM matrix was obtained from the RCSB protein database.Te data were then prepared by removing crystallographic water and crystallised ligands.Molecular docking simulations were performed using AutoDock Vina (Vina) [44].Te intermolecular interactions of the proteinligand complex are inferred using the PoseView accessible via Protein PDB.Optimisation is performed by adding hydrogen atoms and determining the grid box parameters.Te addition of a hydrogen atom is essential for ligand and receptor interactions.Te hydrogen atom, which is automatically added by the program, is polar as it is involved in hydrogen bonding.Determining the grid box parameter produces a three-dimensional map of protein interactions with each type of atom found in the ligand [45].

Statistical Method.
Te statistical analysis of the research results was carried out using the ANOVA normality test, which was performed using the Shapiro-Wilk method.Te normality level of the test is P < 0.05, and the data were analysed using the Statistical Package for the Social Sciences (SPSS).

Result and Discussion
Microdilution is an antifungal and antibioflm testing method that uses the same principle as the liquid dilution method but with small amounts of compounds, media, fungi, and bacteria using a fat-bottomed polystyrene microtiter plate with 96 wells.With research results that are more sensitive and efective in quality, use small samples, and are efcient, this method is preferred for further research because it can test many samples quickly.

Te Results of the Antifungal Test Using the Microdilution
Method.Based on the research results, the C-10 massoia lactone compound provided an antifungal inhibitory activity against C. tropicalis of 84.21% ± 0.01.Although not as great as the positive control which had an inhibitory activity of 87.55% ± 0.01, the C-10 massoia lactone compound provided almost the same inhibitory activity as the positive control nystatin.In addition, at the lowest concentration (0.125%), it could still give an inhibitory activity of 74.21% ± 0.01 against C. tropicalis (Figure 1 and Table 1).According to Zhang et al. [31], the C-10 massoia lactone compound has antifungal activity against Fusarium graminearum spores, where C-10 massoia lactone inhibits the growth of Fusarium graminearum by interfering with the formation of pores in the fungal cell membrane, resulting in increase in intracellular ROS levels, decrease in ergosterol content, and leakage of intracellular components within the fungus, leading to cellular necrosis and fungal cell death.
In addition, according to Zoccolotti et al. [46], masoyi extract reduced Candida albicans cell metabolism from fungal growth to the formation of Candida albicans bioflms, where masoyi extract reduced cell viability, which was better than the control group (nystatin) in its cytotoxicity test.
Te Scientifc World Journal

Determination of Minimum Bioflm Inhibitory Concentration (MBIC 50 ) Using the Microbroth Dilution Method.
Bioflms in healthcare are one of the problems in dealing with infections associated with human diseases, one of which is nosocomial infection [9].Although bioflms are not a direct cause of death, they do cause nosocomial disorders that disrupt the balance of morbidity and mortality in humans [12].
Te determination of MBIC (minimum bioflm inhibitory concentration) values is essential in bioflm research.Tis is because the MBIC value can be used to determine the lowest concentration of antimicrobial agent, which indicates the inhibitory value of the initial bioflm formation.Although this value does not depend on the average number of living cells in the bioflm, the determination of this value becomes a determinant or guide in the treatment of bioflm infections because MBIC is a timeefcient and accurate research method as an antimicrobial agent for bioflm testing [47].
In addition, the MBRC value is determined by the MTT reduction test [40], where the MTT reduction test (MTT assay) is a test that can detect the presence of antibioflm activity of a compound by reducing MTT saltsin living cells (bioflms) to insoluble formazan [48].Te MTT test assesses microbial viability in bioflms formed in vitro.In determining the tendency of microbes to form bioflms in complex media, the MTT test quantifes formazan from the reduction of tetrazolium salts by the electron transport system [49][50][51][52].
Based on the research results obtained, the highest concentration of the C-10 massoia lactone compound extract (1%) gave an inhibitory activity of 80.23% ± 0.01 against C. tropicalis.Meanwhile, the lowest concentration (0.125%) showed an inhibitory activity of 70.21% ± 0.01.Although the inhibition results of this middle phase bioflm were not more signifcant compared to the positive control, which had an inhibitory activity of 85.11% ± 0.01, the C-10 massoia lactone compound was able to inhibit more than 50% (MBIC 50 ) of the bioflm formation, even at the lowest concentration of 0.125%.
Based on the research results, the compound C-10 massoia lactone has the potential as an antibioflm against C. tropicalis up to the maturation phase.Te compound C-10 massoia lactone at 1% concentration was able to inhibit 74.23% ± 0.01.Although this value is not as high as the drug control, i.e., 78.30% ± 0.01, it is almost equal to the inhibition given by the compound C-10 massoia lactone at a concentration of 1%.Furthermore, even at the lowest concentration, 0.125%, the C-10 massoia lactone extract was still able to inhibit the formation of C. tropicalis bioflms by more than 50% (MBIC 50 ) with an inhibition of 78.30% ± 0.01 (Table 2).
In previous studies, the C-10 massoia lactone compound was found to be an antibioflm agent for monomicrobial and polymicrobial organisms.Te C-10 massoia lactone compound can inhibit the formation of bioflms of Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus [34], polymicrobial bioflms of Actinomyces viscosus, Streptococcus mutants, Streptococcus sanguinis, and Lactobacillus acidophilus [35], and polymicrobial bioflms of Candida albicans, Escherichia coli, and Pseudomonas aeruginosa [43].Massoia oil nanoemulsion preparation (C-10 massoia lactone) provides a robust inhibitory activity on Pseudomonas aeruginosa and Staphylococcus aureus bioflms compared to other essential oils [53].In addition, other studies have shown that C-10 massoia lactone may be a potential antibiotic candidate to inhibit bioflm growth, as this compound can disrupt microbial membranes in bacterial and bioflm growth [54].It is known that the C-10 massoia lactone compound works by disrupting the polysaccharide and lipid matrices in bioflms to disrupt the bioflm formation in microbes [55].
In the research results obtained (Figure 2), bioflm inhibition was more signifcant in the middle phase (24 hours) than in the maturation phase (48 hours).Tis is infuenced by the bioflm formation process, where the EPS (extracellular polymeric substances) matrix in the middle phase is  Journal not yet as structured and complex as in the ripening phase.Te microbes themselves incorporate EPS due to changes in growth rates and gene transcription in planktonic cells or free cells where EPS functions to envelop/enshroud/protect the colonies inside [6,7,56].Tis statement is also in line with the opinion of Donlan and Costerton [6], Purbowati et al. [10], Kannappan et al. [57], and Hamzah et al. [1], where there will be a decrease in the inhibitory activity of bioflms in the maturation phase (48 hours) compared to the intermediate phase (24 hours) because bioflm growth in the maturation phase has a more complex and structured bioflm defence system so that bioflms in this phase have formed a bioflm defence system.Te maturation phase bioflm defense is stronger because the bioflm cells continue to develop for several hours and have a longer lifespan compared to the midphase bioflm.
In addition, Homenta [58], Achinas et al. [59], and Muhammad et al. [47] also confrmed this statement in that the structure of the bioflm cells produced in the maturation phase appears thicker and more complex than that in the intermediate phase.Tis is because the mucus layer produced by the bioflm is dense and adheres strongly to the wells.Tis makes it difcult for antibiotics and compounds to penetrate the EPS bioflm layer.EPS on bioflm inhibits the mass transport of antibiotics through bioflm formation.According to Hamzah et al. [60], the bioflm matrix in fungi also acts as the main barrier protecting bioflm cells from attack by antifungal drugs and the body's immune system.
According to Rolli et al. [61], the massoia lactone compound in the masoyi plant is one of the main compounds that can inhibit bioflm formation compared to other compounds.
Te results of statistical analysis using Bonferroni post hoc are used to determine which concentration has a difference.Te results of the data obtained (Figure 2) show that there are diferences or signifcant diferences between each concentration, namely, 1, 0.5, 0.25, and 0.125% w/v, so it is stated (P < 0.05).Based on the calculation of percent inhibition between C-10 massoialactone 1% b/v with nystatin drug control (as a positive control), in vitro results showed no signifcant diference (P > 0.05).

Scanning Electron Microscopy (SEM).
Te compound C-10 massoia lactone at a concentration of 0.5% v/v can cause lysis of C. tropicalis bioflm cells, accompanied by a decrease in cell density.Based on Figure 3, SEM results show that the administration of C-10 massoia lactone 0.5% can cause damage to C. tropicalis fungi, as the C-10 massoia lactone compound attacks the C. tropicalis EPS matrix so that the formation of hyphae is hindered by damage to the fungal cells.Tere was also a reduction in attachment, density, and lysis of C. tropicalis fungal cells.
Te mechanism of inhibition of the C-10 massoia lactone compound on the growth of intermediate and mature bioflms is thought to be by inhibiting the attachment of  Te Scientifc World Journal microbes to the surface so that bioflm development is disrupted.When bioflm development is disrupted, the structure of the bioflm is afected to enhance antimicrobial defence.Te next step is the binding of C-10 massoia lactone compound to bacteria and releasing them from mature bioflms (Figure 4).In addition, the C-10 massoia lactone compound also damages the EPS bioflm so that the communication pathways for cells and nutrients between microbes are cut of, so that the microbes that previously wanted to form bioflms are ultimately unable to form bioflms and cause these microbes to lyse or die due to the loss of nutrients as a component of bioflm formation (Figure 4).Teoretically, the phenolic group is a group that functions in causing antibacterial activity.Te compound C-10 massoia lactone has a phenolic group and shows antifungal and antibioflm activities.

Molecular Docking.
To validate the binding of native ligands, the 3D conformation of natural ligands to receptor proteins is searched for, taking into account the coordinates of the centre of mass of the structure and the grid box arrangement of the binding site pocket in units of Å (angstroms) or number of points.Te docking results were aligned with the natural ligand conformations, and the measurement results were expressed as root mean square deviation (RMSD) values.Te RMSD value for conformational structures that are still acceptable for alignment is less than three, but the most optimal is less than 2 [62].Te closer to 0, the better the alignment value, which means that the output ligand from the docking does not experience much deviation [63].In our study, we validated using the RMSD value.Te following are the RMSD results that we obtained: validity receptor 3HEM (native ligand RMSD: 0.663 (valid, value RMSD <2 Å).Te results of the RMSD calculations between the native ligands and the efects of each docking show a value of 0.663 Å, indicating that the method can be continued for molecular docking tests.Te best RMSD value is the value that is close to 0. Tus, the frst conformation for each ligand compares the conformational value with itself as the best conformation.In addition to looking at the lowest binding energy value, it is also necessary to look at the interaction with the reference compound, as the interaction with the desired residue will afect the role of the combination as an inhibitor.6 Te Scientifc World Journal  Te Scientifc World Journal To propose a molecular explanation for the inhibition of 3HEM enzymes by the compound massoia lactone, the docking score is −6.3.Massoia lactone is primarily associated with hydrogen bonding interactions involving residues TYR 280, TYR 41, PHE 215, and LEU 220.A molecular docking study was performed during the docking procedure, assuming a model in which proteins and ligands are considered rigid and fexible [64].Estimates of the docking scores, number of interactions, interacting residues, and bond lengths are shown in Table 3.
Molecular docking of masoialactone against C. tropicalis can be seen in Figure 5. Proteins are shown as a surface representation.Ligands are shown as a rod representation.A possible explanation could be the limitations of the docking model, namely, how the compounds get to the active site.In all molecular docking experiments, the process starts with the ligand at the active site."Native" is a natural ligand on the target receptor.Te new compound has a strong antibioflm profle, inhibiting bioflm formation without affecting microbial growth.Te interaction shows that the docking massoia lactone showed a signifcant binding and has the potential as an antifungal agent.

Conclusion
Based on the research results obtained, the compound C-10 massoia lactone can inhibit the growth of fungi by 84.21% ± 0.01 * .Meanwhile, the development of C. tropicalis bioflm in the intermediate phase was 80.23% ± 0.01 * , and in the mature phase, it was 74.23% ± 0.01 * .SEM results showed that C-10 massoia lactone damaged the EPS matrix of C. tropicalis, resulting in impaired hyphal formation due to damage to fungal cells, which caused a decrease in attachment, density, and lysis of C. tropicalis fungal cells.Based on molecular docking tests, C-10 massoia lactone could inhibit bioflm formation without afecting microbial growth while docking C-10 massoia lactone showed a signifcant binding and has the potential as an antibacterial agent.In conclusion, the C-10 massoia lactone compound has the potential to be an antibioflm agent against C. tropicalis, so it can become a new antibioflm agent.

Table 2 :
Percentage of antibioflm inhibition of the C-10 massoia lactone compound against C. tropicalis in the middle phase (24 hours) and maturation phase (48 hours).

Table 3 :
Docking score, number of interactions, interacting residues, and bond length from the ligand massoia lactone and native to 3HEM target.