Molecular docking: Bioactive compounds of Mimosa pudica as an inhibitor of Candida albicans Sap 3

Candida albicans (C. albicans) is a commensal microbiota that resides in humans. However, in certain cases, C. albicans can infect and cause several diseases to humans. This study aimed to investigate the interaction between Mimosa pudica bioactive compounds and C. albicans Sap 3. Molecular docking analysis was carried out using YASARA structure. The procedures involved preparation of ligands and target receptor, molecular docking, data analysis and visualization. All 3D ligands were downloaded from PubChem NCBI, while target receptor was downloaded from RCSB PDB. The interaction between Mimosa pudica bioactive compounds against Sap 3 resulted in a binding energies ranges from 5,168 – 7,480 kcal/mol and most of the interactions formed were relatively strong. Furthermore, the test ligands had contact with the catalytic residues and substrate binding site pockets S1/S2/S3/S4 on the target receptor. Bioactive compounds of Mimosa pudica have relatively good interactions in inhibiting C. albicans Sap 3.


Introduction
Most of the human pathogenic fungi of the genus Candida reside in animals and humans 1 . Invasive Candida infection is one of the most common fungal infections globally 2,3 . In western countries, individuals generally have Candida as a commensal microbiota in their gut 1 . In the United States, Candida spp. is reported to be one of the leading causes of healthcareassociated infections 2,3 . Candida infection is also often associated with medical devices such as central venous catheters, cardiovascular machines, and urinary catheters 2,4 . Among the species of Candida sp., Candida albicans (C. albicans) (37%) is the most commonly found in the clinical species 2 . C. albicans is known as a commensal microbiota that lives in normal humans. However, C. albicans is also one of the most common fungal species causing opportunistic infections, such as candidiasis ranging from superficially invasive infections to life-threatening in debilitated patients 5,6 . Invasive candidiasis is associated with a high mortality rate, ranging from 20 to 49%. C. albicans can be found to colonize various mucosal surfaces such as skin, mouth, and vagina; most studies consider the gastrointestinal tract as the main entry point for C. albicans to enter the bloodstream [5][6][7][8][9] . On the other hand, C. albicans is the most common causative agent of oral, vaginal, and disseminated candidiasis. Oral candidiasis is one of the most common opportunistic infectious diseases among patients suffering from HIV infection. C. albicans can multiply rapidly, invade tissues, and cause symptomatic mucosal lesions in people with immune systems disorder [10][11][12] . Therefore, good immune systems are important to maintain the fungus in a commensal state, preventing invasion, epithelial damage, and mucosal infection 1,13 . It is estimated that more than 7.5 million people globally have been infected by invasive candidiasis. There are unwanted side effects, ineffectiveness, and the rapid development of resistance by fungi, therefore there is a need for the development of new antifungals 9 . Many factors and activities have been identified as the cause of the pathogenicity of C. albicans 1,14 , secreted aspartic proteinases (Sap) 1-3 have been observed to play an important role in adhesion and tissue damage in local infections 15,16 . In-depth understanding of the contribution of the Sap family to the pathogenicity of C. albicans, obtained by apprehending the C. albicans strains; Sap 1-3 are involved in mucosal infections, and Sap 4-6 are involved in systemic infections 15 . C. albicans Sap plays a multimodal role in the infection process, therefore the development of inhibiting Sap as targets for a wide variety of infections caused by C. albicans appears to be a good strategy 10 .
Mimosa pudica Linn (M. pudica) is a medicinal plant with pharmaceutical and nutraceutical potential; this plant is also popular among traditional healers to treat various diseases 17 . Herbal plants have been widely used to treat several diseases and are used as traditional medicine 18 . This plant was observed because of its thigmotactic and seismonastic movements. M. pudica is known for its analgesic, anti-inflammatory, diuretic activity, insomnia, and urogenital infections 17,[19][20][21] . Phytochemical compounds in M. pudica are shown in the study of Vijayalakshmi et al. 17 .
The molecular docking method is one of the in silico research methods and is one of the most powerful techniques to help discover new ligands for proteins with known structures, thus playing a pivotal role in structure-based drug design 22 . Therefore, through this study, we investigated the possibility of an interaction between bioactive compounds from M. pudica and the Sap 3 Candida albicans to find new inhibitor candidates.

Receptor preparation
Sap 3 with the code "2H6T" was downloaded from the RCSB PDB website (https://www.rcsb.org/, accessed on 13 July 2022). Preparation was carried out using YASARA structure (Bioinformatics 30.2981-2982 Version 19.9.17) by adding hydrogen atoms, removing water molecules around the receptor, and adjusting the receptor pH value to 7.40 23 .

Ligand preparation
The bioactive compounds of M. pudica was obtained from Vijayalakshmi et al. 17 , then the 3D structure was downloaded from the NCBI PubChem page (https://pubchem.ncbi.nlm.nih.gov/, accessed on 13 July 2022) 24 . The preparation also uses YASARA structure to add hydrogen atoms, adjust the pH value to 7.40, and energy minimization 23,25 .

Molecular docking
To explore the possibility of ligand binding into the pocket area of the substrate binding site or receptor catalytic residue, molecular docking was carried out using YASARA structure software 26 . YASARA structure was set to AutoDock Vina settings, AMBER03 force field, and 25 runs (dock_run). The docking area was set to around all atoms to expand the docking area. Then the other parameters remained unchanged. Finally, the resulting conformers were analyzed to determine hydrogen bonding, hydrophobic interactions, electrostatic interactions, and other interactions 23,27 .

Data analysis and visualization
After docking, the receptor-ligand complex interactions were analyzed using BIOVIA Discovery Studio Visualizer v21.1.0.20298. The results of the analysis were in the form of 2D and 3D structures to help visualize and analyze the interaction pattern of the ligand-protein complex 9 .

Results and Discussion
This research used the molecular docking method to bind compounds (test ligands) from Mimosa pudica (M. pudica) to Sap 3 Candida albicans (C. albicans). The docking process did not target specific residues or limit the docking area, therefore it is hoped that when the tethering process took place, the ligand can have more comprehensive residual contact and still have contact with essential residues of the target receptor.
It is widely known that C. albicans has Sap 1 to Sap 10 genes. Sap is one of the classic virulent factors whose expression is modulated by several conditions such as the influence of pH, temperature, site of infection, and physicochemical environmental conditions 10,28 . This research used one of the Saps, namely Sap 3. We chose Sap 3 with PDB ID code 2H6T because it has been crystallized with pepstatin 15 . The pepstatin used as the inhibitor standard to refer to the standard binding energy, dissociation constant (Kd), and amino acid residues 10 .
Based on the results of this study, interestingly, pepstatin had the highest binding energy of 8.857 kcal/mol and Kd 321908.8438 pM. The test ligands that have binding energy close to pepstatin are turgorin with a binding energy of 7,480 kcal/mol and Kd 3289177.500 pM, then based on the binding energy respectively, D-glucuronic acid with a binding energy of 6.219 kcal/mol and Kd 27632016.000 pM, gallic acid with a binding energy of 6.053 kcal/mol and Kd 36567240.000 pM, L-norepinephrine with a binding energy of 5.770 kcal/mol and Kd 58956804.000 pM, mimosine with a binding energy of 5.599 kcal/mol and Kd 78682576.000 pM, L-ascorbic acid with a binding energy of 5.390 kcal/mol and Kd 111963792.000 pM, and the lowest binding energy is linolenic acid with a binding energy of 5.168 kcal/mol and Kd 162856752.000. The more positive value of the binding energy indicates stronger bond between the ligand and the target receptor whereas 29,30 . The results of the binding energies are presented in Figure 1. The smaller value of Kd indicates that ligand binding affinity againts the receptor is stronger [31][32][33] . We also show the Kd and overall residual contacts in Table 1. It is important to note that through Borelli et al. 15 research, Sap 3 was known to be divided into several pockets of substrate binding sites on S1, S2, S3, and S4. S1 consists of VAL30, TYR84, ASP86, THR88, VAL119, and ILE123 amino acid residues. The S2 consists of GLY85, ASP86, THR221, TYR225, SER301, TYR303, and ILE305 amino acid residues. S3 consists of VAL12, SER13, ASP86, THR88, SER188, ASP120, and GLY220 amino acid residues. S4 consists of VAL12, THR222, ILE223, TYR225, GLN295, LEU297, and GLY299 amino acid residues, while the catalytic residues are located in ASP32 and ASP218. This study usedthese important residues as a criterion. Through the help of the Discovery Studio software, the patterns of interactions that are formed within the complex can be seen. The results of the analysis are presented in Table 2.
Pepstatin tends to make hydrogen bonds with the target receptor ( Figure 2). Hydrogen bonds have a distance ranging between 1.99846 -3.00181 Å. The category of hydrogen bonds formed in the Sap3-Pepstain complex has from chemistry H-donor and to chemistry H-acceptor. SER13, GLY85, ASP86, and THR222 bind with the O atom in pepstatin. GLY220 and THR222 bind with the H atom of pepstatin. Pepstatin also has hydrophobic and unfavorable interactions. The hydrophobic interaction between A:TYR84 -A:UNK1:C was Pi-Alkyl type. Other hydrophobic interactions also formed in Alkyl type between A:UNK1:C -A:VAL119. This study found an unfavorable interaction in the Sap 3-Pepstatin complex at A:GLU193:OE1 -A:UNK1:O under the negative-negative unfavorable type. Pepstatin interacts with amino acid residues at S1/S2/S3/S4 and catalytic residues. In Borelli et al. 15 study, pepstatin interacted with amino acid residues located at S1/S2/S3/S4. However, pepstatin suboptimally filled several binding sites, mainly at S3/S4 15 and his research also explained that pepstatin is a pentapeptide produced by the Streptomyces 15 .

Figure 2.
Visualization using BIOVIA Discovery Studio shows the interaction formed on the pepstatin complex with Sap 3 (a) 2D visualization shows bond distance, amino acid residues, and interaction type (b) 3D visualization shows the semi-transparent complex Turgorin formed ten interactions with the target receptor; out of ten interactions, only one was hydrophobic (Figure 3). The hydrogen bonds formed have a distance between 1.92332 -2.81122 Å. This ligand had contact with the ASP218 catalytic residue formed the OH group. Amino acid residues that interact with the ligand O atom were ASP85 and ASP86 by forming hydrogen bonds. TYR84 interaced with the ligand aromatic ring with a distance of 4.68817 Å, with from chemistry Pi-Orbitals and to chemistry Pi-Orbitals. The strongest hydrogen bond was found on the ASP218 catalytic residue and the ligand OH group with a distance of 1.92332 Å. These ligands interacedt with amino acid residues in S1/S2 and catalytic residues. Visualization using BIOVIA Discovery Studio shows the interaction formed in the turgorine-Sap 3 complex (a) 2D visualization shows bond distance, amino acid residues, and interaction type (b) 3D visualization shows the complex in a semi-transparent manner D-glucuronic acid, gallic acid, and L-ascorbic acid had no interaction with the substrate binding site pocket. They did not even interact with the Sap 3 target receptor's catalytic residue ( Table 2). D-glucuronic acid had ten interactions, with details of nine hydrogen bonds and one unfavorable interaction (Figure 4). Amino acid residues that formed OH groups with ligands are ASN9, GLN11, ARG162, GLN163, and ARG312. The distance created due to the presence of hydrogen bonds ranged from 2.10657 -2.94953 Å. Unfavorable bonds were formed on the ASN9 amino acid residues with ligand H atoms, from chemistry H-donor and to chemistry Hdonor, and the distance is 1.61695 Å.
Abu-Izneid et al. 34 explained that glucuronide or glucuronoside, which are D-glucuronic acid and glucuronide derivatives, is an important class of active pharmaceutical compounds known to have antiviral activity against viruses, such as A/H1N1 and A/H3N2 strains of influenza virus.
Through docking simulations, we looked for whether there was a possibility that Dglucuronic acid ligand has antifungal activity. From the results of the analysis of D-glucuronic acid ligand targeting Sap 3, which showed that nine hydrogen bonds were formed in the receptor-ligand complex, although this ligand had no contact with the catalytic residue or the pocket of the substrate binding site, we predicted this ligand was quite good at inhibiting the C. albicans Sap 3.  35 through an in vitro study, explained that gallic acid has potential as antifungal activity against Candida spp. Another study also reported, through in vitro and in silico studies by Uma Maheshwari Nallal et al. 30 , that gallic acid, which is classified as an active phytochemical compound, is predicted to be an effective inhibitory agent of Sap from Candida species. Therefore, even though in this research gallic acid did not interact with the substrate binding site pocket or the catalytic residue of the target receptor, gallic acid was predicted to have a reasonably good interaction in inhibiting Sap 3.

Figure 5.
Visualization using BIOVIA Discovery Studio shows the interaction formed on the gallic acid-Sap 3 complex (a) 2D visualization shows bond distance, amino acid residues, and interaction type (b) 3D visualization shows semi-transparent complex L-norepinephrine with the target receptor formed three electrostatic bonds, two of which have the same amino acid residue and ligand atom (Figure 6), which also formd hydrogen bonds, namely A:UNK1:H1 -A:ASP218:OD2 and A:UNK1:H3 -A: ASP218:OD2, while A:UNK1:N -A:ASP32:OD1 only formed electrostatic bonds. The distance created by the presence of hydrogen bonds ranges from 2.16417-5.09049 Å. The two catalytic residues formed hydrogen and electrostatic bonds with the ligands in this complex. The hydrophobic interactions formed in this complex are known to form in the amino acid residues VAL30 and ILE123, which had contact with the aromatic ring of the ligand. This ligand had contact with amino acid residues located at S1/S2/S3 and the catalytic residues. Mimosine also formed hydrogen bonds and electrostatic bonds on the identical amino acid residues and ligand atom (Figure 7), namely at A:UNK1:H1 -A:ASP218:OD2 and A:UNK1:H2 -A:ASP32:OD2. An electrostatic bond was also formed at A:UNK1:N -A:ASP32:OD1. Both receptor catalytic residues are formed in the two interactions. The distance created due to the presence of hydrogen bonds ranges from 1.99745 -3.34731 Å. In this complex, the interactions formed at the VAL30, TYR84, and ILE123 amino acid residues had contact with the aromatic ring of the ligand. This ligand had contact with amino acid residues located at S1/S2/S3 and catalytic residues. It was reported that mimosine proved to be more efficient for controlling dermatophyte fungi in a previous study 36 . Mimosine also has antimicrobial and antiviral activity 36 .

Figure 7.
Visualization using BIOVIA Discovery Studio shows the interaction formed on the mimosine-Sap 3 complex (a) 2D visualization shows bond distance, amino acid residues, and interaction type (b) 3D visualization shows semi-transparent complex Dominantly, L-ascorbic acid has hydrogen bond and one unfavorable bond with the target receptor ( Figure 8). The distance generated by the presence of hydrogen bonds ranges from 2.12016 -2.97822 Å. The O atom of the ligand binds with the amino acid residues GLY127, GLU193, and LEU194, while TYR128 forms the OH group. An unfavorable bond is formed between the amino acid residue ASN192 and the O atom of the ligand. The bond distance is 2.69808 Å with an unfavorable acceptor-acceptor type.  The number of hydrogen bonds formed is known to determine binding strength in the receptor-ligand complexes. In addition, the efficiency of ligand binding to enzymes is also influenced by the binding energy 30 . The phytochemicals present in medicinal plants are known to have the potential as antimicrobial and various other biological activities 37 . All analysis data were summarized in Table 2.