Molecular docking of various bioactive compounds from essential oil of Trachyaspermum ammi against the fungal enzyme Candidapepsin-1

The bioactive compounds from essential oil of Trachyaspermum ammi using gas chromatography–mass spectrometry and their inhibition potential against the enzyme Candidapepsin-1 were studied. The research work focuses on the molecular simulation of bioactive compounds against the enzyme that acts as a potential drug target and support the drug discovery process. Candidapepsin-1 has been reported to be the cause for biofilm formation, superficial skin infections, and oral infections. Fifteen active compounds and their interactions with Candidapepsin-1 were studied in this research work. The compounds satisfied Lipinski’s rule of five in order to be used as an oral drug. ADMET properties of the compounds used to determine pharmacodynamic and pharmacokinetic properties which were reported in the study. The compounds were docked against the enzyme with the help of AutoDock 4.2.6 software. Ligustilide has the lowest free binding energy of −5.75 kcal/mol against the Candidapepsin-1 with three hydrogen bond interactions at Ile 223, Tyr 225, and Thr 222 at the active site of the enzyme followed by cedrane with −5.20 kcal/mol. The hydrogen bond interactions, Vander Waals interactions, and two-dimensional and three-dimensional interactions were studied.


INTRODUCTION
Trachyaspermum ammi often known as Ajwain or carom seeds or bishop's weed is a plant seed that are widespread across diverse areas of India and are cultivated predominantly in north-western states of India like Gujarat and Rajasthan, Madhya Pradesh and in other countries like Afghanistan, Bangladesh, Egypt Iran, and Iraq. Trachyaspermum ammi belongs to the family of Apiaceae, dicot and possess high medicinal properties. Trachyaspermum ammi is used by the people traditionally as it possesses several properties to cure diverse liver, lungs, and stomach disorders. Trachyaspermum ammi is commonly used as an herbal medicine by the people. The seed also possesses carminative and antispasmodic properties which make it an effective remedy for various disorders, such as indigestion, abdominal pain, piles, amenorrhea, bronchial inflammation, asthma, hepatoprotective agent, and chronic diarrhea (Bairwa et al., 2012). Research has shown the significant antibacterial properties of T. ammi against a wide variety of bacteria, such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Shigella flexneri, and Salmonella typhi (Kaur and Arora, 2009). Multiple drug resistant fungal strains, such as Candida albicans, Candida krusei, Candida tropicalis, and Candida glabrata, were also inhibited by T. ammi (Khan et al., 2010). Volatile oils of T. ammi has the potential to for antifungal properties against C. albicans and Aspergillus species (Ishwar and Singh, 2000). The plant and its seeds contain several phytochemicals and essential oils, such as thymol, g-Terpinene, isobornylisobutyrate, o-Cymene, p-Cymene, a-Pinene, silphine, verbenene, ionone myrcene, thymyl acetate, etc. (Dhaiwal et al., 2017). Thymol is the major constituent of T. ammi seeds. Trachyaspermum ammi contains 50% of thymol. Thymol is an essential oil obtained from the seeds of T. ammi and finds applications in toothpaste and perfume industries (Joshi, 2000).

Candidapepsin-1 enzyme structure
The Candidapepsin-1 (EC: 3.4.23.24) is an apoenzyme and belongs to the class of Secreted aspartic proteinase (Sap) family Smolenski et al., 1997). The gene responsible for this enzyme is SAP1 and is located on the chromosome 6 of the organism. The Candidapepsin-1 is a proteolytic virulent enzyme from the endophytic polymorphic fungal species C albicans and is responsible for superficial Candida infections like oral and skin infections in immunocompromised individuals (Correia et al., 2010;Meenambiga et al., 2018;Staib et al., 2000). The enzyme invades and adheres to the host tissue by digesting the host cell membrane and small molecules to acquire nutrition. The enzyme digests the cell host cell membrane to inhibit the attack from the host immune system (Naglik et al., 2003). The enzyme attacks the oxygen carrier molecule hemoglobin through its proteolytic activity and releases various antimicrobial hemocidins to combat against various microbes of the same niche (Schaller et al., 2001). The enzyme is very stable and active at pH 5.0 (Aoki et al., 2011). This enzyme is similar in structure and function with its isoenzyme Candidapepsin-5. There are 10 subclasses of acidic hydrolases in the Sap family from Candidapepsin-1 to 10. The structural investigation discloses the highly conserved overall secondary structure of Candidapepsin-1 enzyme (Borelli et al., 2008). However, Candidapepsin-1, Candidapepsin-2, and Candidapepsin-3 differ from Candidapepsin-5 in two aspects, namely, the net overall electrostatic charge and structural conformation of its entrance toward the active site of the enzyme (Borelli et al., 2008). The active site of the enzyme holds a net negative electrostatic charge due to the presence of basic amino acids. The enzyme contains two chains A and B with 391 amino acid residues and molecular weight of 41.6 KDa. The active site is located between the positions at Asp82 and Asp267 of the enzyme. The entry toward the active site of the enzyme is wider. Figure 1 denotes the three-dimensional structure of the Candidapepsin-1 enzyme. The most predominant inhibitor of the enzyme is Pepstatin A. Hence, molecular docking of the enzyme with bioactive plant secondary metabolites supports the existing drug development process as molecular chemical interactions can be comprehensively studied.

Bioactive compounds obtained from gas chromatographymass spectrometry (GC-MS) analysis of Trachyaspermum ammi essential oils
The bioactive compounds from essential oils of T. ammi were obtained from the conventional hydrodistillation process using Dean-Stark apparatus (Dhaiwal et al., 2017;Javed et al., 2012). The essential oils were analyzed using GC-MS (Dhaiwal et al., 2017).
The information about the bioactive compounds, such as IUPAC name, structure, and chemical formula, were retrieved from PubChem database ( Table 1). The bioactive compounds mentioned in Table 1 were used for molecular docking against the enzyme Candidapepsin-1. Lipinski properties and ADMET properties of the compounds were studied.

Enzyme target preparation
The enzyme Candidapepsin-1 (PDB Id: 2qzw) was used as the drug target in this research work with resolution 2.05 Å and the method of incorporation was done using X-ray diffraction. The    (Sasikala and Meena, 2016). The interfering crystallographic water molecules were also removed from the protein for effective ligand binding (Meenambiga et al., 2018).

ADMET properties of bioactive compounds from essential oil
Prediction of absorption, distribution, metabolism, and excretion properties of the compounds were done using the freely available SwissADME software package (Kramer et al., 2017). This was performed to enhance the success of drug discovery and development process.

Molecular docking studies using the AutoDock 4.2.6 software
Molecular docking was performed using the 15 bioactive compounds depicted in the Table 1. The bioactive compounds were docked against the Candidapepsin-1 enzyme using the comprehensive bioinformatics tool AutoDock 4.2.6 software. The AutoDock 4.2.6 is relied on the principle of Lamarckian genetic algorithm and is the most reliable automated tool used by the researchers to understand the protein-ligand interactions and protein-protein interactions (Meenambiga et al., 2015). The ligand-protein structure-based drug designing was performed using this software. AutoDock 4.2.6 is dependent on two techniques, namely, the Rapid-grid based estimation and systematic search of torsional freedom for the ligand-protein molecular docking (Meenambiga et al., 2015).

Grid parameters
Default grid size of 20 Å was set. Total grid points per map were 64,000. Grid spacing was 0.375 Å (default). The center grid box sizes were x center: −16.302, y center: −23.34, and −16.245, respectively.

Discovery studio 3.1-visualizer
Discovery studio 3.1 is a visualizer programmed and developed by the Accelrys. This software is free of cost, provides comprehensive information, and is the most often used by the scientific community to view the ligand-receptor interactions.
The software provides us with the necessary information about the interactions of small and large molecules taking part in the interaction. The software deals with various aspects of molecular docking, such as macromolecule engineering, ligand-receptor interactions, pharmacophore modeling, antibody modeling & optimization simulations, macromolecule design, and proteinprotein interactions (Almagro et al., 2011;Luu et al., 2011;Sutter et al., 2011). The two-dimensional and three-dimensional interactions images displayed in this study were developed through this software.
Molecular docking of ligands against the active site of the enzyme will elucidate the interactions between them. This will pave way for discovery of novel phytomedicines in the field of drug discovery and development.

acceptors (<10), and (5) Molar refractivity (40-130). The
Lipinski's rule of five is utilized to appraise the drug-likeliness of a compound; in other words, it is very essential criteria for a compound to satisfy this rule in order to be administered orally. The Lipinski's rule of five is also used to assess the durability of a drug molecule (Benet et al., 2016). Hence, this is rule is essential for a bioactive compound to be considered an oral drug (Lipinski et al., 2012). Table 2 represents the detailed information about the bioactive compounds with their respective properties.
The in silico analysis through molecular docking revealed the importance of structure-based drug designing strategy toward the development for novel drugs against the inhibition of potential drug target. The virulent enzyme responsible for superficial skin infections like candidiasis and biofilm formation is Candidapepsin-1 (Korting et al., 1998). The enzyme's active site was docked with several bioactive compounds from T. ammi. The binding energy for each bioactive compound against the Candidapepsin-1 enzyme, interaction of hydrogen bonds, Vander Waals interactions, and essential details were listed in Table  3. Ligustilide has the lowest binding energy of −5.75 kcal/mol and has three hydrogen bond interactions with amino acids Ile 223, Tyr 225, and Thr 222 at the active site. Lower the binding energy, greater is the binding efficiency. Greater the hydrogen bonds between the enzyme and ligand determines the strength of binding (Kortemme et al., 2003). The two-dimensional and three-           (Fig. 9) against the enzyme with the same grid parameters. The binding energy of reference inhibitor ligand was found to be +2.77 kcal/mol, which is lower when compared to the binding energies of bioactive compounds from T. ammi (Fig. 11). The binding of bioactive compounds inhibits the activity of enzyme resulting in the neutralization the enzymes virulence . Ligustilide isolated from essential oil of Umbelliferae family has immense antifungal properties against several species of fungi, thus restricting the fungal growth and proliferations   , 2015). This proves that the essential bioactive compounds from the oil has antifungal potential and can be used in therapeutic applications. Also, upon increasing the temperature and pressure had an augmented therapeutic inhibition potential of the essential oil against Candida infection (Pattnaik et al., 1995). Fungal hyphae are an important measure for the fungal growth and virulence. The development of the hyphae is inhibited by thymol, hence disturbing the cell membrane thereby affecting the enzymes responsible for cell wall synthesis (Braga et al., 2007). Figures 2-10 list the two-dimensional and three-dimensional interactions of bioactive compounds with low binding energies against Candidapepsin-1. ADMET profiles of the compounds were depicted in Tables 4-7. All the compounds have high rates of gastrointestinal absorption (Table 4). From Table 5, it is clear that none of the drug penetrates the blood-brain barrier since the logBB value of all the compounds is less than three. For a drug to cross the blood-brain barrier, the logBB value must be greater than three (Muehlbacher et al., 2011).

CONCLUSION
Trachyaspermum ammi is traditionally used in ayurvedic medicine due to its anti-inflammatory, antifungal, antibacterial, anticancer, and antiarthritic potential. The current study revealed the inhibition potential of bioactive compounds from essential oil of T. ammi against the virulent enzyme Candidapepsin-1 of C. albicans. The bioactive compound ligustilide has the lowest binding energy of −5.75 kcal/mol. This proves the antifungal activity of the T. ammi against the Candida biofilm formation, there by inhibiting the virulence of the enzyme. The inhibition of the enzyme leads to novel discovery of plant-based therapeutic products. Computational molecular docking could be used as an effective supporting tool for the drug development process. Computational simulations also provide us with comprehensive results with high accuracy. Hence, their presence is necessary toward the development of drug discovery and development sector.