Molecular Docking Study on Phytochemicals of Alpinia galanga and Their Derivatives as Inhibitors of β-Ketoacyl Reductase (MabA) of Mycobacterium Tuberculosis: An In-silico Study

1. Assistant Professor, Department of Microbiology, Tagore Medical College and Hospital, Chennai, Tamil Nadu, India. 2. Professor, Department of Microbiology, Tagore Medical College and Hospital, Chennai, Tamil Nadu, India. 3. Professor and Head, Department of General Medicine, Tagore Medical College and Hospital, Chennai, Tamil Nadu, India. 4. Associate Professor, Department of Pathology, Tagore Medical College and Hospital, Chennai, Tamil Nadu, India. 5. Associate Professor, Department of Microbiology, Tagore Medical College and Hospital, Chennai, Tamil Nadu, India.

Abstract

Introduction: Tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium Tuberculosis (MTB), continues to be a global health problem. Alpinia galanga (Linn.) of the Zingiberaceae family has antitubercular properties, and their mode of action in in-vitro as well as in-vivo conditions is well established. This knowledge of the active phytochemicals of Alpinia galanga has been utilised to identify new potent drugs for MTB.

Aim: To perform molecular docking studies of various phytochemicals of Alpinia galanga and their derivatives with β-ketoacyl reductase (MabA) of MTB.

Materials and Methods: The present study is an in-silico study conducted in the Bioinformatics facility of the Central Research Laboratory of Tagore Medical College and Hospital, Chennai, Tamil Nadu, India from November 2022 to April 2023. The receptor protein was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) database. The phytochemicals in. sdf format were downloaded from the PubChem database. The derivatives were prepared by Chemsketch software. Docking was performed using AutoDock Vina with PyRx as the GUI (Graphical User Interface). Post-docking analysis was performed in LigPlot+.

Results: Phytochemicals from Alpinia galanga were obtained from the PubChem database and docked with MabA of MTB. The derivatives were further subjected to docking analyses. From the docking study, two molecules, namely, (1E,6Z)-2,4-diamino-6-fluoro-1,7-bis(4-hydroxyphenyl)-1-sulfanylhepta-1,6-diene-3,5-dione and (2E,6Z,10E)-2,6,9,9-tetrakis(hydroxymethyl)cycloundeca-2,6,10-trien-1-one-ethane (1/1), were found to have good binding energy values.

Conclusion: The present study helped us find drug-like molecules that can inhibit the MabA of MTB. Two compounds derived from the phytochemicals of A. galanga were found to have an effective binding capacity to the drug target in-silico. Hence, the outcome of present study has provided a therapeutic strategy for TB, especially for strains of MTB that are drug-resistant.

Antimicrobial activity, Drug resistance, Mycolic acids

Tuberculosis (TB), an infectious disease caused by the bacterium MTB, is one of the global health problems and continues to be a leading cause of morbidity and mortality (1),(2). India led the world in TB infections in 2022, accounting for a startling 27% of the worldwide burden, according to the World Health Organisation (WHO) worldwide TB report 2023. In total, 87% of all TB cases worldwide in 2022 were reported from 30 high burden TB countries, including Pakistan (5.7%), China (7.1%), Indonesia (10%), and the Philippines (7.0%) (3). Studies have shown that A. galanga has antimicrobial activity; however, its active principles are yet to be explored. In the present study, molecular docking is used to identify drug candidates in the plant A. galanga with mycobacterial activity. Due to the increasing resistance to existing drugs, there is an urgent need for the development of new TB drugs (4),(5).

Currently, the cell wall synthesis pathway of the bacteria is a promising target for new anti-TB drug discovery (6). Mycolic acids are the major lipid components of the unique mycobacterial cell wall responsible for protecting the TB bacilli from external threats. Mycolic acids are synthesised in the cytoplasm and transported to the outer membrane. The large size of these unique fatty acids results of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids for mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, which are excellent potential drug targets, several of which show great promise as selective TB therapeutics (7).

During the biosynthesis of mycolic acids, in the second step of the elongation cycle, the resulting β-ketoacyl-ACP product is reduced by MabA, the Nicotinamide Adenine Dinucleotide Phosphate Hydrogen (NADPH) dependent MabA of MTB (8). Mycolic acids impart MTB with unique properties that defy medical treatment. They make the organism more resistant to chemical damage and dehydration, and limit the effectiveness of hydrophilic antibiotics and biocides (9). Mycolic acids also allow the bacterium to grow inside macrophages, effectively hiding it from the host immune system. Mycolate biosynthesis is crucial for the survival and pathogenesis of MTB (10). Thus, this enzyme is a potential drug target for the development of new drugs. Furthermore, the three-dimensional structure of the enzyme has been derived and is available in the protein database. This paves the way for molecular docking studies to identify lead molecules that can inhibit MabA.

Alpinia galangal (L.) belongs to the family Zingiberaceae and is commonly referred to as galangal. It is widely cultivated in Southeast Asian countries, including India (11). The plant is rich in phytochemicals with various pharmacological activities (12). A. galanga, used in various traditional medicines, possesses broad-spectrum antibacterial properties (13). A. galanga exhibits anti-TB activity with multiple modes of action (14). Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non replicating bacteria of latent TB (14). Studies have shown that the extracts of this plant have antimycobacterial activity against MDR strains of MTB (15),(16).

Due to the emergence of multiple drug-resistant strains of MTB, there is a need for new drugs. Therefore, the aim of the present study is to perform molecular docking studies of various phytochemicals of Alpinia galanga and their derivatives with MabA of MTB to find promising drug candidates with anti-mycobacterial activity.

Material and Methods

The present study is an in-silico study conducted in the Bioinformatics facility of the Central Research Laboratory of Tagore Medical College and Hospital, Chennai, Tamil Nadu, India from November 2022 to April 2023.

Protein Preparation: The present in-silico study involves the use of molecular docking software to simulate the interaction between target and drug-like molecules. The study was conducted in the Bioinformatics facility of the Central Research Laboratory of Tagore Medical College and Hospital. The three-dimensional structure of MabA (Table/Fig 1) of MTB was retrieved from the RCSB-PDB database through the search option (www.rcsb.org) and saved in the Protein Data Bank (PDB) format. The 3Diamensional (3D) structure of the protein was visualised in Discovery Studio software.

Active Site Prediction: The possible binding sites of MabA are searched using the binding site prediction tool 3DLigandSite, an online tool (17). The best flexible binding sites were selected for this study.

Generation and optimisation of Ligand: The structures of various flavonoids (quercetin, kaempferide, galangin) were obtained from the PubChem database. Their derivatives in 2D format were generated with the help of the ACD ChemSketch software (18). The ligands were saved in mol 2 format. The Open Babel software was used to convert the mol format to pdb format (19). Rapid virtual screenings of these compounds were performed in the docking tool iGEMDOCK v2.0. A population size of 150 was set with 70 generations and one solution for quick docking. Based on the binding energy, the ligands were selected for further study (20). The selected ligands were then analysed for drug-relevant properties based on Lipinski’s rule of five (molecular weight <500 g/mol, not more than five hydrogen bond donors, not more than ten hydrogen bond acceptors, and a partition coefficient (log P) value <5). Other drug-like properties were analysed using the OSIRIS Property Explorer (21) and Molsoft: Drug-Likeness and Molecular Property Explorer (22). On the basis of binding affinity and drug-like properties, the ligands were selected for further molecular docking studies.

Protein-ligand Docking: The docking of ligands was performed using AutoDock Vina software Vina, using PyRx as the GUI (23). Docking was performed to obtain a population of possible conformations and orientations for the ligands at the binding site, along with their binding energy. Using the software, polar hydrogen atoms were added to the CTB and its non polar hydrogen atoms were merged. All bonds of the ligands were set to be rotatable. All calculations for protein-ligand flexible docking were done using the Lamarckian Genetic Algorithm (LGA) method. A grid box with dimensions of 126×126×126 points was used to cover the entire binding site and accommodate ligands to move freely. The best conformation was chosen based on the lowest docked energy after the docking search was completed.

Results

The phytochemicals from Alpinia galanga were obtained from the PubChem database and docked with MabA of MTB. The best phytochemicals in terms of binding energy were utilised to prepare derivatives. The derivatives were further subjected to docking analyses. From the docking study, two molecules were found to have good binding energy values (Table/Fig 2). The bound ligands were then subjected to LigPlot+ analysis to find the binding forces involved in it (Table/Fig 3).

The ligand (1E,6Z)-2,4-diamino-6-fluoro-1,7-bis(4-hydroxyphenyl)-1-sulfanylhepta-1,6-diene-3,5-dione showed two hydrogen bonds N-O Ile225 and N-O Val268 with bond lengths less than 3.00 Å. This shows to be an excellent tight binding of the ligand to the binding site of the drug target, potentially inhibiting it. Another ligand, (2E,6Z,10E)-2,6,9,9-tetrakis (hydroxymethyl) cycloundeca-2,6,10-trien-1-one-ethane (1/1), showed a hydrogen bond N-O Val268 with a bond length less than 3.00 Å.

The selected two molecules underwent Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) studies in SwissADME and Chemicalise online server tools. Their drug likeness is excellent and fulfills many criteria of Absorption, Distribution, Metabolism and Excretion (ADME) (Table/Fig 4). Both molecules obey the Lipinski’s rule of five (Table/Fig 5).

Discussion

The treatment of TB has become a challenge in recent days due to the Multiple Drug Resistance (MDR) and Total Drug Resistance (TDR) strains of MTB (24). Therefore, new drugs are needed to combat these strains. Molecular docking is a technique that can be exploited to develop new drugs for such strains (25). Not many docking studies are conducted for the development of new drugs from plants for MTB (26). Hence, in the present study, an attempt was done to identify drug-like molecules from the A. galanga plant that can inhibit MabA of MTB, an important drug target.

Molecular docking is least used technique in the identification of drug-like molecules from plants for bacterial diseases, especially MTB (27). Many bacteria contain proteins that are crucial for their survival and pathogenicity (28). Hence, they can be excellent drug targets for the generation of new drugs as alternatives to antibiotics (29). These drugs will be target-specific, meaning they can inhibit or kill only a particular species or genus of bacteria.

The initial step of the study involved retrieving phytochemicals from A. galanga from the PubChem database, which serves as a comprehensive repository of chemical compounds. This approach allowed the researchers to efficiently screen a wide range of compounds and select those with potential therapeutic efficacy against MTB.

Subsequent molecular docking analyses were conducted to evaluate the binding interactions between the selected phytochemicals and the target enzyme, MabA. Molecular docking is a powerful computational technique used to predict the binding mode and affinity of small molecules with protein targets. In this study, the binding energy was used as a key parameter to assess the strength of interaction between the phytochemicals and the target enzyme. The lower the binding energy, the stronger the interaction between the ligand (phytochemical) and the receptor (MabA), indicating a higher likelihood of therapeutic efficacy.

Among the screened phytochemicals, those exhibiting the most favourable binding energies were chosen to design and synthesise derivatives. This rational design approach aimed to optimise the pharmacological properties of the lead compounds, enhancing their binding affinity and specificity towards the target enzyme.

The derivatives synthesised from the selected phytochemicals were then subjected to further docking analyses to evaluate their binding affinities. Interestingly, two molecules emerged from this analysis with particularly promising binding energy values. These molecules exhibited strong interactions with the target enzyme, suggesting their potential as potent inhibitors of MabA and, consequently, as anti-TB agents.

The study has revealed that certain phytochemicals and their derivatives do have the capacity to bind the drug target in-silico effectively. These molecules can be taken for further in-vitro and in-vivo studies to establish their ability to bind to the drug target. Production of such compounds is economical as they are derived from plant sources (30).

Overall, the results of present study highlight the therapeutic potential of phytochemicals derived from A. galanga against MTB. By employing computational approaches such as molecular docking, the study provides valuable insights into the molecular mechanisms underlying the interaction between the identified phytochemicals and the target enzyme. Further experimental validation, including in-vitro and in-vivo studies, will be necessary to confirm the efficacy and safety of these phytochemicals as anti-TB agents. Additionally, structural optimisation and medicinal chemistry efforts could be pursued to enhance the potency and selectivity of the identified lead compounds, ultimately contributing to the development of novel treatments for TB.

Limitation(s)

The study involves molecular docking, which is an in-silico study. It helps to shortlist and identify drug-like molecules. However, further high-throughput studies should be done to confirm the activity of the molecules confirmed by molecular docking.

Conclusion

Tuberculosis is a dreadful disease and remains one of the top infectious diseases worldwide. Throughout history, various plants have been used for treating diseases and for commercial drug preparations. While extensive work is required to explore plant-based drugs traditionally, in-silico studies help in exploring new drugs from medicinal plants against many infectious diseases, reducing the need for animal experiments. The present study helped us find drug-like molecules that can inhibit MabA of MTB. Two compounds, namely (1E,6Z)-2,4-diamino-6-fluoro-1,7-bis(4-hydroxyphenyl)-1-sulfanylhepta-1,6-diene-3,5-dione and (2E,6Z,10E)-2,6,9,9-tetrakis (hydroxymethyl) cycloundeca-2,6,10-trien-1-one-ethane (1/1), were found to have effective binding capacity to the drug target in-silico. Since these molecules are identified from the phytochemicals present in the plant, they can be obtained from plants and used for further studies. Hence, the outcome of this study has provided a therapeutic strategy for TB, especially for the strains of MTB that are drug-resistant.

Acknowledgement

The authors would like to acknowledge the faculties concerned for using the Bioinformatics facility of the Central Research Laboratory of the college where the study was done.

DOI and Others

DOI: 10.7860/JCDR/2024/67937.19242

Date of Submission: Oct 07, 2023
Date of Peer Review: Dec 02, 2023
Date of Acceptance: Feb 13, 2024
Date of Publishing: Apr 01, 2024

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? No
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Oct 08, 2023
• Manual Googling: Dec 13, 2023
• iThenticate Software: Feb 12, 2024 (18%)

ETYMOLOGY: Author Origin

EMENDATIONS: 6