Exploring the synergistic therapeutic potential of Morus alba extract in tuberculosis: A computational analysis

2.1 Hypothesis preparation

TB is primarily caused by Mtb, a Gram-positive, acid-fast bacterium known for its thick, lipid-rich cell envelope that contributes to drug resistance. Even when drugs penetrate this envelope, they can lose effectiveness due to enzymatic or structural modifications. Therefore, evaluating drug efficacy across multiple levels is crucial. This study focus on analyzing how selected metabolites interact with key proteins involved in various metabolic pathways of Mtb. Our objective was to assess the collective impact of these metabolites on Mtb's metabolism.

2.2 Selection of MTb proteins as target

The present study evaluates targets within the mycobacterial cell using five proteins sourced from the RCSB PDB database: Mtb porins (PDB ID: 1UUN), OmpATb (PDB ID: 2KGS) Mycolic acid transporter protein MmpL3 (PDB ID: 7NVH), Ribose oxidase DprE1 (PDB ID: 4P8T), and UgpABCE transporter (PDB ID: 4MFI)) (https://www.rcsb.org/).

2.3 Selection of ligands

From the previous investigation, 22 phytocompounds from Morus alba were screened against five target proteins of Mtb using in-silico tools. Out of these, seven phytocompounds exhibited promising activity against all five target proteins and were selected for further investigation.

2.4 Molecular docking studies

Molecular docking was conducted using Autodock, following the protocol described by Khan et al. (2023). Protein preparation included the removal of heteroatoms, addition of polar hydrogens, and Kollman charges, followed by saving in pdbqt format. Additionally, hydrogen-atoms were added to enhance ligand stability in the selected protein structures. Grid coordinates were defined to specify the docking grid-box. Docking experiments were performed under default settings in normal mode, and binding energy predictions (ΔG-score) were recorded for each ligand against the respective proteins.

2.5 Molecular dynamics (MD) simulation

MD simulation studies over 100 ns, were conducted for the best docking poses of beta-sitosterol, scopolin, and ecdysterone complexes with the proteins LpqN (PDB ID: 6MNA) and DprE1 (PDB ID: 4P8T) using the Schrödinger Desmond 6.9.137 MD simulation program (version 2022–1). The simulations were performed on a Z4 HP workstation with Ubuntu 22.04.2 LTS, Intel Xeon W-2245 @ 3.90 GHz, 8 cores, CUDA 12, and NVIDIA RTX A4000 GPU. MD simulations utilized the OPLS3e force field and SPC solvation model. Simulation details and experimental protocols followed previously published methodologies (Puri et al. 2023; Shaw, 2020-3). Desmond's Simulation Interaction Diagram (SID) was employed to analyze ligand binding orientation and stability based on MD trajectories (Desai et al. 2023).

3.1 Background and hypothesis based on anti-mycobacterial activity of M. alba

The anti-inflammatory, liver and kidney-protective, hypotensive, diuretic, anti-cough, anticancer, and analgesic properties of Morus alba L. extract make it a compelling subject for study. Hence, Dried M. alba leaves were extracted with methanol, ethyl acetate, and chloroform (1:10 ratio) and tested for anti-tubercular activity using the Resazurin microtitre assay (REMA), as described in our previous study (Khan et al. 2023) and compared with the control drugs. Encouraging outcomes led to the hypothesis of synergistic effects of phytocompounds. The idea behind this approach is that diverse compounds can target multiple pathways in the pathogen, potentially preventing resistance development. We explored their potential for combination therapy, detailing our stepwise efforts below.

3.2 Target proteins

Porins and porin-like protein OmpATb were selected for study, as they facilitate hydrophilic nutrient passage across mycobacterial outer membranes. (Faller et al. 2004). The MmpL family facilitates lipid export from mycobacterial cytoplasm to periplasm (Melly and Purdy, 2019), another key focus of the study. The study also targets decaprenylphosphoryl-D-ribose oxidase and the UgpABCE transporter, enzymes crucial for mycobacterial arabinogalactan production (Suma et al. 2020). Additionally, five proteins from our previous study (Khan et al. 2023) were included to explore synergistic phytocompound effects: MTb Mycolic Acid Cyclopropane Synthase CmaA2 (PDB ID: 3HEM), oxidoreductase (PDB ID: 4OTK), A246F mutant of beta-ketoacyl-acyl carrier protein synthase III (FABH) (PDB ID: 2QO0), Enoyl-ACP(CoA) reductase (PDB ID: 2AQ1), and LpqN involved in cell envelope biogenesis (PDB ID: 6MNA).

3.3 Target ligand interaction studies via docking approach

In our earlier work, MTT assay was used to confirm the antitubercular activity of M. alba extracts before in-silico investigations to evaluate their activity against target proteins. Here, we extended our analysis to explore the synergistic effect hypothesis by comparing the activity of the top seven compounds from the prior study against five more proteins and summarized results in Table 1. Selected compounds already have some bioactivity like sitosterol and rutin for antimicrobial, anticancer, anti–inflammatory, hepatoprotective, lipid-lowering effect, anxiolytic & sedative effects, analgesic, immunomodulatory; Ecdysterone increases protein synthesis in skeletal muscle (Khan et al. 2023; Babu, & Jayaraman, 2020).

3.4 Entry of phytocompounds into Mycobacterium

The docking results of Mycobacterium proteins (1UUN, 2KGS, 7NVH, 4P8T, 4MFI) and the phytocompounds of Morus alba are summarized in Table 1. The Mtb cell's thick, hydrophobic wall poses initial penetration challenges. Reseaarch indicates that porins may aid hydrophilic antibiotics' diffusion through this barrier, facilitating cell entry (Gygli et al., 2017). Similarly, OmpATb in Mtb, changes pH, blocking hydrophilic drug entry thus inhibiting OmpATb with specific phytocompounds can facilitate the entry of other phytochemicals (Kwofie et al., 2020). Docking results of the selected phytocompounds range from −8.06 (Scopolin) to −12.11 (Ecdysterone) for porin and −6.93 (Beta-sitosterol) to −1.65 (Petunidin-3-rutinoside) for OmpATb, suggesting high affinity of these phytocompounds for these surface proteins. According to hypothesis, inhibition of OmpATb with Beta-sitosterol can facilitate the entry of other phytochemicals showcasing a synergistic effect. The interactions of ecdysterone, beta-sitosterol and rutin with porins are summarised in Fig. 1A and 1B suggesting the significant role of covalent interactions in the attachment of these phytocompounds to the porin protein.

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Fig. 1

A &Fig. 1b: Interaction of ecdysterone, beta-sitosterol and rutin with porins.

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Fig. 1

A &Fig. 1b: Interaction of ecdysterone, beta-sitosterol and rutin with porins.

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Hydrophobic compounds can pass through bacterial membranes by dissolving in the lipid bilayer, but hydrophilic compounds require protein channels like porins (MspA in M. smegmatis). Mtb uses porins like OmpATb in its outer membrane to facilitate hydrophilic solute entry (Niederweis, 2003). OmpATb is crucial for Mtb's survival in acidic environments. Kwofie et al. (2020) identified natural products that inhibit OmpATb, potentially disrupting Mtb's nutrient uptake and survival, offering new avenues for tuberculosis treatment development.

3.5 Action on different metabolic pathways

After entering, phytocompounds target vital biosynthetic pathways, crucial for the pathogen's structural and functional integrity. The absence of targeting proteins/pathways in the host or gut microbes will minimize the side effects (Maurya et al. 2022). The cell wall of Mtb, absent in humans and distinct from normal gut flora, is a prime target for drugs like isoniazid and ethionamide (Bolla, 2020).

3.6 Cell envelope biogenesis

The study also emphasizes Mycobacterial membrane protein large-3 (MmpL3; PDB ID: 7NVH), crucial for transporting precursors (trehalose monomycolates) in cell-envelope biogenesis. The docking results indicate that only Beta-sitosterol (−7.1) Petunidin-3-rutinoside (−6.9), Quercitrin (−6.9) and Quercetin-3′-glucoside (−6.6) exhibit a good affinity towards this protein, showcasing their potential to inhibit Mycobacterial cell envelope biogenesis. Fig. 2 summarizes the interaction of these phytocompounds with MmpL3, highlighting the significant role of covalent interactions. MmpL3 facilitates the transport of TMM across the inner membrane and has been a critical target for various preclinical agents (Bolla, 2020).

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Fig. 2

Interaction of beta-sitosterol, Petunidin-3-rutinoside, quercitrin and Quercetin-3′-glucoside with Mmpl3.

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3.7 Arabinogalactan biosynthesis

DprE1 (Decaprenylphosphoryl-β-D-ribose 2′-epimerase), crucial for mycobacterial cell-wall synthesis, is a significant target. DprE1, a flavoprotein, catalyzes the oxidation of decaprenylphosphoryl-D-ribose (DPR) to decaprenylphosphoryl-2-ketoribose (DPX), which subsequently reduced to decaprenylphosphoryl arabinose (DPA) by enzyme, DprE2. Arabinosyltransferase converts DPA into arabinogalactan and lipoarabinomannan, key components of Mtb cell-wall (Mali et al., 2022). Out of seven, two phytocompounds- Beta-sitosterol and Ecdysterone with docking energy −11.21 and −9.15 Kcal/mole, respectively show their affinity to bind with DprE1 enzyme (4P8T) (Table 1) indicating their potential to inhibit the normal functioning of DprE1. In both cases, PHE and GLN residues of the active site form conventional hydrogen bonds (Fig. 3). Mali et al. (2022) reported that non-covalent inhibitors (2-carboxyquinoxalines) possess 2-carboxylate moiety crucial for hydrogen-bond formations with the sidechain of Lysine-418 and the hydroxyl group of Tyrosine-60. In the current work, tyrosine and leucine are also found to form hydrogen bonds with Petunidin-3-rutinoside, indicating a similar inhibition mechanism as for 2-carboxyquinoxalines.

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Fig. 3

Interaction of beta-sitosterol and ecdysterone with DprE1.

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3.8 Peptidoglycan biosynthesis

The absence of peptidoglycan biosynthesis in humans makes it a promising target to inhibit Mtb (Isa and Mohammed 2021). This study focuses on the final steps of the UDP-N-acetylmuramic acid formation, catalyzed by UDP-N-acetylenolpyruvoylglucosamine reductase (MurB). MurB is a Flavoprotein with a flavin adenine dinucleotide (FAD) binding structure, belonging to the superfamily of FAD-binding proteins (Murzin, 1996). Hence the peptidoglycan synthesis regulated by MurB is supported by the nutrient uptake facilitated by UgpABCE, highlighting a complex interplay between cell wall biosynthesis and nutrient acquisition in bacterial physiology (Soni et al., 2020). Ecdysterone with good binding affinity −9.51 kcal/mol with UgpABCE transporter (PDB ID: 4MFI) suggests indirect control on peptidoglycan biosynthesis.

3.9 Mycolic acid biosynthesis

Our previous research demonstrated the affinity of these compounds towards crucial proteins of Mycolic acid biosynthesis, including CmaA2 (3HEM: ΔG 6.7–9.0 kcal/mol), FaB (2QO0: ΔG 7.4–9.1 kcal/mol), and LpqN (6MNA: ΔG −6.2 to −7.8 kcal/mol). Petunidin-3-rutinoside, Beta-sitosterol, and Scopolin showed affinity for 3HEM, while Petunidin-3-rutinoside, Ecdysterone, and Rutin had affinity towards the 2QO0 protein. Additionally, these phytocompounds exhibited affinity towards 2AQI (ΔG −7.4 to −8.3 kcal/mol) enzyme having important role in FAS II cycle (Khan et al., 2023). β-sitosterol is well-documented for its antitubercular activity. Otari et al (2012) reported the antitubercular activity of ethanolic extract of plant Vitex negundo Linn containing betulinic acid, ursolic acid and β −sitosterol. Similarly, Sasikumar et al., (2018) reported the antimycobacterial potentials of quercetin and rutin.

3.10 MD trajectory analysis

The Glide docking protocol is flexible for small molecules but overlooks receptor structural dynamics in docking. Beta-sitosterol, Scopolin, and Ecdysterone were studied in complex with LpqN (6MNA), and DprE1 (4P8T) proteins to validate the docking results and assess their conformational stability. Each system underwent a 100 ns MD simulation, evaluating root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and hydrogen bond interaction using MD trajectories. The values of each trajectory analyzing parameter including maximum, minimum, and average values are summarised in Table 2.

The RMSD of alpha-carbon atoms helps to understand their stability (Kikiowo et al. 2022). Fig. 4A and 5A, for the 6MNA-Beta-sitosterol complex, the protein backbone's RMSD peaked at approximately 2.33 Å within the initial 24 ns, and stabilized around 2 Å for the remaining period. Similarly, Beta-sitosterol-4P8Tcomplex exhibited stable RMSD values around 3.2 Å throughout the simulation. For 6MNA-Scopolin Complex, an initial rise in RMSD around 2.97 Å occurred within 32 ns, stabilizing with minor fluctuations. The alpha-carbon of 4P8T-Ecdysterone complex exhibited stable variations with RMSD of below 3 Å with some initial fluctuations upto 21 ns.

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Fig. 4

MD simulation Trajectory analysis of ligand-6MNA complexes A. Time dependent RMSD plot; B. Individual amino acids RMSF plot; C. Time dependent Hydrogen bond analysis; D. Time dependent Radius of gyration plot.

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Fig. 5

MD simulation Trajectory analysis of ligand-4P8T complexes A. Time dependent RMSD plot; B. Individual amino acids RMSF plot; C. Time dependent Hydrogen bond analysis; D. Time dependent Radius of gyration plot.

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The initial RMSD rise may reflect structural changes or rearrangements in the protein backbone during phyto-compound interactions, possibly involving conformational adjustments or binding events. However, the subsequent RMSD stabilization indicates the complex settled into a relatively stable conformation, showing minor fluctuations in the simulation.

The 6MNA and 4P8T backbone complexes with phyto-compounds remained stable throughout the simulation, showing no significant deviations. The average protein backbone RMSD were 1.87 Å (6MNA-Beta-sitosterol), 2.28 Å (6MNA-Scopolin), 2.34 Å (4P8T-Beta-sitosterol), and 2.61 Å (4P8T-Ecdysterone). The maximum RMSD across all frames was 3.24 Å, indicating no extreme deviation during the MD simulation. The low average and RMSD support the stability of each complex in dynamic states (Fig. 4A and 5A).

Pandey et al. (2022) elucidated the role of amino acids in maintaining protein–ligand complexes during dynamic phases. The amino acid deviation of individual 6MNA and 4P8T proteins are shown in Fig. 4B and 5B. In the 6MNA protein complex, a maximum fluctuation of 3.89 Å and 3.321 Å is detected in Ser100 and Ala113, respectively. The visual analysis of MD simulation trajectories suggests that both Beta-sitosterol and Scopolin engaged in significant binding interactions with residues, Ala184, Ala203, Ala217, Ile121, Ile220, Leu201, Leu213, Met214, Ser154, Thr182, and Thr210 of the 6MNA protein. Residues with low RMSF values (0.43 Å and 1.95 Å) indicate stable Beta-sitosterol and Scopolin structures during simulation, suggesting higher protein stability. Amino acids of the 4P8T protein, including, Ala244, Asn385, Gln334, Gln336, Gly117, His132, Leu115, Leu317, Lys134, Lys418, Phe320, Pro116, Pro316, Ser228, Trp230, Trp296, Tyr297, Tyr303, Tyr314, and Val365, interact with Beta-sitosterol and Ecdysterone which have RMSF values less than 2.5 Å.

From Table 2, the average RMSF of 6MNA-Beta-sitosterol, 6MNA-Scopolin, 4P8T-Beta-sitosterol, and 4P8T-Ecdysterone complexes was found to be 0.96, 1.15, 0.89, 0.99 Å respectively. Loop regions distant from the binding pocket show higher fluctuations in RMSF. These residues lack binding interactions with the studied phyto-compounds, as indicated by their above-average values, explanation, and Fig. 4B and 5B. This suggests that during dynamic phases, all amino acids in the 6MNA and 4P8T proteins consistently and strongly bind the phyto-compounds.“.

Radius of gyration (RGyr) assesses structural compactness in complexes by measuring mass concentration distance from an axis, crucial for understanding macromolecules' 3D structure, folding behavior, and conformational changes during simulations. (Maurya et al. 2022; Patel et al. 2023). Large RGyr values correspond to more flexible conformations, while small RGyr values indicate more constrained conformations (Ayipo et al. 2022). The minimal Deviations for 6MNA (20.82–21.36 Å) and 4P8T (20.73–21.23 Å) complexes with phyto-molecules (Fig. 4C, 5C) suggest stable structures with limited conformational changes during simulations, indicating protein stability. RGyr deviation for 6MNA-Beta-sitosterol, 6MNA-Scopolin, 4P8T-Beta-sitosterol, and 4P8T-Ecdysterone, was 0.36, 0.54, 0.51, and 0.29 Å, respectively suggesting the firmly defined structure of proteins with few conformational changes.

In MD simulations, hydrogen bonds (HBs) ≤ 3 Å between ligands and proteins enhance complex stability (Tabti et al. 2023). Beta-sitosterol and Scopolin showed significant HB formation profiles over 100 ns, influencing binding affinity by establishing multiple specific interactions, as depicted in Fig. 4D and 5D.