Combining Molecular Docking and Molecular Dynamics Simulations to Explore the Binding Affinity and Interactions of Berberine with Wild-Type and Mutated Phosphatase and Tensin Homolog ( PTEN )

Noorullah Ismail Oghli ¹, Abdul Musawer Bayan ¹, Rafiullah Shirzadi ¹, *Mohammad Esmail Ahmadyar ¹, Ahmadzia Anwari ², Sayed Najib Atif ²

Introduction

Cancer remains one of the most severe health challenges worldwide, characterized by uncontrolled cell growth, invasion, and the potential for metastasis, making it a leading cause of death globally (1). Tumor suppressor genes are essential elements of cellular systems that control cell growth, division, and programmed death, ensuring cellular balance. These genes encode proteins that act as regulators, halting excessive cell proliferation, repairing DNA damage, and triggering apoptosis when damage is beyond repair (2). By maintaining these control mechanisms, they prevent the uncontrolled cell growth that characterizes cancer development. Mutations or dysfunction in critical tumor suppressor genes like PTEN significantly contributes to cancer development and progression (3).

PTEN (Phosphatase and Tensin Homolog) is essential for regulating cell proliferation, growth, and survival by negatively controlling the PI3K/AKT signaling pathway. Loss of PTEN function due to mutations, deletions, or epigenetic modifications disrupts these regulatory mechanisms, leading to unchecked cell division and resistance to programmed cell death (4). This dysfunction is observed in several aggressive cancers, including breast, prostate, endometrial, and glioblastomas (5). PTEN mutations are also linked to increased tumor invasiveness and metastasis, further worsening the prognosis. PTEN is a vital tumor suppressor gene integral to maintaining cellular balance and regulating numerous biological processes. PTEN is situated on chromosome 10q23 and produces a lipid phosphatase enzyme essential for controlling cell growth, division, survival, and mobility. Its tumor-suppressive function is primarily mediated through inhibition of the PI3K/AKT signaling pathway, which is central to cell proliferation and survival (6). PTEN counteracts this pathway by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PIP3) back to phosphatidylinositol 4,5-bisphosphate (PIP2), effectively halting AKT activation and downstream tumor-promoting effects. In addition to regulating the PI3K/AKT pathway, PTEN contributes to other crucial cellular functions, such as maintaining genomic stability, orchestrating cell cycle progression, and inducing apoptosis (7). By interacting with proteins involved in DNA repair, PTEN helps avert the accumulation of genetic mutations (8). Furthermore, it plays a role in cell migration and invasion by modulating the cytoskeleton, processes essential for normal tissue repair but detrimental when dysregulated, as seen in cancer metastasis. Several mutations in the PTEN gene have been identified, such as Val119Ile (V119I), Val158Ile (V158I), and Arg234Gln (R234Q), linked to various types of cancer (9).

Berberine is a potent bioactive compound found in various plants, including Berberis vulgaris (barberry), Coptis chinensis (golden thread), Phellodendron amurense, and Hydrastiscanadensis (goldenseal) (10). This alkaloid is widely studied for its potential synergistic effects when combined with other therapeutic compounds (11). A notable example is its combination with curcumin, derived from turmeric, to enhance therapeutic outcomes, especially in treating cancer and inflammatory conditions. Berberine and curcumin work together to inhibit tumor growth, promote apoptosis (programmed cell death), and reduce inflammation. Additionally, berberine is investigated for its ability to improve the bioavailability of other drugs, particularly chemotherapy agents. It does so by inhibiting enzymes likecytochrome P450 and modulating intestinal transporters, which may boost the effectiveness of these drugs when used in combination therapies. Berberine also has its positive impact on the cardiovascular system (12), antidiabetic action (13), anti-obesity action (14), neurodegenerative and neuropsychiatric disorders (15), as well as sensitization and drug resistance (16).

Considering the importance of identifying PTEN activators in the treatment of various types of cancers and the importance of natural compounds of berberine, we aimed to investigate the activating role and function properties of berberine inside the binding site of PTENs by using computational methods of molecular docking and molecular dynamics simulation.

Materials and Methods

Computational methods

The combination of computational biology and molecular modeling has proven to be a valuable and effective strategy in drug discovery and design. By employing virtual assessment techniques, researchers can efficiently search and screen vast structural libraries within virtual and computer environments. As a result, the use of computational biology and molecular modeling presents promising opportunities for advancing drug discovery and design processes (17). This crucial investigation took place at the Bioinformatics Center of Ghalib University, Kabul, Afghanistan in 2024.

Molecular docking

The affinity and interactions of berberine with Wild-type PTEN and Mutated PTEN were investigated through the utilization of Autodock 4.2.2 software in molecular docking analysis (18). Access to the x-ray crystallographic structures of PTEN, identified by pdb code (1D5R), was made possible through the RCSB protein data bank (19). As no specific mutated PTENs required were available in protein data bank, VMD software was used to make mutations to the wild-type PTEN pdb file by introducing three mutation points, V119I, V158I, and R234Q. In order to enhance docking calculations, non-polar hydrogen atoms were incorporated into the PDB file instead of water molecules and the original ligands. The PubChem server was utilized to download Berberin's 3D structure with CID 2353 in SDF format, which was then converted to PDB format using the OpenBabel program (18). The AMBER99SB force field was employed to minimize the energy of the enzyme using the GROMACS 2019.6 package (20). Docking calculations were performed with the macromolecules held rigid, while Berberin was allowed to rotate freely. By utilizing co-crystallized ligands from the enzyme's pdb files, the active site of the macromolecule was determined. Following the identification of the active site, a grid map consisting of 50 points and a grid point spacing of 0.375 was selected. To conduct the docking calculations, the Lamarckian genetic algorithm (LGA) approach was utilized, running a total of 200 calculations with 25 million energy evaluations for each complex. Ultimately, the conformation in the cluster with the lowest binding energy was chosen as the optimal docking mode.

Molecular dynamic simulation

Molecular dynamic simulations were performed on the enzyme in both its free state and in complex with Berberine, contained within a cubic box solvated by the tip3p water molecule model using the GROMACS 2019.6 program on the Kubuntu 2020.4 Linux operating system (20). Systems force field parameters were generated utilizing the Python-based ACPYPE tool (21). To maintain system neutrality, ions were introduced, and the dissolved systems were initially minimized using the steepest descent method to alleviate excessive forces. Subsequent stabilization of the systems at 310 K and 1 bar was achieved through 1 ns simulation in nvt and npt ensembles. Following thorough system equilibration, a molecular dynamics simulation was conducted with a time step of 2 fs over a duration of 200 ns. The molecular structures of enzymes, ligands, and intermolecular interactions were then analyzed based on the simulated trajectories.

Results

Molecular docking

The molecular docking performed for Wild-type and mutant PTEN suggests that the ligand has well positioned within the active site of the Proteins. The key residue in active site of PTEN and its interaction with atoms of berberine exhibited in Figure 1. Key residues are Ala 126, His 93, Gly129, Lys128, Lys 125, Arg 130, Asp 92, Ile 168, Gin 171, Cys 124Gly 165, Lys 130 and Thr 167.The carboxyl group of berberine formed hydrogen bond with Wild-type PTEN carboxyl atoms of Thr 167 and carbonyl atoms of Lys 330 and Arg 130. Additionally, carboxyl group of berberine formed bivalent H-bond with Lys 332 cabonyl group of Mutated PTEN.

Binding energies and inhibition constants of berberine with Wild-type and mutant PTEN exhibited in Table 1. Notably, the Wild-type and mutant PTEN system exhibits the suitable binding energy, suggesting high affinity between berberine and the wild-type PTEN.

Figure 1a and b: The best docking pose and molecular interactions of the Beberine and the residues of the Wild-type PTEN and Mutant PTEN respectively. The C, N and O atoms are indicated in black, blue and red respectively. Hydrogen bonds are identified by green drops and hydrophobic interactions are shown by red curves with spokes radiating towards the ligand atoms they interact. The atoms in contact are shown with spokes radiating back. Figures provided by VMD1.9.3 and Ligplot+ programs.

Table : The obtained docking results, binding energies and inhibition constants predicted by AutoDock program.

Analysis of the Root Mean Square Deviation (RMSD)

RMSDused to find the structural changes and stability of all four system. Free wild PTEN has higher stability than mutated PTEN shown in Figure 2a. Moreover, wild-type PTEN/ berberine complex has slightly higher stability during simulation compared to Free wild-type PTEN, achieving equilibrium within 20 ns exhibited in Figure 2b. Furthermore, mutated PTEN with berberine also showed unstable equilibrium and high structural fluctuation during simulation time than free mutated PTEN represented in Figure 2c. Significantly, the average RMSD values for the last 20 ns exhibited in Table 2, referring that wild-type PTEN with berberine forms a stable system.

Figure 2a,b,c: RMSD plots of free and complex systems as a function of time.

Table 2: The average and standard deviations of RMSD, Rg, RMSF and SASA for free and complex enzyme during the last 20ns

Analysis of the root mean square fluctuation (RMSF)

RMSDanalysis used to calculate the average deviation of a residue, over time. Figure 3 depicts the RMSF values for all free enzymes and when it is complexed with Berberine. According to Figure3a free mutated PETN offered low residual fluctuations rather than free Wild-type PTEN. When berberine binds to Wild-type PTEN, there’s a noticeable decline in the system’s fluctuations compared to free Wild-type PTEN displayed on Figure 2b. In addition, when berberine binds to Mutated PTEN, there’s also major decline in the system’s fluctuations compared to free Mutated shown in Figure 2c. Additionally, Table 2, confirming above interpretation about residual integrity.

Figure 3a, b, c: RMSF plots of free and bound enzyme.

Analysis of the radius of gyration (Rg)

The Rg measurement is utilized to evaluate the structural compactness. Findings stated Rg of the free Wild-type PTEN has smaller RG compared to other three system displayed in Table 2. The graphic representation of rg for Free mutated PTEN showed up less fluctuation of structural integrity rather than Free wild-type PTEN displayed in Figure4a.In presence of berberine the RG for Wild-type PTEN has decreased compared to free Wild-type PTEN and the presence of berberine with Mutated PTEN showed small decrease average value for RG compared to Free mutated PTEN represented in Figure4b and c. All systems shown to be equilibrated in 180 ns except for Mutated PTEN/ berberine complex which reached within 160 ns. As a result, in absence of berberine, mutated PTEN shown to be more compactness less than wild-type PTEN.

Figure 4: A-C: RG plots of free and bound enzyme as a function of time

Analysis of the solvent accessible surface area (SASA)

SASA ease to understand surface area of a biomolecule that is accessible to a solvent and used to find molecular interactions, stability, and function in duration of the simulation. The SASA diagrams is illustrated in Figure 5. The average SASA for the Free mutated had diminished compared to free Wild-type PTEN shown in Figure5a. The SASA plot for Wild and Mutated complexes seem to be in same insane displayed Figure 5b and c. The average SASA value for wild-type PTEN has diminished when berberine binds to it, because the ligand replaces the active site surrounded by solvent (Table 2). That also seem to be same in case of Mutated complexes.

Figure 5: SASA plots of free and bound enzyme as a function of time.

Hydrogen bonds analysis

Hydrogen bond analysis is essential for investigating molecular interactions, specifically in the study of molecular dynamics simulations. The mutated PTEN combined with berberine formed more stable hydrogen bonds with a higher number’s bonds compared to wild-type PTEN/ Berberine. Additionally, the mutated PTEN with berberine exhibited a maximum of six hydrogen bonds over the simulation period as shown in Figure 6. Furthermore, the presence of berberine led to an increase in the number of hydrogen bonds between Enzyme-Enzyme interactions of Wild-type PTEN, while the number of hydrogen bonds between Enzyme-solvent interactions of Wild-type PTEN decreased. The average number of hydrogen bonds between protein-protein and protein-solvent can be found in Table 3. The peak number of hydrogen bonds observed between berberine and Wild-type PTEN was four, confirming molecular docking outcomes and indicating the structural stability of complexes. The average hydrogen bond count among the enzyme's atoms of both PTENs has increased in the presence of berberine represented in Figure 7. In contrast, there was a fall in hydrogen bonding between enzyme and solvent molecules for both PTENs when berberine was bounds as shown in Figure 8.