Structure-Guided Design and Development of Novel Cyclophilin A Inhibitors and Ganoderiol-F Derivatives: An In-Silico Approach

Summary

Here, we present a protocol for the structure-guided design of protein-ligand binding interaction between Sanglifehrin A and Cyclophilin A/Ganoderiol-F, essential in discovering new drugs. The stability of the ligand-receptor complex was evaluated using molecular dynamics simulation.

Abstract

The cyclin Ds-CDKs axis (Cyclophilin A (Cyp /Ganoderiol F)) plays a critical role in cancer through various processes, such as controlling proliferation and inhibiting cancer cells. Ganoderiol F derivatives can serve as oncogenic targets for multiple cancer types. This study demonstrated that the structure-guided design and development of novel Cyp A inhibitors and Ganoderiol F derivatives can be employed to select calculated positions for increased effectiveness in chemical inhibitors and their interaction with the chosen receptors. For this approach, numerous software applications were utilized to achieve optimal molecular interactions. Overall, 117 novel cyclophilin inhibitor ligand molecules were constructed using Chemsketch software. The converted PDB files of the novel Cyp A inhibitor ligands were prepared for introduction to the Sanglifehrin A enzyme receptor. Ligand-protein docking interactions were performed using AutoDock 4.2.6 software to reveal the best geometric interactions. The docking confirmations were carried out using PyMOL 2.5 software. Finally, hydrogen bonds were detected using Chimera and Maestro software. The selected ligand-receptor docked complex was subjected to molecular dynamics simulation. The MD simulation process demonstrated the stability of the complex and indicated that the chosen ligand can be utilized as a drug inhibitor for cancer cells. In the results, the active amino acids in binding site were identified as Arginine 55 that in motion of 1.53Å, Asparagine (Asn), Glycine (Gly), Threonine (Thr), Lysine (Lys), Isoleucine (Iso), Histidine (Hid), Phenylalanine (Phe), and Cysteine (Cys) with the highest number of hydrogen bond ligand of 45 hydroxymangiferonic. That connected with 1nmk receptor in grid box coordinate of X 38.86, Y of 10.987, and Z of 40.734. The 1nmk receptor-ligand complex evaluation in molecular dynamics (MD) simulation in Gromacs version CHARMM 36 force field has optimum degrees.

Introduction

Cyclophilin A is a strong inhibitor of human Cyp A. Cyclophilin is a robust inhibitor that does not impair the immune system, possesses better pharmacological qualities, and reduces transporter inhibition. Poor prognosis has been linked to the overexpression of cyclophilins, a family of proteins with peptidyl-prolyl isomerase activity¹. Cyclophilins have been reported to be overexpressed in numerous cancers, including hepatocellular carcinoma. In hepatocellular carcinoma (HCC), cyclophilins are crucial for both cancer drug resistance and proliferation². The inhibition acquires essential information to identify new immunosuppressors based on quinoxaline derivatives by employing atomic-level molecular docking and MD simulation to determine the precise binding site of DC838 to Cyp A³.

Additionally, in vivo research showed that cyclophilin DC838 prevents concanavalin-induced mouse spleen cell proliferation. Several studies have reported a series of macrocyclic cyclophilin inhibitors synthesized based on Sanglifehrin A (SFA), a natural substance⁴. Initial compound optimization revealed that the hydroxyl group of the m5 tyrosine residue, the stereo-centres at C14 and C15, and the valine5m5tyrosine5piperazic acid tripeptide in the Sanglifehrin core were essential factors influencing compound potency⁵. The C185C21 diene unit of SFA could be replaced with a styryl group to create potent compounds that show a novel binding mode, in which the styrene moiety interacts with Arginine (Arg)55⁶ of Cyp A via a stacking interaction. Non-immunosuppressive cyclophilin inhibitors are effective in treating hepatitis C virus. Cyclosporin A¹, alisporivir, and most other cyclosporins are strong inhibitors of MRP2, MDR1, OATP1B1, and other crucial drug transporters^7,8.

On the other hand, Ganoderiol F purified from Ganoderma leucocontextum retards cell cycle progression by inhibiting CDK4/CDK6⁹ cyclins, and cyclin-dependent kinases (CDKs) are pivotal in controlling the mammalian cell cycle. Cyclin D-CDK4/CDK6 and Cyclin E-CDK2 are the critical players in Ganoderiol F's control of cell cycle progression. Therefore, Ganoderiol F could be used as a potential CDK4/CDK6 inhibitor in the treatment of breast cancer^10,11. FA inhibits the G1 phase of the cell cycle, which prevents M-CSF-dependent macrophage growth without affecting cell viability¹². The immunosuppressor inhibits proliferation by deactivating the activity of CDK2. In the context of cancer, the role of Cyclin D-CDK4/6, along with other Cyclins and CDKs, is well-established. Cancer is characterized by the abnormal deregulation of the cyclin-CDK pathway. Specifically, when the cell is ready to initiate DNA synthesis, CDK4 mediates passage through the G1 phase in conjunction with the D-type cyclins. This process is crucially linked to cancer development and progression⁹.

The active site of Cyp A is characterized by the invariant catalytic arginine (Arg55) and a highly conserved mixture of hydrophobic, aromatic, and polar residues, such as Arg55, Glutamine (Gln), Glycine (Gly), and Threonine (Thr)^1,13,14. The active site of the cyclophilin family also consists of Alanine (Ala) and Asparagine (Asn)¹. Meanwhile, the SFA active site consists of the amino acids Arg55¹⁵, Gln, Gly, Thr, Ala, and Asn, which form a hydrophobic pocket that encases the piperazine acid moiety of SFA. In contrast to unliganded Cyp A, the linker between the macrocycle and spin bicycle of SFA forces Trp121 to realign its side chains¹⁶.

1nmk is selected as a specific PDB code of Sanglifehrin A, as mentioned in previous studies¹⁷. SFA, an immunosuppressive natural substance, was first discovered in Streptomyces sp. A92-3081^5,18. SFA exhibits high affinities for Cyp A and has a distinct structure compared to CsA, employing a unique method to suppress the immune system. The complex molecular structure of SFA comprises a 22-membered macrocycle with a nine-carbon tether at position 23, terminating in a highly substituted spiro-bicyclic moiety. In vitro studies with fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis demonstrated that SFA increases the production of cyclophilin B and inhibits the secretion of collagen type I^19,20.Overall, 20 different sanglifehrins were used to isolate SFA (C16). Its affinity for cyclophilins is approximately 60 times higher than that of the other members of the SFA group in a cell-free competitive binding experiment¹⁵. For instance, SFA significantly reduces the quantity of bioactive IL-12p70, the primary source of IL-12 released by dendritic cells, thereby inhibiting dendritic cell activity. This study suggests that SFA is a potent new immunosuppressive medication¹.

In this protocol, novel ligand structures for Ganoderiol F/Cyp A were generated using in silico methods with Chemsketch software and then converted to PDB files using compatible software. Protein-ligand binding conception is a significant issue in the drug discovery and design process^21,22. The ligand-receptor interactions were docked using AutoDock 4.2.6 software. Computational docking systems typically generate several potential protein-ligand complex configurations. One or a few candidates with the lowest energy binding to the 1nmk receptor can be selected for the next stage of drug design to ensure effective in silico drug design trials. Chimera software was used to identify the optimal hydrogen-binding ligand. Finally, molecular dynamics (MD) simulations were performed on the ligand-receptor complex to measure its stability. This method is described in the protocol below.

Protocol

1. In silico generation of ligands and ligand preparations in ChemSketch software

NOTE: This software will be used to draw the structure of a ligand/molecule, i.e., molecular editing.

1. Draw the ligand/structure as indicated in the drawing process followed in the software, as shown in Supplementary File 1 (Screenshot 1).
2. Press f9 to clean the structure after drawing the complete structure.
3. Click on File and then Save As. (Navigate to the folder, e.g., molecule 1 will be saved in folder 1, and so on).
4. Change the Save As type to mdl molfiles, then click Save (no need to specify a filename; use the default filename provided by the software, e.g., noname).
   NOTE: One file will be created in the folder, and each file will have the mdl format of a 2D structure ligand.
5. Repeat the above steps for all molecules.

2. Conversion of file format (Open Babel software)

NOTE: The software converts file formats. Here, the .mol file will be converted to .pdb format, as shown in Supplementary File 1 (Screenshot 2).

1. On the left-hand side, in the input format, please select mol--mdl mol format from the drop-down menu. Similarly, on the right-hand side, in the output format, select pdb--protein data bank format.
2. Under the input format (on the left-hand side), click ... (the browse button).
3. Navigate to the folder (e.g., folder 1), click on the Chemsketch file just drawn, and open it.
4. Under the output format, click ....
5. A default window will open. Type 1.pdb here to name the file. For structure 1, type 1.pdb and click Save. Repeat this step for molecule/structure 2, typing 2.pdb , and so on.
6. Finally, from the middle of the software interface, click Convert. This will convert the coordinates from the mdl mol format to the PDB format.
   NOTE: Now, one more file will be added to the folder. Each file will have 1 ligand pdb file that will be used in the next steps.
7. Right-click on the newly created file, select Open With, and then choose Notepad.
8. Repeat the same steps for all molecules.
   NOTE: AutoDock 4.2.6 will appear on the desktop after installing the MGL 1.5.7 tools.
9. Prepare the input 1nmk.pdb file as described below.
10. In the 1nmk.pdb file, remove all external ligands by visiting the pdb.org website and selecting the 1nmk structure. Look for the ligands under the small molecule title on the 1nmk structure, as shown in Supplementary File 1 (Screenshot 3).
11. Find the ligand named SFM, as shown in Supplementary File 1 (Screenshot 4), and open the 1nmk file. Download the 1nmk PDB file from the PDB website. Locate the termination residue (TER.), as shown in Supplementary File 1 (Screenshot 5).
12. Delete the residues of the external ligands (SFM) in the 1nmk structure, starting from the residue after TER and ending before the residue listed before the end. Save the modified 1nmk PDB file on the system.
    NOTE: The 1nmk PDB file will be ready, and preparation of the receptor active coordinate site file in Table 1.
13. Open the 1nmk file and locate Arg at amino acid number 55.
14. Find the coordinates of x, y, and z, as shown in Supplementary File 1 (Screenshot 6).

3. Preparation of the docking parameters using AutoDock 4.2.6 software.

1. Open the AutoDock 4.2.6 tools software from the desktop.
2. Select AutoDock 4.2.6 4.0 for 1nmk receptor preparation, as shown in Supplementary File 1 (Screenshot 7).
3. Open the File menu, select Read Molecules, navigate to the folder, select 1nmk, and press Open.
4. Press Edit, choose Hydrogen, and click Add. Select Polar only, and press OK. (Supplementary File 1, Screenshot 8).
5. Press Edit, choose Charges, and then select Kollman Charges.
6. Press Edit, choose Atoms, and then select Assign ad4 type (choose any one of them).
7. Open the File menu, choose Save, and type pdbqt. A window will open. Browse folder 1, type the filename 1nmk.pdbqt, and click Save.
8. Select from Atom, and then Add as shown in Supplementary File 1 (Screenshot 9). Check the boxes for sort nodes, save transform coords, write all records, and then click OK.
9. Open Ligand, select input, and then Open. Navigate to folder 1, change the file type to all files, select 1.pdb, and click Open. A small window will appear, providing information about the selected Ligand, as shown in Supplementary File 1 (Screenshot 10).
10. Open the Ligand, then the Torsion Tree, and then Detect the Root, as shown in Supplementary File 1 (Screenshot 11) .
11. Open Ligand, then Output, and then save as pdbqt. Navigate to folder 1, type the filename 1. pdbqt, and click Save, as shown in Supplementary File 1 (Screenshot 12) .
    NOTE: All ligand pdbqt files will be prepared.
12. Open Grid, select Macromolecule, choose 1nmk, and then select Molecule. A warning will appear; click OK, as shown in Supplementary File 1 (Screenshot 13). Navigate to folder 1, type the filename 1nmk.pdbqt, click Save, and when a replace window opens, click Yes, as shown in Supplementary File 1 (Screenshot 14).
13. Open Grid and then set map types, choose Ligand, click on 1, and select Ligand, as shown in Supplementary File 1 (Screenshot 15).
14. Prepare the GPF receptor active coordinate sites file as shown in Table 1.
    NOTE: The file GPF represents the active coordinate site of the receptor (Table 1).
15. Click on Grid, then go to Grid Box, choose Centre, pick an atom, and input the Centre grid box ( Supplementary File 1, Screenshot 16).
    1. Choose File, then Close, saving the current settings.
    2. Choose Grid, then Output, and save as GPF. Go to folder 1, and type the file name 1nmk.gpf.
    3. Choose Docking > Macromolecule > Set Rigid File Name, navigate to folder 1, and click on 1nmk.pdbqt, then Open, as shown in Supplementary File 1 (Screenshot 17).
    4. Choose Docking, find 1, and Select Ligand (Supplementary File 1, Screenshot 18). A window with default parameters will open. Accept the default parameters, as shown in Supplementary File 1 (Screenshot 19) .
    5. Then choose Docking, go to Search Parameters, select Genetic Algorithm, which opens a window with default parameters, and then choose Accept, as shown in Supplementary File 1 (Screenshot 20) .
    6. Choose Docking, then Output, Lamarckian GA, as shown in Supplementary File 1 (Screenshot 21) .
    7. Go to folder 1, type the file name " 1nmk.dpf," and click Save, as shown in Supplementary File 1 (Screenshot 22).
       NOTE: Close the AutoDock 4.2.6 software window, reopen it, and repeat the process for the next molecule. Besides, the 1nmk.pdbqt, 1nmk.gpf, and 1nmk.dpf will be ready for the docking process step.

4. Cygwin-I (Docking process)

NOTE: To create GLG and DLG files, run the algorithm. The algorithm (AutoGrid) runs in this Cygwin terminal. In Cygwin, navigate to the folder. For example, if folder 1 is located on the C drive, it should contain one folder named "project," followed by another folder named "molecules," and then all the folders within it. Follow these steps.

1. Open the Cygwin terminal.
2. Type cd c:/ and press Enter. Then type cd project and press Enter.
3. Type cd molecules and press Enter, then type cd 1 and press Enter. Now folder 1 is in the Cygwin terminal.
4. Type ./autogrid4.exe -p 1nmk.gpf -l 1nmk.glg & and press Enter, as shown in Supplementary File 1 (Screenshot 23).
   NOTE: In "./autogrid4.exe -p 1nmk.gpf -l 1nmk.glg &", -l is a minus small l.
5. Type tail -f 1nmk.glg and press Enter, as shown in Supplementary File 1 (Screenshot 4).
6. If AutoGrid completes successfully, close this Cygwin window, open a new Cygwin window, and repeat the above process for molecule 2.
7. Repeat this step for all molecules and run the algorithm (AutoDock 4.2.6).
   NOTE: For AutoDock 4.2.6, repeat the process of navigating to the folder, and when the folder is accessed, type the and press Enter for AutoDock 4.2.6.
8. Type ./autodock4.exe -p 1nmk.dpf -l 1nmk.dlg & and press Enter, as shown in Supplementary File 1 (Screenshot 24) .
   NOTE: In the above step "-l" is a lowercase letter "L" with a negative sign.
9. Type tail -f 1nmk.dlg
   NOTE: If AutoDock 4.2.6 completes, close the Cygwin window, and repeat the process for molecule 2.
10. Follow this Cygwin procedure for all molecules.
11. After creating DLG files for molecules, go to folder 1 and open the DLG file in WordPad. Press Ctrl + F, type, and press Enter three times to reach the RMSD table, as shown in Supplementary File 1 (Screenshot 25) .
12. Open a Microsoft Excel window or note down the page and create three columns : No., Min. Binding Energy, and Run.
13. Write down the data according to the RMSD table in the log file. The binding energy is at the top of the table, along with the run in which it was observed. Do this for all molecules.
    NOTE: The molecule with the lower binding energy is considered better than the others.
14. Create another spreadsheet listing the details (serial number, min. binding energy, and run) of the top 20 molecules.
    NOTE: The following procedure will be performed for the top 20 molecules only.

5. Cygwin-ii

1. Open the Cygwin terminal, type cd c:/ and press Enter.
2. Type cd project and press Enter, type cd molecules and press Enter.
3. Type cd (folder number of the top molecule which is selected) and press Enter.
   NOTE: The number should correspond to the folder in the Cygwin terminal.
4. Type grep '^docked' 1nmk.dlg |cut -c9- > 1nmk_run.pdbqt and press Enter. Unlike AutoGrid and AutoDock, no successful completion message will appear for this and press Enter, as shown in Supplementary File 1 (Screenshot 26).
5. Press Enter, and the file will be created.
6. Type cut -c-66 1nmk_run.pdbqt > 1nmk_run.pdb and press Enter. The file will be created, as shown in Supplementary File 1 (Screenshot 27) .
7. Close the Cygwin window.
   ​NOTE: To visualize the interaction of ligands and receptors after the docking using PyMOL 2.5 software
   1. Open PyMOL 2.5 software, then open the file, then select receptor.
   2. Open the file, then select the chosen ligand configuration, as shown in Supplementary File 1 (Screenshot 28) .
   3. In PyMOL 2.5, select Hide Molecule and Hide Receptor. In the Receptor menu, select Show Surface, as shown in Figure 1A.
      NOTE: To visualize the hydrogen bond in ligand-1nmk receptor complex UCSF Chimera Software will be used in 20 lowest binding energy ligands.

6. UCSF Chimera software for visualization of H-bond and analysis of results

1. Open UCSF Chimera, and then Select File, then Open, and navigate to drive c:/, then Project, Molecules), select 1nmk.pdb, and open it.
2. Select the drive where the molecules are located, then open the protein in the UCSF program.
3. Select Residue, then choose UNL Zoom.
4. Select Zone, then press OK, then select Action, then choose Label, select Residue, and choose Name + Specifier.
5. Select Tools, then choose Structure Analysis, and then choose Find H-Bond, as shown in Supplementary File 1 (Screenshot 29) .
6. Open the default window and check the boxes for color H-bonds that do not meet precise criteria, and only find H-bonds with selected.
7. Select Clear Selection to start a new survey, then Zoom in and search for the blue/orange color lines, which represent H-bonds, as shown in Figure 3.
8. Check how many bonds are to be counted.
   NOTE: The zoom should show the H-bond interactions with Arg55, Asn149, Gly150, Lys151, Thr 152, Lys54, Lys155, Isoleucine 156²³, Histidine 54²⁴, Phenylalanine (Phe)53, Cysteine (Cys)52, Gly72, Asn71, and Hid70, and indicate whether they are within the active site of the protein, as shown in Figure 3A - D.
9. Note the number of H-bonds and the molecule number.
10. Put the mouse over the line showing the H-bond, indicating interactions with Arg55 and Methionine, Gln, Gly, and Thr.
11. Press the Print Screen key.
12. Open the Paint application, paste the Supplementary Figure, and save it in the named folder.
    NOTE: To show H-bonds and active site amino acids clearly, the 2D structure of the chosen ligands and receptors should be as follows:

7. Preparing the ligand interaction diagram for docking confirmation and showing the H-bond

1. Open AutoDock 4.2.6 software.
   1. Select Analysis and open Macromolecule, then choose 1nmk, as shown in Supplementary File 1 (Screenshot 30).
   2. Select Analysis and open Docking, then choose Open, navigate to the dlg.pdb file, and select it, as shown in Supplementary File 1 (Screenshot 31).
   3. Select Analysis and open Docking, then choose to Write AutoDock Virtual Screening Ligand and save it as a PDB file, as shown in Supplementary File 1 (Screenshot 32) .
2. Open PyMOL 2.5 software as shown in Supplementary File 1 (Screenshot 33).
   1. Open the saved dlg.pdb file, then open the 1nmk.pdb file.
   2. Open File, select Export Molecule, then Save, as shown in Supplementary File 1 (Screenshot 34).
   3. Open Maestro software, open File, then choose Import Structure as shown in Supplementary File 1 (Screenshot 35).
   4. Import the Ligand Interaction Diagram, save the 2D structure, as shown in Supplementary File 2.

8. MD Simulation

NOTE: To open GROMACS in Linux follow these steps

1. Download Ubuntu 20.04.6.
   1. Turn on the Windows Features application on the PC. And then Choose Windows Subsystem for Linux and press OK.
   2. Follow these and press Enter to install GROMACS:
      1. Type sudo apt-get update and press Enter, then type sudo apt-get upgrade and press Enter.
      2. Type sudo apt-get install gcc and press Enter, and type sudo apt-get install cmake and press Enter.
      3. Type sudo apt-get install built-essential and press Enter, then type sudo apt-get install liDevfftw3-d and press Enter.
      4. Type sudo apt-get install GROMACS and press Enter, then press shift + Ctrl + T.
   3. Open the folder on the personal computer PC and name it GROMACS, as shown in Supplementary File 1 (Screenshot 36) .
   4. Open the GROMACS folder and press Shift + Ctrl + Right key to enter Linux.
      NOTE: The simulations used the water model and GROMACS version 4.0, Win64-multicore version, which incorporated the CHARMM 36 force field. The timestep was set to 2 fs, the cutoff distance of 1.0 to 1.2 nm was chosen.

9. Creating ligand topology using GROMACS pdb2gmx modules (Supplementary File 1, Screenshot 37)

1. Create lig.gro file, extract the ligand from the protein structure by typing grep UNL 135_clean.pdb > lig.pdb and pressing Enter, then rename "UNL" to "LIG" in the file.
2. Open lig.pdb in PyMOL 2.5, remove unwanted atoms by pressing select the atoms and pressing delete the atom. Click on save to save the cleaned version as lig_clean.pdb.
3. Open the cleaned file in Avogadro to add hydrogen using the Build tool and save it as lig.mol2. Edit the top of the file in a text editor to ensure the residue name is set to LIG.
4. Type perl sort_mol2_bonds.pl lig.mol2 lig_fix. mol2 and press Enter.
5. Generate the ligand topology using CGenFF: upload lig_fix. mol2, process the input, and download lig_fix.pdb. Activate the Python environment and run python cgenff_charmm2gmx_py3_nx1.py LIG lig_fix. mol2 lig.str charmm36-jul2022.ff and press Enter.
   NOTE: This step will generate four ligand files (lig.prm, lig.itp, lig.top, and lig_ini.pdb).
6. Generate the ligand.gro file, type gmx editconf -f lig_ini.pdb -o lig.gro and press Enter.
7. Separate the protein by saving only the non-ligand atoms into prot_clean.pdb and ensure UNL atoms are listed at the top of the combined complex.

10. Creating protein topology in GROMACS pdb2gmx modules (Supplementary File 1, Screenshot 38).

1. Prepare the protein topology, Type gmx pdb2gmx -f prot_clean.pdb -o pro.gro -ter and press Enter .
2. Create the complex of protein and ligand after adding lig.gro file to the end of pro.gro file. Then type gmx editconf -f complex.gro -o box.gro -c -d 1.0 -bt cubic and press Enter.
3. Generate solvate using the specific point charges water model to maintain equilibrium, type gmx solvate -cp box.gro -cs spc216.gro -o solv.gro -p topol.top and press Enter.
4. Create ions files, type gmx grompp -f ions.mdp -c solv.gro -p topol.top -o ions.tpr and press Enter. Type gmx genion -s ions.tpr -o solv_ions.gro -p topol.top -pname NA -nname CL -neutral, and press Enter, then choose group 15 to add ions.
5. Generate energy minimization em.gro.
6. Type gmx grompp -f em.mdp -c solv_ions.gro -p topol.top -o em.tpr and press Enter, and type gmx mdrun -v -deffnm em and press Enter.
   NOTE: The solvated system will energy-minimized using a maximum of 50,000 steps with a force convergence criterion of 1,000.0 kJ/mol/nm.
7. Create the index files, type gmx make_ndx -f lig.gro -o index_lig.ndx, press Enter.
8. Type gmx genrestr -f lig.gro -n index_lig.ndx -o posre_lig.itp -fc 1000 1000 1000 and press Enter.
9. Generate index.ndx file, type gmx make_ndx -f em.gro -o index.ndx and press Enter.
10. Generate NVT equilibration nvt.gro file, type gmx grompp -f npt.mdp -c nvt.gro -t nvt.cpt -r nvt.gro -p topol.top -n index.ndx -o npt.tpr and press Enter. Then, type gmx mdrun -deffnm nvt and press Enter.
    NOTE: The equilibration of the system was performed at a temperature of 27.35 °C (300.5 K) for the ensemble of the system's number of atoms (N), volume (V), and temperature (V) (NVT) for 100 ps.
11. Update the topology file topol.top to include ligand topology and parameters to make NPT.
12. Generate NPT equilibration.
13. Create second phase NPT equilibration, type gmx grompp -f npt.mdp -c nvt.gro -t nvt.cpt -r nvt.gro -p topol.top -n index.ndx -o npt.tpr and press Enter, and type gmx mdrun -deffnm npt and press Enter.
    NOTE: Equilibrated NVT refers to the number of particles, volume, and temperature, while equilibrated NPT refers to stability for the number of particles, pressure, and temperature. The NVT.mdp and NPT.mdp files are needed to produce the results of the MD simulation as follows.

11. Releasing the position restraints and running the production molecular dynamics simulation to collect data

1. Type gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -n index.ndx -o md_0_10.tpr and press Enter. Then, type gmx mdrun -deffnm md_0_10 and press Enter.
2. Subject the system to a 1 ns MD simulation to test the stability of the complex.
   NOTE: Root mean square deviation (RMSD) quantifies the average structural deviation of atoms compared to a reference structure, and fluctuation mean square deviation (FMSD) measures the time-averaged atomic fluctuations around their mean positions. The two values are needed to finalize the MD simulation runs.
3. Perform structural analyses, including RMSD, FMSD, H-bonds, and free energy.
4. Draw the analysis graphs using qtgrace_v026_win7 software and md_0_10.gro file to show the trajectory box using VMD software.

Results

As mentioned in the introduction, in vivo and in vitro studies demonstrate the importance of cyclophilin inhibitors in simplifying the structure of Sanglifehrin, thereby reducing mouse spleen proliferation by interfering with the mouse cell cycle⁵. In addition, Cyclophilin A, along with Ganoderiol F derivatives, plays vital roles in protein folding, immune responses, and cell signaling through its PPIASE activity. Its upregulation in various cancers suggests its potential as a th...

Discussion

A new immunosuppressive natural substance called SFA or 1nmk molecular receptor was extracted from Streptomyces. Molecular docking analysis was used to determine the binding energies of the constructed ligands with the target protein. The results showed that, of all the designed ligands, SFA differs structurally from CsA and exhibits a considerable affinity for Cyp A¹⁶. Cyp A, which possesses peptidyl-prolyl cis-trans isomerase activity, controls a variety of cellular processes by attachi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autodock	Autodock	v4.2.6	http://autodock.scripps.edu/downloads/autodock-registration/autodock-4-2-download-page/
CGenFF	CGenFF	v2024	https://cgenff.umaryland.edu
Chemsketch	Chemsketch	v2024	http://www.acdlabs.com/resources/freeware/
Cygwin terminal	Cygwin terminal	v3.6.3	http://www.cygwin.com/
Gromacs tutorial	Gromacs tutorial	v25.04 (Plucky Puffin)	http://www.mdtutorials.com/gmx/complex/index.html
MGL tools	MGL tools	v1.5.7	mgl tools 1.5.6 rc3
Open babel	Open babel	v3.1.1	http://openbabel.org/wiki/main_page
PyMOL	PyMOL	v3.1.6.1	PyMOL | pymol.org
Ubuntu	Ubuntu	v25.04l	https://ubuntu.com/download/desktop
UCSF chimera	UCSF chimera	v1.19	chimera-1.17.3-win64.exe