Name: Structure-Guided Design and Development of Novel Cyclophilin A Inhibitors and Ganoderiol-F Derivatives: An In-Silico Approach (Video) | JoVE Journal
Uploaded: 2026-06-23T12:30:00.000+00:00
Duration: 10 min 1 s
Description: Discover computational drug design methods using molecular docking and dynamics simulations to develop novel Cyclophilin A inhibitors targeting the Cyclophilin D-CDK axis for cancer therapy.

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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...

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 transporters7,8.
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 cancer10,11. 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. A92-30815,18. 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 I19,20.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 process21,22. 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.

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

structure-guided design and development of novel cyclophilin a inhibitors and ganoderiol-f derivatives: an in-silico approach

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

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 1.53&#197;, 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.

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...

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.

The cyclin Ds-CDKs axis (Cyclophilin A (Cyp /Ganoderiol F)) plays a critical role in cancer through various processes, such as controlling proliferation ...

This research focus on designing novel Cyclophilin A inhibitors and gan-it-role antiproliferative to target the Cyclophilin DCDK axis and inhibit cell cancer proliferation. Key challenge include achieving optimal ligand-receptor geometric interaction and maintaining a stable ligand-protein complexes under dynamic experimental and physiological condition. To begin, open the 1NMK"file.Locate the amino acid arginine"at position number 55, and record the X, Y, and Z coordinates in the AutoDock"interface. Open the File"menu, select Read Molecules. Choose the file 1NMK"and click Open.Add hydrogen atoms by selecting Edit, choosing Hydrogen, and clicking Add. Then choose Polar Only"and confirm with OK.Then go to the Edit"menu again, choose Atoms, and select Assign AD4 Atom Type. Next, choose Charges"and select Kollman Charges.Save the prepared receptor as 1NMK. pdbqt"by selecting File, choosing Save, setting the file type, and confirming the save location. Next, open the Ligand"menu, select Input.Click Open, choose the PDB file, and click Open"again. When the information window appears, review the ligand details. To detect the ligand root, select Ligand, then choose Torsion Tree"and click Detect Root.Save the ligand by selecting Ligand, choosing Output, and clicking Save as PDBQT. Prepare the receptor grid by selecting Grid, choosing Macromolecule, selecting 1NMK, and clicking Molecule. Confirm the warning and save as 1NMK.pdbqt.For setting the map types, select Grid, followed by Set Map Types, Ligand. Choose Ligand"and finally select Ligand"again. Then define the grid box center by selecting Grid, choosing Grid Box, and clicking Center.Then select an atom and enter the grid box coordinates. Save the grid parameter as a GPF file. Configure docking by selecting Docking, followed by Macromolecule"and Set Rigid File Name.Navigate to the required folder and click on 1NMK.pdbqt. to open it, select Docking, choose Output, click Lamarckian GA, and save the docking parameter file as 1NMK.dpf. In the Cygwin"terminal, type cd C:and press enter;then type cd project"and press enter again.Next, type cd molecules"and press enter. Then type cd 3"and press enter. Type the AutoGrid"command to generate grid maps and press enter to execute AutoGrid.After the execution, type tail f 1NMK. glg"and press enter to view the log progress, type the command for molecular docking simulation. To generate the docking log file, and press enter to start AutoDock.Then, type tail f 1NMK. dlg"and press enter to monitor the docking process. After creating all DLG files, go to folder 1 and open the DLG file in WordPad, press Ctrl+F.Type the search keyword and press enter three times to reach the RMSD table. Copy the binding energy and corresponding run information for each molecule from the RMSD table, compile the top 20 molecules in a spreadsheet with columns created for serial number, minimum binding energy, and run. In the Cygwin"terminal, navigate to the specific molecules folder within the project directory.Type the command to extract docked ligand coordinates from DLG and trim the data. Press enter once more to finalize, confirming the creation of the 1NMK run PDBQT"file. Verify that the 1NMK run PDB"file is created in the same folder.Next, open the PyMOL 2.5"software and the AutoDock"from the desktop, and open the receptor file to select the receptor structure for visualization. Open the corresponding ligand configuration file for the chosen molecule and display the loaded ligand in the 3D workspace. Select Hide Everything"from the menu, import the file to Maestro"to visualize the structure, and then click on Ligand Interaction"to get the ligand interaction diagram.Open the UCSF Chimera"software from the desktop. Select File, followed by Open. Navigate to drive C, project, and molecules.Select the file 1NMK. pdb"and click Open. From the menu, select Residue"and choose UNL Zoom"to focus on the UNL residue region.Select Zone. Then press OK"to confirm. Next, select Action.Choose Label, then select Residue"and choose Name Plus Specifier"to label the residues. Open Tools, select Structure Analysis, and then click FindHBond. In the default FindHBond"settings window, check the boxes for Color H-bonds That Do Not Meet Precise Criteria"and Only Find H-bonds with Selected.Clear the zone selection to begin a new survey. Then select the zone parameters and zoom into the molecular display, and observe the blue and orange lines representing hydrogen bonds. Finally, examine the visualized structure and count the total number of visible hydrogen bonds.The docking confirmation of the ligand-receptor complex was visualized using PyMOL 2.5, showing 109 hydroxy six hydroxyethyl bound within the active pocket of the 1NMK"receptor. The 4-hydroxymandelic acid ligand formed hydrogen bonds with the 1NMK"receptor, particularly with arginine 55 at a distance of 1.53 angstroms. Two-dimensional structural analysis illustrated the amino acids with which the ligands interacted through hydrogen bonding.Molecular dynamics simulations confirm the stability of the ligand-receptor complex through the analysis of the radius of gyration over 1000 picoseconds. The RMSD graph showed consistent stabilization of the complex after one nanosecond of simulation, indicating steady protein-ligand binding. The average density of the system was approximately 1019 kilograms per cubic meter, confirming equilibrium under NVT conditions, and the pressure of the system remains stable during NPT equilibration for 100 picoseconds.The temperature stabilized around 300 kelvin during the NVT phase, confirming successful thermal equilibration. The ligand-protein complex trajectory box was visualized after the simulation, showing the distribution of water molecules surrounding the docked complex. Few studies investigate gan-it-role antiproliferative as an oncogenic therapeutic target.This protocol addresses that gap by designing and validating inhibitor using molecular docking and dynamics analysis. Our protocol interrogate multiple computational tool into a unified workflow, ensuring comprehensive ligand-protein or receptor analysis. Our future research will focus on validating these inhibitor in vitro and in vivo by exploring border application of Cyclophilin A inhibitors across multiple cancer cell type.

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Discover computational drug design methods using molecular docking and dynamics simulations to develop novel Cyclophilin A inhibitors targeting the Cyclophilin D-CDK axis for cancer therapy.

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

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