A Comprehensive Guide to Virtual Screening with PyRx: Docking Multiple Ligands with a Macromolecule

Introduction:

PyRx is a widely used bioinformatics tool for performing virtual screening and molecular docking. In a previous article, we demonstrated how to conduct simple protein-ligand site-specific docking using PyRx. In this tutorial, we will expand on that by performing virtual screening, docking multiple ligands with a macromolecule using PyRx’s AutoDock Vina.

In this guide, we will walk through docking three ligands—Sodium Octanoate (SO), Naphthalene (NAPH), and Salicylic Acid (SA)—with the protein human serum albumin (HSA) to demonstrate the process. While this tutorial uses three ligands for illustration, PyRx allows docking a database of compounds, providing flexibility for large-scale virtual screening projects.

Step-by-Step Guide:

1. Downloading Protein

We will use human serum albumin (HSA), a protein previously explored in our docking tutorials, with the PDB ID 2BXA. Follow these steps to download the protein:

• Visit PDB and search for the protein by its PDB ID (2BXA).
• Download the structure in either PDB or PDBx/mmCIF format.
• Save the file in a designated folder for easy access.

2. Downloading Ligands

For this tutorial, we will dock Sodium Octanoate (SO), Naphthalene (NAPH), and Salicylic Acid (SA) with HSA. Here’s how to download the ligands:

• Go to PubChem and search for each compound.
• Download their 3D conformers in SDF format.
• Alternatively, ligands can be downloaded from databases such as ZINC if working with a larger compound set.

3. Preparing the Protein

Before performing docking, the protein must be prepared:

• Remove HETATOMS: HSA is complexed with CMPF in its crystal structure. To ensure accurate docking, remove HETATOMS (bound ligands) from the structure using PyMOL.
• Remove Extra Chains: If the protein contains multiple chains, keep only the relevant chain (e.g., Chain A) and remove the others to avoid complexity.
• Save the modified structure as “protein.pdb” for further use.

4. Preparing the Ligands

Convert the downloaded ligands from SDF format to PDB format:

• Open each ligand in PyMOL.
• Go to “File” -> “Save Molecule” and select the ligand to save it in PDB format.
• Repeat the process for all three ligands.
• Alternatively, OpenBabel can be used for batch conversion if dealing with large numbers of ligands.

5. Performing Docking with PyRx

Now we proceed to the virtual screening using PyRx with the following steps:

Loading Protein and Ligands:

• Open PyRx and select ‘File’ -> ‘Load Molecule’ to load the protein structure (“protein.pdb”).
• Load the ligands by repeating the same step or selecting all ligands at once.

Defining Protein and Ligands:

• Right-click on the loaded protein under the ‘Molecules’ tab -> ‘Autodock’ -> ‘Make Macromolecule.’
• Right-click on each ligand -> ‘Autodock’ -> ‘Make Ligand.’ PyRx will generate PDBQT files automatically.

Defining the Grid Box:

• In the ‘Controls’ panel, select all ligands for docking.
• Click ‘Forward’ and adjust the grid box around the protein’s binding site by visualizing the binding residues.
• Use PyMOL to identify and label the binding residues if necessary, and adjust the grid box accordingly.

Running Vina for Docking:

• Set the exhaustiveness parameter for the Vina algorithm to refine the docking results.
• Click ‘Forward’ to start the docking process. Once completed, you will see the binding affinities and poses in the bottom panel.

6. Analyzing Results

Once docking is complete:

• Open the PDB or PDBQT files in PyMOL to visualize the poses and binding modes.
• Analyze the binding affinities and poses, using software like PyMOL or Discovery Studio Visualizer for detailed visualization.

Conclusion:

This tutorial provided a comprehensive walkthrough of performing virtual screening using PyRx. By docking multiple ligands with a macromolecule like HSA, we demonstrated how PyRx’s AutoDock Vina can be used for efficient molecular docking. While this tutorial focuses on three ligands, PyRx is versatile enough to handle larger ligand sets, making it a valuable tool for virtual screening in drug discovery.