Reproducible Computational Workflow for Drug Discovery to Standardize Network Pharmacology and Molecular Docking Analyses

Summary

This standardized protocol unifies network pharmacology and molecular docking with Molecular Dynamics (MD) simulations for drug discovery. It establishes quantitative screening criteria and reproducible steps, suitable for multi-target drug screening using public datasets and improving result reliability.

Abstract

Network pharmacology and molecular docking are widely applied in drug discovery, yet fragmented workflows and inconsistent operations frequently undermine result reproducibility. Here, a standardized protocol integrating these approaches into a reproducible framework for drug screening and mechanism exploration is described, with the workflow organized into three sequential phases: data preparation, computational analysis, and validation. In the preparation phase, compound libraries from public databases are filtered via Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) criteria, including oral bioavailability, drug-likeness, and toxicity prediction, while potential therapeutic targets are obtained through target prediction and integration of disease-related databases to comprehensively identify drug-disease interaction candidates. In the computational analysis phase, overlapping targets undergo Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and protein-protein interaction network topological analysis to identify core targets; molecular docking is configured with two standardized optional strategies with distinct advantages. The two-step progressive strategy employs AutoDock Vina for high-throughput preliminary screening of the compound library, followed by precise re-docking with YASARA, which eliminates false positives from high-throughput screening and generates protein-ligand complexes natively compatible with subsequent YASARA molecular dynamics (MD) simulations to avoid structural deviations caused by cross-software format conversion. The one-step strategy completes the full docking process via YASARA alone, which simplifies the operation workflow, improves experimental efficiency, and is fully applicable for specific research goals. In the validation phase, standardized MD simulations evaluate ligand-protein complex stability via core metrics of root mean square deviation (RMSD) and root mean square fluctuation (RMSF). This unified, reproducible pipeline enhances the reliability of network pharmacology and docking studies and facilitates cross-study comparisons in computational drug discovery.

Introduction

Network pharmacology represents a research approach that deciphers the interaction patterns between drugs and the organism from a holistic network perspective¹. By constructing and analyzing the interaction network encompassing drug-component-target-disease-biological pathway, it quantitatively identifies the key molecules, core pathways, and synergistic mechanisms through which drugs exert their effects. This analytical framework aligns with the international standard for network pharmacology, which emphasizes multi-omics data integration and topological network analysis, and ultimately elucidates the overall therapeutic effects of drugs, pred....

Protocol

This protocol involves only computational analyses of publicly available databases and does not involve the use of human subjects, vertebrate animals, or biological tissues. All the summary workflows described in this section are illustrated in Figure 1.

Figure 1: Summary of the workflow.

Results

Following the network pharmacology analysis of loratadine against allergic rhinitis (AR), the interaction between loratadine and PTGS2 was selected as a representative case study to illustrate the step‑by‑step application of the molecular docking and MD simulation protocol. This example is intended to demonstrate workflow execution and data interpretation, rather than to provide biological validation of the specific interaction. For quantitative assessment against experimental data, users are encouraged to apply the prot.......

Discussion

Significance and critical steps
This protocol combines network pharmacology, molecular docking, and molecular dynamics simulation, which offers distinct advantages over standalone methods or dual-combination workflows, and can help address key inefficiencies and reliability gaps in current drug discovery. The entire process relies on three critical steps that ensure its reliability, each addressing a core challenge in computational drug screening. First, multi-database integration (e.g., PubChem fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADMETlab 3.0	Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences	N/A	Online platform for ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) property prediction; used for evaluating the pharmacokinetic and toxicological profiles of ligands (URL: https://admetlab3.scbdd.com/)
AutoDock Tools (AutoDock 4)	The Scripps Research Institute	AutoDock 4.2.6	Software suite for molecular docking simulations; includes AutoDock 4 for docking and AutoDockTools (ADT) for preparing protein and ligand input files (adding hydrogens, assigning charges, setting rotatable bonds), defining docking grids, and analyzing docking results.
AutoDock Vina	The Scripps Research Institute	AutoDock Vina 1.1.2	Open-source molecular docking software; used for predicting binding affinities and poses between small-molecule ligands and protein receptors
Chem3D	PerkinElmer Informatics	Chem3D 2024	Molecular modeling software; used for constructing, optimizing, and visualizing 3D structures of small-molecule ligands
Cytoscape	Cytoscape Consortium (Institute for Systems Biology)	Cytoscape 3.10.3	Open-source software for visualizing and analyzing biological networks; used for constructing and editing gene/protein interaction networks
DAVID (Database for Annotation, Visualization and Integrated Discovery)	National Institute of Allergy and Infectious Diseases (NIAID), USA	N/A	Online tool for functional annotation and enrichment analysis; used for performing GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of target genes (URL: https://david.ncifcrf.gov/)
DisGeNET Database	Barcelona Supercomputing Center (BSC)	N/A	Database of gene-disease associations; used for identifying genes linked to specific diseases (URL: https://disgenet.com/)
GeneCards Database	Weizmann Institute of Science	N/A	Integrative database of human genes; used for retrieving comprehensive gene information (e.g., expression, function, disease associations) (URL: https://www.genecards.org/)
LigPlus	European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI)	LigPlus 2.3	Software for automatically generating 2D protein-ligand interaction diagrams from 3D coordinate files . It schematically depicts hydrogen bonds, hydrophobic contacts, and the binding site residues . Available upon registration with an academic email at https://www.ebi.ac.uk/thornton-srv/software/LigPlus/ .
OMIM Database	Johns Hopkins University School of Medicine (in collaboration with NCBI)	N/A	Online Mendelian Inheritance in Man; used for retrieving information on genetic disorders and their associated genes (URL: https://www.omim.org/)
OpenBabel	OpenBabel Development Team	N/A	Open-source chemical toolbox; used for converting molecular file formats (e.g., from .mol2 to .pdb) between different software platforms
PharmGKB Database	Stanford University	N/A	Pharmacogenomics Knowledge Base; used for retrieving information on gene-drug interactions and pharmacogenomic variants (URL: https://www.pharmgkb.org/)
Prism	GraphPad Software	Prism 9	Used for scientific graphing, data analysis (e.g., plotting binding energy distribution curves, analyzing error bars) and generating publication-quality figures.
ProTox 3.0	Charité - Universitätsmedizin Berlin, Germany	N/A	Online tool for predicting toxicological endpoints of small molecules; used for assessing potential toxicity of candidate ligands (URL: https://tox.charite.de/protox3/index.php?site=home)
PubChem Database	National Center for Biotechnology Information (NCBI), USA	N/A	Public database of chemical information; used for retrieving 2D/3D structures and physicochemical properties of small-molecule ligands (URL: https://pubchem.ncbi.nlm.nih.gov/)
PyMOL	Schrödinger, LLC	PyMOL 2.6.1	Molecular visualization software; used for viewing, editing, and generating high-quality images of protein-ligand complexes
R Studio	Posit, PBC	Rstudio 2025.09.1+401	Integrated development environment (IDE) for R programming; used for statistical analysis of biological data and generation of GO/KEGG plots
RCSB PDB Database	Research Collaboratory for Structural Bioinformatics (RCSB)	N/A	Database of protein structures; used for retrieving 3D structures of protein receptors in PDB format (URL: https://www.rcsb.org/)
SEA (Similarity Ensemble Approach)	The Scripps Research Institute	N/A	Online tool for target prediction based on chemical similarity; used for complementing SwissTargetPrediction to confirm ligand targets (URL: https://sea.bkslab.org/)
STRING	STRING Consortium (EBI, SIB, etc.)	N/A	Database of known and predicted protein-protein interactions; used for constructing gene/protein interaction networks (URL: https://string-db.org/)
SwissTargetPrediction	Swiss Institute of Bioinformatics (SIB)	N/A	Online server for predicting potential protein targets of small molecules; used for identifying candidate receptors for ligands (URL: http://swisstargetprediction.ch/)
TTD Database	Institute of Drug Discovery and Development (IDRBL), Sun Yat-sen University	N/A	Therapeutic Target Database; used for retrieving information on validated and potential drug targets (URL: https://db.idrblab.net/ttd/)
UCSF Chimera	Resource for Biocomputing, Visualization, and Informatics (RBVI), University of California, San Francisco	UCSF Chimera 1.19	Molecular visualization and analysis software; used for protein structure preparation including missing loop reconstruction (via Modeller interface), side chain optimization (Dunbrack rotamer library), protonation state adjustment, and energy minimization with AMBER ff14SB force field. Version 1.19 (released March 2025) fixes PDB structure fetching capabilities . Available free of charge for non-commercial use at https://www.cgl.ucsf.edu/chimera/ .
UniProt Database	UniProt Consortium (EBI, SIB, PIR)	N/A	Comprehensive database of protein sequence and function; used for retrieving protein sequences, structures, and functional annotations (URL: https://www.uniprot.org/)
Venny 2.1.0	Centro Nacional de Biotecnología (CNB-CSIC), Spain	N/A	Online tool for generating Venn diagrams; used for visualizing overlaps between gene sets (e.g., target genes from different databases) (URL: https://bioinfogp.cnb.csic.es/tools/venny/)
YASARA	YASARA Biosciences	YASARA 10.3.16	Molecular modeling and simulation software; used for molecular docking (Step 3.7) and subsequent molecular dynamics simulations to validate docking results