Network Pharmacology and In Vitro Cell Experimental Study for Exploring the Therapeutic Potential of Thiazolone Derivatives in Rhabdomyosarcoma

Summary

We present a study that systematically investigates the therapeutic potential of thiazolone derivatives against rhabdomyosarcoma based on network pharmacology and computational chemistry, with validation and evaluation performed through in vitro cell experiments.

Abstract

Rhabdomyosarcoma (RD) is a malignant tumor originating from rhabdomyoblasts, which poses a serious threat to the health of children and adolescents. Based on the potential of thiazolone derivatives in a variety of biological activities, this study intends to systematically explore the mechanisms of thiazolone derivatives in the treatment of RD by combining network pharmacology and in vitro cell experiments. Through network pharmacology, high-throughput data were integrated to predict the potential targets of thiazolone. Additionally, AutoDock Vina software was used to calculate the binding energy of the compound and the target protein molecule, revealing its signaling pathway related to RD. Finally, in vitro cell experiments further verified the tumor suppressive effect of the compounds. Network pharmacology was used to analyze the targets of thiazolone derivatives in the treatment of RD, and 172 target genes were finally obtained by combining and deduplicating. Five protein targets with low binding energy were screened by docking between the compound and the target protein molecule, PIK3CD, AKT1, GSK3B, HSP90AB1, and ITGB1. The results of in vitro cell culture experiments showed that a specific thiazolone derivative had a significant inhibitory effect on RD cell lines, providing a scientific basis for the discovery of new targets and effective compounds for the treatment of RD. This study revealed the mechanisms of thiazolone derivatives in the treatment of RD through a combination of network pharmacology and in vitro experiments and provided a new perspective for future drug development and treatment strategies.

Introduction

Rhabdomyosarcoma (RD), a highly aggressive pediatric soft tissue sarcoma that represents a significant threat to pediatric health, with limited therapeutic options. Epidemiological studies indicate that while RD exhibits relatively low incidence rates, its high malignancy and poor prognosis present substantial clinical challenges. RD is classified into four distinct histological subtypes: embryonal, alveolar, spindle cell/sclerosing, and pleomorphic, with embryonal rhabdomyosarcoma (ERMS) being the most prevalent form^1,2,3. Current therapeutic strategies for newly diagnosed low-risk ERMS patients primarily involve surgical resection of localized tumors combined with chemotherapeutic agents, including vincristine, dactinomycin, and cyclophosphamide. High-risk patients typically receive intensified multi-agent chemotherapy regimens incorporating ifosfamide and etoposide^4,5. While the multimodal approach combining surgery, chemotherapy, and radiotherapy has demonstrated efficacy in achieving local tumor control and eradicating micrometastases⁵, significant challenges remain. The substantial toxicity profiles of current chemotherapeutic agents, including neurotoxicity and irreversible renal and bladder damage⁶, pose particular risks to pediatric and adolescent patients. Furthermore, the inherent heterogeneity of RD and its propensity for relapse underscore the critical need for developing more effective and targeted therapeutic agents⁷.

In this context, the research here focuses on a class of thiazolone derivatives with distinct pharmacological properties. Thiazolone derivatives exhibit significant anti-inflammatory, antioxidant, and apoptosis-regulating activities⁸, demonstrating broad biological activity and promising potential for drug development^9,10. Molecular docking simulations by Elgubbi et al. revealed strong binding affinities of thiazolone derivatives to key targets such as human carbonic anhydrase IX (CA IX), a hypoxia-inducible enzyme overexpressed in aggressive tumors, suggesting a mechanism for their antiproliferative effects¹¹. The computational study elucidates the molecular interactions between thiazolone derivatives and cancer-associated proteins. In 2024, El-Helw et al. reported that benzoquinoline-based thiazolone derivatives exhibit potent activity against cancer cell lines, with thiazolethione and thiazole analogs demonstrating the highest efficacy¹². Mechanistic studies^10,13 suggest these compounds disrupt tumor cell viability through dual pathways: (1) induction of oxidative stress through the antioxidant pathway modulation and (2) direct inhibition of proliferation. These findings emphasized the broad pharmacological profile of thiazolones, including anti-inflammatory and antimicrobial properties. Notably, compounds such as pioglitazone¹⁴ and troglitazone¹⁵ are primarily employed in the treatment of type 2 diabetes¹⁶, while 4-thiazolone derivatives exhibit anti-proliferative effects against leukemia cells¹⁷ (Figure 1). These compounds not only demonstrate significant therapeutic promise in clinical studies but also specifically target multiple cell signaling pathways, including the inhibition of tumor cell proliferation, induction of apoptosis, and regulation of the cell cycle. The anticancer mechanisms of thiazolone derivatives involve several signaling pathways that effectively hinder tumor cell growth and survival by modulating critical pathways, such as the PI3K/Akt and NF-κB pathways. Furthermore, thiazolone derivatives have also exhibited regulatory effects on the tumor microenvironment, including the inhibition of angiogenesis and modulation of immune responses¹⁸. The multifaceted mechanistic actions of thiazolone derivatives position them as ideal candidates for the development of novel cancer therapeutics.

In previous research, we efficiently constructed thiazolone derivatives with adjacent chiral centers through a copper-catalyzed distal asymmetric propargylation reaction. Using propargyl esters and thiazolones as substrates, the reaction was catalyzed by CuI in the presence of a phenyl-substituted pyridine bis(oxazoline) ligand. By optimizing the reaction conditions, we achieved high efficiency in the synthesis of these thiazolone derivatives¹⁹. Network pharmacology, as a systems biology approach, reveals complex interactions among drugs, targets, and disease pathways, revolutionizing the understanding and development of therapies for complex diseases like cancer²⁰. This study preliminarily aims to reveal the potential therapeutic value of novel thiazolone derivatives in the treatment of RD. Through integrated network pharmacology and cellular experiments, compounds with superior activity will be identified, which may exert multi-target therapeutic effects by coordinately regulating five core targets, including PIK3CD and AKT1, thereby influencing key signaling pathways such as PI3K-Akt. Molecular docking and molecular dynamics simulations will be used to demonstrate binding characteristics between the compound and target proteins. Furthermore, a preliminary structure-activity relationship model will be established, providing a reference for subsequent structural optimization. This work not only offers new candidate molecules for the drug development of rhabdomyosarcoma but also highlights the positive role of multidisciplinary strategies in exploring multi-target therapeutic mechanisms of drugs.

Protocol

All the network pharmacology procedures were carried out in accordance with the Guidelines for Network Pharmacology Evaluation Methods²¹. The comprehensive network pharmacology workflow adopted in this study is schematically illustrated in Figure 2.

Synthesis of Thiazolone derivatives
The thiazolone derivatives in this study were synthesized in our previous work¹⁹, through a copper-catalyzed remote asymmetric propargylation reaction. The reaction utilized CuI as a catalyst and a chiral Pybox ligand (L1) as the stereocontrol core, in a DIPEA base and methanol solvent system at -10 °C for 48 h, achieving high enantioselective coupling between propargyl esters and thiazolones. The target product was obtained with a yield of up to 89%, an enantiomeric ratio (er) of 97.8:2.2, and a diastereomeric ratio (dr) of 17.8:1. For detailed information on the synthesis methods of this series of compounds, please refer to previous studies¹⁹. This method demonstrated broad substrate applicability, accommodating aryl (ortho-, meta-, para-substituents), aliphatic chains, and fused-ring propargyl esters, as well as various substituted thiazolones, with yields ranging from 51% to 98%. This strategy efficiently constructs multi-chiral-center thiazolone frameworks in a one-step process for the first time, providing a highly selective and straightforward approach for the synthesis of chiral drug molecules with significant potential applications in medicinal chemistry.

Network pharmacological prediction
Target prediction of the thiazolone derivative: The structural representations of thiazolone derivatives were generated using KingDraw software, and the corresponding structural files were subsequently converted into SMILES ID through Open Babel software. To predict potential therapeutic targets against RD, we employed a comprehensive computational approach utilizing three distinct target prediction databases: SwissTargetPrediction (http://swisstargetprediction.ch/), TargetNet (http://targetnet.scbdd.com), and SuperPred (https://prediction.charite.de). For TargetNet analysis, we implemented a stringent filtering criterion by selecting genes with Probability > 0. In the SuperPred database, we applied more rigorous selection parameters, retaining only those genes exhibiting Model accuracy > 90% and Probability > 60%, ensuring high-confidence target predictions.

Prediction of RD targets: Based on the GeneCards database (https://www.genecards.org/) and the OMIM database (https://omim.org/), we conducted a search for Rhabdomyosarcoma. For the data sourced from the GeneCards database, we filtered the results with a score greater than 20 to obtain genes associated with rhabdomyosarcoma.

Construction and analysis of protein-protein interaction (PPI) networks: The potential targets of thiazolone derivatives and the genes associated with RD were intersected. The potential targets for the treatment of human embryonal rhabdomyosarcoma by thiazolone derivatives were uploaded to the STRING database (https://www.string-db.org/) for high-confidence target PPI (protein-protein interaction) relationship analysis. The PPI network was constructed in Cytoscape software (http://www.cytoscape.org/), and the core targets were screened based on degree values.

Compound-disease-target pathway network construction: The drug-target-pathway network can clearly see the targets of compounds and diseases and the pathways involved in these targets, so as to identify possible key targets and pathways for compound therapy for RD. First, build the network table and attribute table in the spreadsheet. The content of the network table mainly includes the correspondence between the compound and the core target, the correspondence between the core target and the pathway involved, and the correspondence between RD and the main pathway. The content of the attribute table is mainly to classify and name all the information in the network table. After that, open the Cytoscape software, upload the netlist to the loading location, set the start point, end point, upload the attribute table, and finally modify the image shape.

Integrated functional annotation and network pharmacology analysis - Construction Gene Ontology (GO) Enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG): GO and KEGG enrichment analysis of core targets was performed by the DAVID database (https://david.ncifcrf.gov/). According to the order of P value from small to large, the top 10 items in biological process (BP), cellular component (CC) and molecular function (MF) in GO were selected for analysis, and the top 20 items in KEGG were analyzed, and the GO and KEGG bubble maps of core targets were created based on the online micro-bioinformation platform.

Molecular docking: Core target proteins were selected from the PDB database (https://www.rcsb.org/), and the structural files of the core targets were downloaded. Initially, the proteins were dehydrated using PyMol software, followed by the separation of ligands and receptors. Subsequently, the proteins were hydrogenated using ADFRSuite software, and the grid box parameters for the AutoDock molecular docking software were obtained. The pdbqt files of thiazolidinone derivatives and core target proteins were prepared using AutoDock Vina software. Molecular docking simulations of thiazolone derivatives and core target proteins were then conducted. Finally, the molecular docking models were visualized using PyMol software.

Molecular dynamics simulation: The PDB file was converted to GROMACS-compatible GRO format using the amber99sb-ildn force field and the TIP3P water model:
gmx pdb2gmx -f Pro.pdb -o Pro_temp_H.gro -ff amber99sb-ildn -water tip3p -ignh
A cubic periodic boundary box was added with a distance of 1.2 nm from the protein:
gmx editconf -f Pro_temp_H.gro -o Pro_temp_H_box.gro -c -d 1.2 -bt cubic
The box was filled with TIP3P water molecules:
gmx solvate -cp Pro_temp_H_box.gro -o Pro_temp_H_box_water.gro -p topol.top
The system was neutralized by adding Na ions:
gmx grompp -f ions.mdp -c Pro_temp_H_box_water.gro -p topol.top -ions.tpr
gmx genion -s ions.tpr -o Pro_temp_H_box_water_ion.gro -p topol.top -neutral
Energy minimization was performed:
gmx grompp -f minim.mdp -c Pro_temp_H_box_water_ion.gro -p topol.top -o em.tpr
gmx mdrun -v -deffnm em
NVT equilibration was carried out:
gmx grompp -f nvt.mdp -c em.gro -r em.gro -p topol.top -o nvt.tpr
gmx mdrun -deffnm nvt -v
NPT equilibration was carried out:
gmx grompp -f npt.mdp -c nvt.gro -r nvt.gro -t nvt.cpt -p topol.top -o npt.tpr
gmx mdrun -deffnm npt -v
The production molecular dynamics simulation was initiated:
gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -o md_0_1.tpr
gmx mdrun -deffnm md_0_1 -v

Upon completion of the simulations, the resulting trajectories were analyzed using Visual Molecular Dynamics (VMD) and PyMOL, and the binding free energy analysis between the proteins and small molecule ligands was performed employing the g_mmpbsa program.

Assessment of drug effects on RD cell viability
RD cells (Embryonal Rhabdomyosarcoma Cell; STR Authenticated) in the logarithmic growth phase were trypsinized to create a cell suspension at a concentration of 1 x10⁵ cells/mL. This suspension was then seeded into a 96-well plate at a density of 1 x 10⁴ cells/well (100 µL per well) and incubated at 37 °C with 5% CO₂ to allow cell adhesion. Following adhesion, the medium was replaced with 100 µL of serum-free medium containing 1% FBS, and cells were starved for 12 h. Subsequently, the medium was exchanged for 100 µL of the respective medium containing varying concentrations of the test compound. Control wells were treated with either solvent-containing medium or normal medium, and blank wells containing only the corresponding medium without cells were included. The plates were incubated at 37 °C with 5% CO₂ for 24 h (duration was determined by preliminary experiments to be optimal for observing the maximal inhibitory effect of this class of compounds on RD cells. That is, the time required for the OD value of the cell density to reach approximately 1.0.). Thereafter, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for an additional 1-4 h. Finally, the absorbance at 450 nm was measured using a microplate reader. The solvent-treated cells served as the control group, and the blank wells were used for baseline correction. The cell viability was calculated based on the absorbance readings.

Survival rate% = [(control group-blank)-(experimental group-blank)]/(control group-blank) x 100%

Results

In this study, 13 thiazolone derivatives (compounds a-m; Figure 3, Figure 4) were previously synthesized¹⁹ were selected for evaluation of their inhibitory activity against cancer cells (Figure 5). Among these compounds, compound m at 100 µM showed the highest inhibition rate against RD cells (Embryonal Rhabdomyosarcoma Cell) (STR Authenticated)²². Subsequent experiments involving vary...

Discussion

Network pharmacology is commonly used to predict and screen potential active molecules within complex traditional Chinese medicine components, and to elucidate the synergistic mechanisms of multi-component, multi-target, and multi-pathway actions in herbal formulations. In this study, we applied a network pharmacology approach to systematically investigate the potential mechanism of our newly synthesized thiazolone derivative against RD by constructing a compound-target-pathway-disease network. In this study, we systemat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CCK-8 Assay Kit	Beyotime Biotechnology Co., Ltd.	C0038	A kit used for measuring cell viability and proliferation based on the reduction of a water-soluble tetrazolium salt to form a colored product.
Clean Bench	Suzhou Antai Airtech Co., Ltd	SW-CJ-1FD	A laminar airflow workstation that provides a sterile environment for handling cell cultures and other sensitive materials.
CO2 Incubator	Heal Force	HF90	A temperature, humidity, and CO2-controlled environment is used for cell culture to maintain optimal growth conditions.
DMEM High Glucose	cytiva	SH30022.01	A cell culture medium with high glucose concentration (4.5 g/L), suitable for a variety of cell types, including fast-growing mammalian cells.
FBS	YEASEN	40130ES76	A common supplement in cell culture media, providing essential growth factors, hormones, and nutrients for cell proliferation.
Inverted Microscope	OLYMPUS	IX51	A microscope designed with the objective lens below the stage, allowing observation of cell cultures in flasks or plates from below.
Microplate Reader	Diatek	DR-200Bs	An instrument used to detect biological, chemical, or physical events in microplate samples by measuring absorbance, fluorescence, or luminescence.
PBS	Jinuo Biomedical Technology Co., Ltd.	GNM20012	A buffer solution is commonly used in biological research for washing cells, diluting solutions, and maintaining pH stability.
RD (Embryonal Rhabdomyosarcoma Cell) (STR Authenticated)	Shanghai FuHeng Biotechnology Co., Ltd.	FH0712	The cell line has been authenticated by short tandem repeat (STR) analysis, possesses a well-defined genetic background and clear cell identity, thereby ensuring the reliability and reproducibility of experimental data.
RMPI Medium Modified (1640)	cytiva	SH30809.01	A widely used cell culture medium originally developed for human leukocyte cells, is now applied for various mammalian cell types.
Trypsin-EDTA	Jinuo Biomedical Technology Co., Ltd.	GNM25200	A solution used to detach adherent cells from culture surfaces by digesting extracellular proteins and chelating calcium ions.