Discovery of the Inhibitor Targeting the SLC7A11/xCT Axis through In Silico and In Vitro Experiments
<p>SLC7A11/xCT maintains intracellular GSH/GSSG balance mechanism.</p> "> Figure 2
<p>Process of discovering the potential inhibitors targeting the SLC7A11/xCT axis through molecular docking. Eint represents the interaction energy.</p> "> Figure 3
<p>Oxidative/antioxidant balance within the HeLa cells influenced by compound 1. (<b>A</b>) After 24 h of drug treatment, the effect of 25 μM compound 1 and erastin on intracellular GSH levels in HeLa cells (n = 6). (<b>B</b>) After 24 h of drug treatment, the effect of 25 μM compound 1 and erastin on intracellular relative glutamate levels in HeLa cells (n = 6). (<b>C</b>) Cytotoxicity assay results of compound 1 incubated with HeLa cells for 12 h, 24 h, 36 h, and 48 h, respectively (n = 6). (<b>D</b>) Cytotoxicity assay results of compound 1 incubated with HeLa cells for 36 h in the presence or absence of 2.5 mM L-cysteine (n = 6). (<b>E</b>) Cytotoxicity assay results of compound 1 and erastin at concentrations of 50, 25, 12.5, 6.25, and 3.13 μM, respectively, after 36 h of incubation (n = 5). (<b>F</b>) The effect of 25 μM compound 1 and erastin on intracellular ROS levels in HeLa cells (n = 4). (<b>G</b>) Cytotoxicity assay results of 5 or 10 mM NAC combined with compound 1 treatment or compound 1 treatment alone after 36 h of incubation (n = 6). (<b>H</b>) Calcein-AM staining results of 5 or 10 mM NAC combined with compound 1 treatment or compound 1 treatment alone after 36 h of incubation. (<b>I</b>) The 2D structure of compound 1. The magnification is 40×. (<b>J</b>) The 2D structure of erastin. The data are presented as mean ± S.D. *, <span class="html-italic">p</span> < 0.05; ***, <span class="html-italic">p</span> < 0.001, “ns” indicates “non-significant.”</p> "> Figure 4
<p>Inhibition of HeLa cell migration by compound 1 at low concentrations. (<b>A</b>) Wound healing images. (<b>B</b>) Quantitative analysis of scratch wounds treated with different concentrations of compound 1 (0.5, 1, 2, 4 μM) and erastin (0.5, 1, 2, 4 μM) for 24 h (n = 4). (<b>C</b>) Transwell migration images. (<b>D</b>) Quantitative analysis of Transwell migration after treatment with compound 1 (4 μM) and erastin (4 μM) for 24 h (n = 4). The data are presented as mean ± S.D. *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01, ***, <span class="html-italic">p</span> < 0.001, “ns” usually indicates “non-significant.”</p> "> Figure 5
<p>Toxic effects of compound 1 on HeLa three-dimensional spheroids. (<b>A</b>) HeLa spheroids were treated with 10 μM erastin or 5 μM and 10 μM compound 1 in a 96-well round-bottom ultra-low-attachment plate. The treatment started on day 0, and images were captured and HeLa spheroid volumes were measured daily from day 0, with six consecutive recordings. (<b>B</b>) Changes in HeLa spheroid volumes over 6 days (n = 5). (<b>C</b>) Proliferation rates of HeLa spheroid volumes over 6 days (n = 5). The data are presented as mean ± S.D. *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01 and ***, <span class="html-italic">p</span> < 0.001.</p> "> Figure 6
<p>Structural analyses of APO, SLC7A11-c1, and SLC7A11-erastin. (<b>A</b>) RMSD of APO, SLC7A11-c1, and SLC7A11-erastin. (<b>B</b>) RMSD of compound 1 and erastin. (<b>C</b>) Radius of gyration of APO, SLC7A11-c1, and SLC7A11-erastin. (<b>D</b>) RMSF of APO, SLC7A11-c1, and SLC7A11-erastin. (<b>E</b>) The 2D structure of SLC7A11. APO, SLC7A11-c1, and SLC7A11-erastin (<b>A</b>–<b>D</b>) are outlined in green, red, and blue lines, respectively.</p> "> Figure 7
<p>Principal component analysis (PCA) of APO, SLC7A11-c1, and SLC7A11-erastin. (<b>A</b>) Essential subspace projection of PC1 vs. PC2 of APO, SLC7A11-c1, and SLC7A11-erastin. The continuous color scale (from blue to white to red) indicates that there are periodic jumps between these conformers throughout the trajectory. (<b>B</b>) Porcupine plots represent the motions captured in PC1 of APO, SLC7A11-c1, and SLC7A11-erastin, color scale from blue to red depict low to high atomic displacements. (<b>C</b>) Line plots represent the degree of motions captured in PC1 for APO, SLC7A11-c1, and SLC7A11-erastin. The solid box in (<b>B</b>) highlights the ligand-binding pocket within the structure.</p> "> Figure 8
<p>The dynamics cross-correlation matrix (DCCM) plots for APO (<b>A</b>,<b>D</b>), SLC7A11-c1 (<b>B</b>,<b>E</b>), and SLC7A11-erastin (<b>C</b>,<b>F</b>). Red (−1) and blue (+1) correspond to correlated and anti-correlated motions, respectively.</p> "> Figure 9
<p>H-bond analysis results. (<b>A</b>) Analysis of hydrogen bond interactions between erastin and SLC7A11. (<b>B</b>) Analysis of hydrogen bond interactions between compound 1 and SLC7A11. (<b>C</b>) H-bond occupancy of each interacting residue in their relevant systems, including SLC7A11-c1, and SLC7A11-erastin.</p> "> Figure 10
<p>Representative conformation of compound 1(orange) and erastin(yellow) bound to SLC7A11. The key residues of SLC7A11 are in green.</p> "> Figure 11
<p>Residue binding energy contribution of SLC7A11-c1 and SLC7A11-erastin. (<b>A</b>) Overall binding free energies of compounds with SLC7A11. (<b>B</b>) Binding energies of compounds to SLC7A11 residues. (<b>C</b>) The positions of residues with high energy contribution bound to erastin. (<b>D</b>) The positions of residues with high energy contribution bound to compound 1. The deeper the color, the higher the energy contribution.</p> ">
Abstract
:1. Introduction
2. Results
2.1. The Discovery of Potential SLC7A11/xCT Axis Inhibitors
2.2. In Vitro Evaluation of the Effect Compound 1 Has on the Oxidative/Antioxidant Balance within HeLa Cells or Similar
2.3. In Vitro Evaluation of the Inhibitory Effects of Compound 1 on HeLa Cell Migration at Low Concentrations
2.4. Assessment of the Toxic Effects of Compound 1 on HeLa Three-Dimensional Spheroids
2.5. Molecular Dynamics Simulations
2.5.1. Structural Mobility and Compactness of SLC7A11 Systems
2.5.2. Collective Motions of SLC7A11 Systems
2.5.3. Local Correlation Motion Patterns
2.5.4. Hydrogen Bond Analysis
2.5.5. K-Means Clustering Analysis
2.5.6. Free Energy Calculations
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Molecular Docking
4.3. Molecular Dynamics Simulations
4.4. Cell Culture
4.5. Cell Viability Assay
4.6. Cell Scratch Assay
4.7. Migration Assay
4.8. ROS Assay
4.9. Toxicity Assessment of 3D Tumor Spheroids
4.10. Calcein-AM Staining
4.11. Intracellular GSH Level Assay
4.12. Intracellular Glutamine Level Assay
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Yue, J.; Yin, Y.; Feng, X.; Xu, J.; Li, Y.; Li, T.; Liang, S.; He, X.; Liu, Z.; Wang, Y. Discovery of the Inhibitor Targeting the SLC7A11/xCT Axis through In Silico and In Vitro Experiments. Int. J. Mol. Sci. 2024, 25, 8284. https://doi.org/10.3390/ijms25158284
Yue J, Yin Y, Feng X, Xu J, Li Y, Li T, Liang S, He X, Liu Z, Wang Y. Discovery of the Inhibitor Targeting the SLC7A11/xCT Axis through In Silico and In Vitro Experiments. International Journal of Molecular Sciences. 2024; 25(15):8284. https://doi.org/10.3390/ijms25158284
Chicago/Turabian StyleYue, Jianda, Yekui Yin, Xujun Feng, Jiawei Xu, Yaqi Li, Tingting Li, Songping Liang, Xiao He, Zhonghua Liu, and Ying Wang. 2024. "Discovery of the Inhibitor Targeting the SLC7A11/xCT Axis through In Silico and In Vitro Experiments" International Journal of Molecular Sciences 25, no. 15: 8284. https://doi.org/10.3390/ijms25158284