N-Degron-Based PROTAC Targeting PLK1: A Potential Therapeutic Strategy for Cervical Cancer
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemistry
2.1.1. General Methods
2.1.2. Peptide Synthesis
2.2. MALDI-TOF Mass Spectrometry Analysis of Synthesized Peptides
- NC1: calculated mass for C116H216N57O23P, m/z 2806.72; observed m/z 2807.82 [M + H]+.
- Arg1-K(biotin): calculated mass for C22H41N9O4S, m/z 527.30; observed m/z 528.38 [M + H]+.
- Arg4-K(biotin): calculated mass for C40H77N21O7S, m/z 995.60; observed m/z 996.71 [M + H]+.
- Arg8-K(biotin): calculated mass for C64H125N37O11S, m/z 1620.01; observed m/z 1621.22 [M + H]+.
- Arg10-K(biotin): calculated mass for C76H149N45O13S, m/z 932.21; observed m/z 1933.522 [M + H]+.
- Arg12-K(biotin): calculated mass for C88H173N53O15S, m/z 2244.41; observed m/z 2245.569 [M + H]+.
- Ala4Arg8-ahx-4j: calculated mass for C104H188N45O23S, m/z 2467.87; observed m/z 2468.97 [M + H]+.
2.3. Pull-Down Assay
2.4. Protein Expression and Purification
2.5. Crystallization, Data Collection, Structure Solution, and Refinement
2.6. Isothermal Titration Calorimetry (ITC)
2.7. Compound Treatment and Immunoblotting
2.8. In Vivo Antitumor Activity
2.9. Cell Viability Assay
2.10. Morphological Assessment of NC1 Treated Cancer Cells
2.11. Cellular Uptake of FITC-Conjugated Compounds
2.12. Analysis of G2/M Phase Cell Cycle Arrest by Flow Cytometry
2.13. Evaluation of Cancer Cell Apoptosis Using Fluorescence Apoptosis Assay
2.14. Apoptosis Detection by Flow Cytometry
2.15. Statistical Analysis
3. Results
3.1. Design and Synthesis of PROTAC, NC1
3.2. Validation of Arginine Recognition by UBR1
3.3. Recognition of UBR Box by NC1
3.4. Evaluation of PLK1 PBD Binding Affinity
3.5. PLK1 Degradation
3.6. Complex Crystal Structure of PROTAC with PLK1 PBD
3.7. Cell Viability Assay and Cancer Cell Morphology Analysis
3.8. Cell Penetrating Ability of FITC-Conjugated NC1 in Cancer Cells
3.9. G2/M Phase Cell Cycle Arrest Using a Fluorescence-Activated Cell Sorter
3.10. Apoptosis Effects of NC1 on Cancer Cells
3.11. Anti-Tumorigenic Effect of NC1 on Tumor-Bearing Mice
4. Discussion and Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gunasekaran, P.; Shin, S.C.; Hwang, Y.S.; Lee, J.; La, Y.K.; Yim, M.S.; Kim, H.N.; Kim, T.W.; Yang, E.; Lee, S.J.; et al. N-Degron-Based PROTAC Targeting PLK1: A Potential Therapeutic Strategy for Cervical Cancer. Pharmaceutics 2025, 17, 1027. https://doi.org/10.3390/pharmaceutics17081027
Gunasekaran P, Shin SC, Hwang YS, Lee J, La YK, Yim MS, Kim HN, Kim TW, Yang E, Lee SJ, et al. N-Degron-Based PROTAC Targeting PLK1: A Potential Therapeutic Strategy for Cervical Cancer. Pharmaceutics. 2025; 17(8):1027. https://doi.org/10.3390/pharmaceutics17081027
Chicago/Turabian StyleGunasekaran, Pethaiah, Sang Chul Shin, Yeon Sil Hwang, Jihyeon Lee, Yeo Kyung La, Min Su Yim, Hak Nam Kim, Tae Wan Kim, Eunjung Yang, Soo Jae Lee, and et al. 2025. "N-Degron-Based PROTAC Targeting PLK1: A Potential Therapeutic Strategy for Cervical Cancer" Pharmaceutics 17, no. 8: 1027. https://doi.org/10.3390/pharmaceutics17081027
APA StyleGunasekaran, P., Shin, S. C., Hwang, Y. S., Lee, J., La, Y. K., Yim, M. S., Kim, H. N., Kim, T. W., Yang, E., Lee, S. J., Yoon, J. M., Kim, E. E., Jeon, S., Ryu, E. K., & Bang, J. K. (2025). N-Degron-Based PROTAC Targeting PLK1: A Potential Therapeutic Strategy for Cervical Cancer. Pharmaceutics, 17(8), 1027. https://doi.org/10.3390/pharmaceutics17081027