Stapled peptide PROTAC induced significantly greater anti-PD-L1 effects than inhibitor in human cervical cancer cells
Yu-Ying Shi1,2,3, An-Jin Wang1,2,3, Xue-Lian Liu1,2,3
1Department of Gynecological Oncology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China.
Introduction:
Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that target immune checkpoints that suppress immune cell activity. Low efficiency and high resistance are currently the main barriers to their clinical application. As a representative technology of targeted protein degradation, proteolysis-targeting chimeras (PROTACs) are considered to have potential for addressing these limitations.
Methods:
We synthesized a stapled peptide-based PROTAC (SP-PROTAC) that specifically targeted palmitoyltransferase ZDHHC3 and resulted in the decrease of PD-L1 in human cervical cancer cell lines. Flow cytometry, confocal microscopy, protein immunoblotting, Cellular Thermal Shift Assay (CETSA), and MTT assay analyses were conducted to evaluate the effects of the designed peptide and verify its safety in human cells.
Results:
In cervical cancer celllines C33A and HeLa, the stapled peptide strongly downregulated PD-L1 to < 50% of baseline level at 0.1 μM. DHHC3 expression decreased in both dosedependentand time-dependent manners. MG132, the proteasome inhibitor, can alleviate the SP-PROTAC mediated degradation of PD-L1 in human cancer cells. In a co-culture model of C33A and T cells, treatment with the peptide induced IFN-γ and TNF-α release in a dose-dependent manner by degrading PD-L1. These effects were more significant than that of the PD-L1 inhibitor, BMS-8.
Conclusions:
Cells treated with 0.1 μM of SP-PROTAC or BMS-8 for 4 h revealed that the stapled peptide decreased PD-L1 more effectively than BMS-8. DHHC3-targeting SP-PROTAC decreased PD-L1 in human cervical cancer more effectively than the inhibitor BMS-8.
Insights
A novel stapled peptide-based PROTAC effectively degrades PD-L1 in cervical cancer cells, enhancing immune responses. This targeted protein degradation approach shows promise for overcoming resistance to immune checkpoint inhibitors.
Area of Science:
- Oncology
- Immunology
- Biochemistry
Background:
- Immune checkpoint inhibitors (ICIs) face challenges of low efficiency and high resistance in clinical applications.
- Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) offers a potential strategy to overcome ICI limitations.
Purpose of the Study:
- To synthesize and evaluate a stapled peptide-based PROTAC (SP-PROTAC) targeting ZDHHC3 for PD-L1 degradation in cervical cancer.
- To assess the efficacy and safety of the SP-PROTAC in human cervical cancer cell lines and a co-culture model.
Main Methods:
- Synthesis of SP-PROTAC targeting ZDHHC3.
- Evaluation using flow cytometry, confocal microscopy, protein immunoblotting, CETSA, and MTT assays.
- Co-culture model with C33A and T cells to assess immune response.
Main Results:
- SP-PROTAC significantly downregulated PD-L1 (<50% of baseline) in C33A and HeLa cells at 0.1 μM.
- DHHC3 expression decreased in a dose- and time-dependent manner.
- SP-PROTAC treatment induced IFN-γ and TNF-α release, outperforming the PD-L1 inhibitor BMS-8.
Conclusions:
- DHHC3-targeting SP-PROTAC is a potent strategy for decreasing PD-L1 in human cervical cancer.
- The SP-PROTAC demonstrated superior efficacy in PD-L1 reduction compared to the PD-L1 inhibitor BMS-8.
- This approach holds potential for improving cancer immunotherapy by overcoming resistance mechanisms.
