Poster T10 - Identification of highly potent oncolytic peptides NF27 via structure-activity relationship analysis

Introduction: Oncolytic peptides induce cell death in cancer cells without significant toxicity to healthy cells. Oncolytic peptides have an amphipathic, helical structure that is positively charged on one side and neutral on the other. This allows the peptides to damage the cell membrane due to a detergent-like effect. The specificity of oncolytic peptides towards cancer cells stems from the difference in cell surface charge between cancer cells and healthy cells. Most healthy cells maintain a neutrally charged cell surface. In contrast, due to multiple factors, such as the exposure of phosphatidylserine, elevated glycosylation of surface proteins, and the overproduction of lactate, the cell surface of cancer cells is typically negatively charged. This allows interactions with the positively charged oncolytic peptides. Furthermore, the cell membrane of cancer cells is usually more fluid than healthy cells, which increases the vulnerability to damage in the cell membrane. We previously reported that CKS1, a peptide originally identified as an anti-angiogenic peptide, possesses oncolytic activity. Although it is widely acknowledged that amphipathic and cationic structures are indispensable for oncolytic activities, the precise structure-activity relationship (SAR) that determines the potency of oncolytic peptides remains unclear. CKS1 was not designed as an oncolytic peptide and therefore has room for optimization. Here, we produced and analyzed multiple peptides derived from CKS1 to understand the underlying SAR of oncolytic peptides and to optimize the structure of CKS1 to improve its cancer cell lysis activity. We further assessed the therapeutic potential of NF27, the most potent peptide obtained in the process.

Materials and

Methods: We designed 39 peptides and assessed their oncolytic activities against 4T1 murine triple-negative breast cancer cells by LDH cytotoxicity assay. We identified NF27 as the most potent peptide and evaluated the cytotoxicity against multiple murine and human cell lines by LDH cytotoxicity assay and alamarBlue cell viability assay. We evaluated the efficacy of NF27 to induce cell death in 59 human cancer cell lines derived from 9 different cancer types by NCI60 screening. To identify the mechanism of NF27-induced cell death, we observed the morphology of cancer cells treated with NF27 by live-cell imaging. Furthermore, we investigated the localization of NF27 using fluorescently tagged peptides. We utilized caspase-3 assay and JC-1 imaging to assess the effect of NF27 on mitochondria and the involvement of the apoptotic pathway in the cell death induced by NF27. To assess the toxicity of NF27, we incubated NF27 with human red blood cells and evaluated the hemolysis capability of NF27. Finally, we assessed the oncolytic capability in vivo in CT26 murine colorectal cancer model and HT29 xenograft colorectal cancer model.

Results, Conclusions, and Discussions: We identified multiple peptides with higher oncolytic potency compared to CKS1. NF27, which was the most potent oncolytic peptide, was nearly 15-fold more potent against 4T1 cells compared to the original CKS1. The EC50 measured by LDH assay was in the range of 5 µM to 10 µM in multiple cancer cell lines, while the EC50 was approximately 150 µM for normal human endothelial cells and fibroblasts. We unexpectedly discovered NF22, a peptide that completely lost its oncolytic activity. By comparing the structures of the peptides, we identified the regions and the characteristics important for the oncolytic activity. Among the 59 cell lines tested in the NCI60 screening, NF27 demonstrated an IC50 of below 10 µM in 58 cell lines. The one cell line that was relatively non-responsive had an IC50 of 26 µM which is still significantly lower than the IC50 for healthy cells. When treated on cancer cells, NF27 quickly ruptured the cell membrane and localized in specific organelles within the cell. NF27 also diminished mitochondrial membrane potential and activated caspase-3, indicating that NF27 induces cell death via two mechanisms, cell membrane rupture and activation of the apoptotic pathway. NF27 did not show hemolysis even at a concentration of over 1000 µM. In both CT26 and HT29 tumor models, NF27 demonstrated significant inhibition of tumor growth. Furthermore, NF27 eradicated 8 out of 10 tumors and 9 out of 11 tumors in CT26 tumor model and HT29 tumor model, respectively. In conclusion, our SAR study contributed to the understanding of how oncolytic peptides work and led to the discovery of a highly potent oncolytic peptide, NF27. NF27 showed cytotoxic activity against multiple cancer cell lines regardless of their origin. NF27 demonstrated anti-tumoral activity in vivo and has the potential to become a novel treatment for cancer.

Acknowledgements (Optional):