(F-213) Discovery of a highly potent oncolytic peptide via structure-activity relationship analysis of CKS1 mutants

Introduction:: Oncolytic peptides are cationic, amphipathic, and selectively target cancer cells via electrostatic interactions, causing membrane pore formation and cell death. Oncolytic peptides offer key advantages over traditional chemotherapeutics in terms of safety and drug resistance. Cancer cell surfaces are more negatively charged than normal cell surfaces, in part due to the production of large quantities of lactate. Unlike chemotherapeutics, oncolytic peptides do not target proliferating cells and therefore do not induce side effects such as hair loss and anemia caused by damage to stem cells. Moreover, the half-lives of peptides are generally short in the bloodstream, and they pose a minimal risk of inducing systemic side effects. While acquired genetic and epigenetic alterations in cancer cells can lead to resistance to chemotherapy, oncolytic peptides do not target a specific biomarker or signaling pathway. Consequently, the risk of developing drug resistance to oncolytic peptides is low.

We previously discovered a novel oncolytic peptide, CKS1, which is composed of an α-helical region and a loop region. CKS1 elicits cytotoxic effects against multiple cancer cell lines but not against normal endothelial cells or fibroblasts. Here, we sought to engineer a peptide with a higher potency than existing oncolytic peptides and to investigate the biochemical properties important for potency. To this end, we designed multiple peptides by introducing mutations to CKS1 and performed a structure-activity relationship (SAR) analysis. Finally, we evaluated the efficacy of the most potent peptide to treat cancer in vivo.




Materials and Methods:: We first designed 23 peptides (NF4-26) by introducing mutations to CKS1 to alter the charge, hydrophobicity, and length of the α-helical region, the parameters we postulated to be important based on the mechanism of action. We treated 4T1 murine triple-negative breast cancer cells with 5 μM to 200 μM of each CKS1 mutant. We then performed a lactate dehydrogenase (LDH) assay to measure the concentration of extracellular LDH, an indicator of necrotic cell death. From this data, we calculated each peptide’s half maximal effective concentration (EC50). To identify the properties that are important for oncolytic potency, we plotted the EC50s against the charge, hydrophobicity, and length of the α-helix. We noted that at 5 μM, NF10 exhibited the highest oncolytic activity out of the 23 peptides. However, NF10 contained a highly reactive cysteine residue, which we sought to replace with a less reactive amino acid to improve the ease of production and long-term storage of the peptide. Hence, we designed four additional peptides (NF27-30) based on the structure of NF10. Based on an LDH assay of 4T1 cells treated with each NF10 mutant, we identified NF27 as the highest potency peptide lacking cysteine. Furthermore, we performed an LDH assay on NF27-treated CT26 murine colorectal carcinoma cells, as well as on human umbilical vein endothelial cells (HUVECs) to verify that NF27 exhibits low cytotoxicity against normal mammalian cells. Lastly, we performed intratumoral injection of NF27 in immunocompetent mice with established CT26 tumors to verify the potency of NF27 in vivo.




Results, Conclusions, and Discussions:: Results:

Our SAR analysis of CKS1 mutants revealed a weak negative correlation between EC50 and hydrophobicity (Fig. 1). We did not find any appreciable correlation between EC50 and charge. Additionally, increasing the length of the peptide did not meaningfully improve the oncolytic potency.

We identified NF27 as the highest potency cysteine-lacking mutant, possessing an EC50 of 5.0 µM and 0.96 µM for 4T1 and CT26 cells, respectively. Furthermore, we confirmed that NF27 was minimally cytotoxic against HUVECs, with an EC50 of 94.6 µM. Lastly, intratumoral injection of NF27 into tumor-bearing immunocompetent mice effectively inhibited the growth of CT26 tumors and induced complete tumor regression in 60% of the mice when treated with 40 mg/kg of the peptide (Fig. 2).



Discussion:

Our in vivo study demonstrated that NF27 has the potential to serve as an effective treatment against tumors that are endoscopically or transdermally accessible. Due to its mechanism of action, NF27 has the potential to be used in treatment applications for a wide range of cancer types. Specifically, NF27 could be used as a drug in neoadjuvant therapy for skin and breast cancers. Moreover, for cancer types such as rectal cancer, for which surgery should be avoided, combination therapy using NF27 with chemotherapy or radiotherapy may potentially eliminate the need for any surgical intervention. To the best of our knowledge, out of the oncolytic peptides being developed for clinical application, NF27 has the widest therapeutic window and may be the optimal peptide for treating cancer.



Conclusion:

We engineered a novel oncolytic peptide, NF27, with higher potency compared to CKS1 and other existing oncolytic peptides. We showed that high hydrophobicity is important for oncolytic potency, while the charge and the length of the helix has less impact on the oncolytic activity.

Supported by NIH grant R01CA138264.


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