Dual functional piscidin 1: a natural chelating ligand for ⁶⁴Cu and an anti-cancer peptide

Objectives: Piscidins (PCD) were the first antimicrobial peptides discovered in vertebrates. Our recent investigation confirmed that one of the family members, PCD-1, possesses two unique properties: it shows decent cancer cell killing efficacy (IC₅₀: 4.8 µM for MDA-MB-231 cells), and it bears a Cu²⁺-binding motif inside its structure. In this study, we hope to increase the cancer cell selectivity and accumulation of PCD-1, and use PCD-1 as a binding ligand for ⁶⁴Cu to achieve chelator-free radiolabeling of other tumor-targeting molecules. The goal is to use this ⁶⁴Cu-labeled PCD-1 derivative as both cancer detection and therapeutic agent for in vivo applications.

Methods: A fused peptide containing PCD-1 sequence and another tumor-targeting peptide F3 (against nucleolin) was synthesized (gapped by poly-glycine) and named PCD-1-F3. The cytotoxicity of PCD-1, ^natCu-PCD-1 (^natCu stands for non-radioactive copper), PCD-1-F3, and ^natCu-PCD-1-F3 was measured in MDA-MB-231 cells and L929 fibroblasts by the CCK-8 assay. Fluorescein was site-specifically introduced onto PCD-1-F3 and PCD-1 to study their interaction with cancerous and normal cells by flow cytometry and confocal fluorescence microscopy. Both PCD-1-F3 and PCD-1 were radiolabeled with ⁶⁴Cu and the product stability was subsequently assessed by HPLC. ⁶⁴Cu-PCD-1-F3 and ⁶⁴Cu-PCD-1 was injected into MDA-MB-231 tumor bearing mice with serial PET imaging being conducted to examine their pharmacokinetics and tumor accumulating behavior. Biodistribution studies were also carried out to correlate with PET findings, and histological analysis was used to determine the sub-tumor distribution profile of these PCD-1 derivatives. In the end, a treatment study (n = 5 per group) was done in MDA-MB-231 tumor-bearing mice by using ^natCu-PCD-1-F3 (5 mg/kg intravenously every other day for four weeks).

Results: Both PCD-1 and PCD-1-F3 showed strong binding affinity for copper, and ^natCu-PCD-1-F3 had the most potent killing efficacy for MDA-MB-231 cells (IC₅₀: 508 nM). None of these PCD-1 derivatives showed significant impact for L929 growth and status. Flow cytometry and fluorescence microscopy both confirmed that the internalization of fluorescein-PCD-1-F3 into MDA-MB-231 cells was significantly stronger than that of fluorescein-PCD-1, and these PCD-1 derivatives showed clear accumulation in mitochondria. Judged from HPLC analysis, ⁶⁴Cu-PCD-1-F3 was sufficiently stable to be used in vivo (<15% ⁶⁴Cu detached from it after a 4 h incubation with mouse serum). ⁶⁴Cu-PCD-1-F3 (~0.7 GBq/mg) had a circulation half-life of ~ 15 min in mice (based on dynamic PET scans) and possessed a more potent uptake in MDA-MB-231 (4.1 ± 0.9 %ID/g at 4 h post-injection) than that from ⁶⁴Cu-PCD-1 (1.3 ± 0.8 %ID/g at 4 h p.i.), and this uptake could be successfully blocked (to 1.1 ± 0.4 %ID/g at 4 h p.i.) by co-injection of excessive amount of F3 peptide (10 mg/kg). Histological analysis of MDA-MB-231 tumors indicated that PCD-1-F3 indeed bound to nucleolin (overexpressed in tumor cell membrane) from tumors. At the treatment dose, ^natCu-PCD-1-F3 could significant delay the growth of tumor burden.

Conclusion: Our study confirmed the uniqueness of PCD-1 as an anti-cancer agent and a natural “chelator” for copper (radioactive or non-radioactive). With the attachment of a tumor-targeting molecule (e.g. F3 in this report), PCD-1 showed satisfactory tumor detection/therapy properties. This report can encourage us to seek peptides with different metal-binding motifs as labeling agents for various radiometals, especially for those currently without a preferrable chelating ligand (e.g. Mn). Research Support: This work is supported, in part, by the NIH/NCI P01 CA085878, and the National Natural Science Foundation of China (81201696)

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