Copper(II) Cyclopeptides with High ROS-Mediated Cytotoxicity

Abstract

Cu(II) coordination complexes are emerging as promising anticancer agents due to their ability to induce oxidative stress through reactive oxygen species (ROS) generation. In this study, we synthesized and characterized two novel Cu(II) metallopeptide systems, 1/Cu(II) and 2/Cu(II), derived from the oligocationic bipyridyl cyclopeptides 1 and 2, and designed to enhance the transport of Cu(II) into cells and increase ROS levels. Spectroscopic and electrochemical analyses confirmed the formation of stable metallopeptide species in aqueous media. Inductively coupled plasma mass spectrometry (ICP-MS) studies demonstrated that both metallopeptides significantly increase intracellular Cu(II) accumulation in NCI/ADR-RES cancer cells, highlighting their role as efficient Cu(II) transporters. Additionally, ROS generation assays revealed that 1/Cu(II) induces a substantial increase in intracellular ROS levels, supporting the hypothesis of oxidative stress-induced cytotoxicity. Cell-viability assays further confirmed that both 1/Cu(II) and 2/Cu(II) exhibit strong anticancer activity in a number of cancer cell lines, with IC₅₀ values significantly lower than those of their free cyclopeptide counterparts or Cu(II) alone, showing an order of activity higher than that of cisplatin. Finally, molecular modeling studies provided further insights into the structural stability and coordination environment of Cu(II) within the metallopeptide complexes. These findings suggest that these Cu(II) cyclometallopeptide systems hold potential as novel metal-based therapeutic agents, leveraging Cu(II) transport and ROS increase as key strategies for cancer treatment.

1. Solid-Phase Peptide Synthesis of Oligocationic Bipyridyl Cyclopeptides cyclo-(βAlaBpy-Arg₃)₂ (1) and cyclo-(βAlaBpy-Arg₃)₃ (2)

1

(a) Normalized emission spectrum of a 2 μM solution of cyclopeptide 1 in 1 mM phosphate buffer, 10 mM NaCl, pH 6.5, thick black line, and spectra of the same solution in the presence of increasing concentrations of Cu­(II) ions (progressively lighter shades of gray). Inset: titration profile showing the emission at 410 nm (λ_exc = 308 nm) of three independent fluorometric titrations and best fit according to the 1:2 model (cyclopeptide:metal) in DynaFit. , (b) Same as in (a), but with cyclopeptide 2 and best fit according to a simplified model, including 1:1 and 2:1 species (see the text for details).

2

Top: UV–vis spectra of cyclopeptides 1 (a) and 2 (b), in 1 mM phosphate buffer, 10 mM NaCl, pH 6.5, before (dashed lines) and after (continuous lines) the addition of Cu­(II) ions (1/Cu­(II): 3 equiv; 2/Cu­(II): 5 equiv). Bottom: CD spectra of 10 μM solutions of 1 (c) and 2 (d), in 1 mM phosphate buffer, 10 mM NaCl, pH 6.5, before (dashed lines) and after (continuous lines) the addition of Cu­(II) ions (1/Cu­(II): 3 equiv; 2/Cu­(II): 5 equiv) in 1 mM phosphate buffer, 10 mM NaCl, pH 6.5. Spectra in parts b and c on the right are in the same scale as (a) in part b on the left.

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DFT-optimized model of the most stable structure of the 1/Cu­(II) metallopeptide system based on experimental data.

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Time course of ascorbate oxidation monitored by the absorbance at 265 nm. The reaction was started by the addition of free Cu­(II) (circles) ions or the preformed Cu­(II) metallopeptide system to a solution of ascorbate in 100 mM HEPES at pH 7.4 after 10 min. The equivalents of each ligand (cyclopeptide or 5DMB) in the medium were fixed to 1.1 (dashed lines) or 2.1 (solid lines) with respect to the concentration of Cu­(II) ions. The final concentration of Cu­(II) ions and ascorbate were 300 nM and 100 μM, respectively.

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Cyclic voltammetry of 1 mM Cu­(II) metallopeptide systems 1/Cu­(II) (3 equiv of Cu^II) and 2/Cu­(II) (5 equiv of Cu^II) in an aqueous solution of 50 mM AMPD/HCl pH 5 + 0.1 M NaClO₄, on a glassy carbon disk (diameter 1 mm). Scan rate 0.5 V·s^–1. (a) Cyclopeptide 1 (black line) and metallopeptide system 1/Cu­(II) (blue line); (b) cyclopeptide 2 (black line) and metallopeptide system 2/Cu­(II) (blue line).