Periodic Reporting for period 1 - pepRu4PACT (Ruthenium Peptide Bioconjugates for Photoactivated Chemotherapy)
Reporting period: 2023-03-01 to 2025-02-28
Conventional anticancer therapies suffer from severe side effects due to their lack of selectivity, often damaging healthy tissues alongside tumours. A promising strategy to overcome this limitation is photoactivated chemotherapy (PACT), in which prodrugs remain inactive until triggered by light at the tumour site, thereby achieving spatial and temporal control over toxicity. Metal complexes, particularly polypyridyl-ruthenium (Ru(II)) compounds, are well-suited for this approach due to their ability to undergo ligand exchange upon irradiation. However, challenges remain, including insufficient light penetration, reliance on oxygen-dependent mechanisms, and non-selective cellular uptake. To address these issues, this proposal introduces a novel multimetallic Ru(II)-peptide bioconjugate strategy that integrates the benefits of metal-based PACT with the biological activity of anticancer peptides (ACPs).
The core innovation of pepRu4PACT lies in coordinating one or multiple Ru(II) complexes to methionine residues of biologically active peptides. This dual-component system enables mutual caging in the dark, minimizing off-target toxicity, while light activation cleaves the Ru-thioether bond(s), simultaneously releasing both cytotoxic components, i.e. the ruthenium fragment and the ACP. By leveraging red or near-infrared light, which penetrates more deeply into tissues, this approach ensures effective activation even in hypoxic tumour environments where conventional photodynamic therapy (PDT) is less effective. Additionally, peptide-based targeting enhances selective cellular uptake through receptor-mediated pathways, further increasing therapeutic precision.
In order to achieve our general goal, intermediate objectives had to be established first: (a) tuning the coordination sphere of Ru(II)-polypyridyl complexes bound to thioethers to achieve light activation in the red or near-IR spectral region; (b) synthesize and characterize these Ru(II)-peptide conjugates; (c) validate their activation by red/near-infrared light; and (c) assess their combined phototoxic effects in cancer models. This work will provide fundamental insights into peptide-metal interactions while paving the way for more effective, side-effect-free chemotherapies.
The core innovation of pepRu4PACT lies in coordinating one or multiple Ru(II) complexes to methionine residues of biologically active peptides. This dual-component system enables mutual caging in the dark, minimizing off-target toxicity, while light activation cleaves the Ru-thioether bond(s), simultaneously releasing both cytotoxic components, i.e. the ruthenium fragment and the ACP. By leveraging red or near-infrared light, which penetrates more deeply into tissues, this approach ensures effective activation even in hypoxic tumour environments where conventional photodynamic therapy (PDT) is less effective. Additionally, peptide-based targeting enhances selective cellular uptake through receptor-mediated pathways, further increasing therapeutic precision.
In order to achieve our general goal, intermediate objectives had to be established first: (a) tuning the coordination sphere of Ru(II)-polypyridyl complexes bound to thioethers to achieve light activation in the red or near-IR spectral region; (b) synthesize and characterize these Ru(II)-peptide conjugates; (c) validate their activation by red/near-infrared light; and (c) assess their combined phototoxic effects in cancer models. This work will provide fundamental insights into peptide-metal interactions while paving the way for more effective, side-effect-free chemotherapies.
The work performed has been divided into working packages (WPs). WP1 consisted in the design of a new family of thioether-ruthenium(II) polypyridyl complexes that can be activated by ligand photosubstitution using red light irradiation. We used amide groups and extended π-conjugation to fine tune the excited state energies and shift light absorption towards the red region. Interestingly, we did not find the expected correlation between the observed photochemical properties of the complexes and the accepted electronic description of the process. In order to elucidate the mechanism of photosubstitution, we performed, in collaboration with theoreticians from the group, advanced computational simulations which provided valuable insights in the structure-activity relationship of the complexes. These results have been described in a combined manuscript that is currently under revisions.
In WP2, we developed a Ruthenium-peptide conjugate incorporating the anticancer peptide MPX from the Mastoparan family. MPX exhibits its cytotoxic effect through membranolytic activity, driven by its amphipathic nature when folding into an α-helical secondary structure. In this part of the project, we demonstrated that coordination with a Ru(II) complex effectively inhibits the peptide’s membrane-disrupting properties in the dark, thereby reducing its inherent toxicity. Upon light irradiation, the peptide’s original activity is restored. To optimize this modulation strategy, we explored various structural modifications, with designs that disrupt α-helix formation proving the most effective in inhibiting activity in the absence of light. These results are currently being written in the form of another manuscript which will be submitted in 2025.
In the final part of the project, WP3, we focused on investigating the self-aggregating properties of a short peptide coordinated to a Ru complex, both in the dark and under light irradiation. This approach aimed to exploit red light to control the formation of aggregates. For this project, we collaborated with Prof. Marchesan’s group from Trieste University, which provided their self-aggregating small peptide containing a methionine residue in its sequence, enabling effective Ru coordination. We synthesized and characterized two distinct Ru-peptide conjugates. Although we successfully demonstrated the efficient red light-induced dissociation of the Ru complexes from the peptides, restoring the original self-aggregating properties, we could not achieve any aggregation. This could be attributed to the presence of Ru fragments in solution, which probably interfere with the aggregation process.
In WP2, we developed a Ruthenium-peptide conjugate incorporating the anticancer peptide MPX from the Mastoparan family. MPX exhibits its cytotoxic effect through membranolytic activity, driven by its amphipathic nature when folding into an α-helical secondary structure. In this part of the project, we demonstrated that coordination with a Ru(II) complex effectively inhibits the peptide’s membrane-disrupting properties in the dark, thereby reducing its inherent toxicity. Upon light irradiation, the peptide’s original activity is restored. To optimize this modulation strategy, we explored various structural modifications, with designs that disrupt α-helix formation proving the most effective in inhibiting activity in the absence of light. These results are currently being written in the form of another manuscript which will be submitted in 2025.
In the final part of the project, WP3, we focused on investigating the self-aggregating properties of a short peptide coordinated to a Ru complex, both in the dark and under light irradiation. This approach aimed to exploit red light to control the formation of aggregates. For this project, we collaborated with Prof. Marchesan’s group from Trieste University, which provided their self-aggregating small peptide containing a methionine residue in its sequence, enabling effective Ru coordination. We synthesized and characterized two distinct Ru-peptide conjugates. Although we successfully demonstrated the efficient red light-induced dissociation of the Ru complexes from the peptides, restoring the original self-aggregating properties, we could not achieve any aggregation. This could be attributed to the presence of Ru fragments in solution, which probably interfere with the aggregation process.
This research presents significant advancements beyond the current state of the art, particularly in the development and application of Ru(II)-polypyridyl-peptide conjugates. A major innovation of this work is the improved ability to rationally design the coordination environment of Ru(II) complexes bound to thioethers, allowing fine-tuning of their light-induced photosubstitution properties in the red spectral region. At the same time, we demonstrated that coordinating Ru complexes to anticancer peptides, without altering their natural sequence, effectively inhibits their cytotoxic properties in the dark, while light irradiation restores the peptide’s original structure and activity. Additionally, we showed that multiple Ru(II) complexes can be coordinated to a single peptide through methionine residues, opening new possibilities for functionalization and for modulating the chemical and physical properties of the peptide. Another key aspect of this work is the ability to harness the synergistic cytotoxic activity of both the Ru complexes and the peptide, enhancing their overall therapeutic potential. To further improve the suppression of peptide toxicity in the dark, we successfully developed multimetallic Ru-peptide conjugates and cyclic constructs that progressively disrupted the peptide’s original secondary structure, refining control over its activity. We finally demonstrated that next to using several ruthenium photocages bound to the same peptide to disrupt its supramolecular properties in the dark, it is the ability of the ruthenium photocage to modify the shape of the peptide, and hence its ability to make holes in membranes, that matters most. This result was unexpected and is part of the fundamental discoveries made in this project.