The Discovery of a New and Effective Series of Anti-Cancer Agents Acting through Metal Sequestration and Reactive Oxygen Generation

Cancer cells exhibit constitutive oxidative stress due to overproduction of reactive oxygen species (ROS), which is closely linked to their metabolic rewiring and oncogenic transformation.
This oxidative stress is intricately connected to metal homeostasis, particularly redox-active metals, for these reasons the development of metal-based anticancer drugs has shown promise, but their efficacy remains a significant challenge due to various factors such as toxicity, resistance, and limited clinical success. Ongoing research and innovative approaches are crucial to overcoming these challenges and realizing the full potential of these therapies.
The main scope of this thesis was to focus on the development of novel anti-cancer drugs to target oxidative stress (ROS) through metal chelation as a key stress pathway in cancer cells. Our primary focus was to develop methods to exploit oxidative stress to induce preferential and selective cytotoxicity against cancer cells but with greater safety to normal cells.
In this study we developed three potential drug classes [termed Pyrimindylhydrazone (PH, series one), Terminal amines (series two) and pincer (series three)] which may coordinate metal ions in a similar geometry to the well characterised thiosemicarbazone (TSC) Dp44mT. We replaced the sulfur (S) atom of the TSC with a nitrogen that was part of a pyrimidine ring to alter the nature of the metal-sulfur interaction that was a key component of the TSC- metal association in our series one compounds (Akladios et al., 2016b; Santoro et al., 2019). Since it is reported that a key site of action for TSC is the lysosome, we then investigated the potential for directing drugs to acidic cell vesicles through the addition of an amine terminus (series two compounds) and, finally, investigated the impact of expanding the number of metal coordination sites on the ligand to create a more lipophilic complex (series three compounds).
Our Pyrimindylhydrazone (PH, series one) compounds demonstrated potency against an ovarian cancer (A2780) cell line (IC50 < 0.5 µM), followed by a slightly lesser cytotoxicity against melanoma (MelRM) cells (IC50 < 1.5 µM) and a further reduction in potency against pancreatic cancer cells (MiaPaca-2) with IC50 < 4.3 µM. The PH exhibited extremely low toxicity (no effect at 25 µM) against normal (non-cancerous) cells (MRC-5) resulting in high safety profiles.
The following key points were demonstrated in this study:
• In the absence of metal ion supplementation, drug administration alone in cells showed promising cytotoxicity against cancer cells but was safe against normal cells even at the highest concentration studied (> 25 µM).
• Iron (Fe(III)) or zinc (Zn(II))- supplementation either decreased drug efficacy or cytotoxicity remained unchanged.
• Copper (Cu(II)) supplementation slightly improved or did not alter drug cytotoxicity, in contrast to the observations related to TSC and Elesclomol.
• Ligand size or bulkiness reduced efficacy.
Our series two compounds (terminal amine) employed the chloroquine (CQ) concept utilising an amine terminus to have the drugs charge trapped into the acidic vesicles of cells, these vesicles including the lysosome. The cytotoxicity results of these drugs demonstrated improved potency against the three cancer cell lines studied but also increases in toxicity to the normal cells (MRC-5) resulting in smaller therapeutic windows than our series one compounds.
The study of our series two compounds suggested that the addition of an amine terminus to the ligand does direct the ligand to the acidic vesicles and results in generally increased potency against cancer cells. This, however, came at the expense of increased toxicity against non-cancerous cells. We felt that these data were suggesting that the increased lysosomal population and size within cancerous cells was driving uptake into vesicles of this type within the cancer cell – the size and population of lysosomes being a differentiating factor between cancerous and non-cancerous cells. We further hypothesised that the implication of charge trapping was to direct the drug to the lysosomes of the non-cancerous cells too efficiently – resulting in toxicity. To offset the cytotoxicity of these drug sets in future studies, placement of the amine group in other regions of the drug structure may be beneficial and may lessen its toxicity impact against the normal cells.
Our series three compounds were developed with the aim of increasing the ligand’s metal coordination sites and create a more lipophilic vehicle for metal mobilisation. Our cytotoxicity data demonstrated that these pincer compounds displayed only poor activity against the cancer cell lines used in this study albeit without toxicity against the normal cell line (MRC-5). Like our preceding two drug sets, the results also demonstrated that supplementation of metal ions does not contribute to the cytotoxicity profile of the ligand.
In conclusion, the central research theme of this study was the development of novel anti-cancer drugs with metal chelating abilities that may induce ROS generation through the Fenton reaction. At the same time, these drugs were required to exert potency against cancer cells but be safe for normal cells. We also sought to develop potential drugs with improved localization to acidic vesicles within the cell such as the lysosome. In this research project we demonstrated that our series one and two compound are potent as the drug alone against the three cancer cell lines studied, series one compounds are also safer for normal cells. Our terminal amines (series two) compounds suggested that amine terminus does aid in the accumulation of the drug in the acidic vesicles and both drug sets (one and two) have metal chelating and ROS generating properties.
We feel that further investigations of these drug classes are essential because these drugs do show promising signs as a new and perhaps more efficacious approach to the chemotherapy of various cancer cell lines.