Design of Tetrazolium Cations for the Release of Antiproliferative Formazan Chelators in Mammalian Cells

Abstract

Cancer cells generally present a higher demand for iron, which plays crucial roles in tumor progression and metastasis. This iron addiction provides opportunities to develop broad spectrum anticancer drugs that target iron metabolism. In this context, prochelation approaches are investigated to release metal-binding compounds under specific conditions, thereby limiting off-target toxicity. Here, we demonstrate a prochelation strategy inspired by the bioreduction of tetrazolium cations widely employed to assess the viability of mammalian cells. We designed a series of tetrazolium-based compounds for the intracellular release of metal-binding formazan ligands. The combination of reduction potentials appropriate for intracellular reduction and an N-pyridyl donor on the formazan scaffold led to two effective prochelators. The reduced formazans bind as tridentate ligands and stabilize low-spin Fe(II) centers in complexes of 2:1 ligand-to-metal stoichiometry. The tetrazolium salts are stable in blood serum for over 24 h, and antiproliferative activities at micromolar levels were recorded in a panel of cancer cell lines. Additional assays confirmed the intracellular activation of the prochelators and their ability to affect cell cycle progression, induce apoptotic death, and interfere with iron availability. Demonstrating the role of iron in their intracellular effects, the prochelators impacted the expression levels of key iron regulators (i.e., transferrin receptor 1 and ferritin), and iron supplementation mitigated their cytotoxicity. Overall, this work introduces the tetrazolium core as a platform to build prochelators that can be tuned for activation in the reducing environment of cancer cells and produce antiproliferative formazan chelators that interfere with cellular iron homeostasis.

Figure 1.

Iron binding in medicine: a) Selected iron chelators evaluated as anticancer agents in clinical trials; b) General prochelation strategy; and c) Example of disulfide-based prochelator activated by intracellular reduction.

Figure 2.

A prochelation strategy inspired by the chemistry of tetrazolium cations: a) Bioreduction of the MTT reagent employed to assess the viability of eukaryotic cells; b) Schematic of the activation of a triaryltetrazolium prochelator to release a formazan ligand carrying a metal-binding donor.

Figure 3.

Structures of the formazan and tetrazolium scaffolds investigated in this study.

Figure 4.

Crystal structures of Fe(MTF–H)₂, Fe(2a–H)₂, and Fe(4a–H)₂ showing a partial atom labeling scheme. Thermal ellipsoids are scaled to the 50% probability level. In each complex, one of the ligands is shown as capped sticks, and carbon-bound hydrogen atoms are omitted for clarity (CCDC, Fe(MTF–H)₂ 2217720, Fe(2a–H)₂ 2217721 and Fe(4a–H)₂ 2217722).

Figure 5.

Effects of prochelators 2b and 4b on cell cycle and cell death as assessed by flow cytometry in A2780 ovarian cancer cells. (a) Cell cycle distribution after treatment with the test compounds (40 μM) for 24 h; (b) Apoptotic cell death after incubation with the test compounds (40 μM) for 48 h. Experiments were conducted in triplicate and the values shown are mean ± standard deviation. All T-tests relative to vehicle only (DMSO): ** p < 0.01, *** p <0.001, **** p < 0.0001.

Figure 6.

Relative extent of intracellular iron binding assessed through the calcein assay after incubation with the test compounds (40 μM) in A2780 ovarian cancer cells (a) and MRC-5 normal lung fibroblasts (b). Experiments in triplicate shown as mean ± standard deviation. All T-tests relative to vehicle only (DMSO): * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7.

Effects of the prochelators on the expression of transferrin receptor 1 (TfR1) and ferritin heavy chain (ferritin H): Western blot analysis (n=4) and representative gel images after treatment with test compounds for 24 h in A2780 cancer cells (a) and normal MRC-5 cells (b). β-Actin was used as a loading control. All T-tests relative to vehicle only (DMSO): * p < 0.05, ** p < 0.01.

Figure 8.

Effects of iron supplementation on toxicity in A2780 cells. The percentage of dead cells was determined using the LIVE/DEAD kit as measured by flow cytometry. Quantification of dead cells after treatment with test compounds (40 μM, 48 h) in the presence or absence of (a) ferric ammonium citrate (FAC, 50 μM) or (b) holo-transferrin (holo-Tf, 50 μM). Experiments in triplicate and values shown as mean ± standard deviation. T-tests relative to sample without iron supplementation: ** p < 0.01, *** p < 0.001, **** p < 0.0001.