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.

Targeting misfolding and aggregation of the amyloid-β peptide and mutant p53 protein using multifunctional molecules

“…In addition, dysregulated metal ion binding is hypothesized to influence Aβ peptide misfolding in AD, thereby showing similarities to the impaired Zn binding and aggregation properties of mutant p53. 142 This link is further demonstrated by the success of targeted metal ion binding and redistribution as an anticancer strategy, 67,146–148 and in modulating amyloidogenic protein aggregation. 48,149 There continues to be significant interest in the development of therapeutic interventions targeting the p53 pathway, including gene therapy, 150,151 RNA silencing, 152 peptide vaccines, 153 and small molecules targeting p53.…”

“…The use of masked chelation agents is not constrained to AD, and many recent breakthroughs have been reported in cancer treatment. 66,67…”

Quinoline-based tetrazolium prochelators: formazan release, iron sequestration, and antiproliferative efficacy in cancer cells

“…5,11,12 In addition, bioconjugation strategies, for instance to carbohydrates 13 or serum albumin, 14 have been pursued to facilitate accumulation in malignant cells relative to normal cells. Thiol-reactive 15 and reductively activatable prochelators 16–18 are generally advantageous because they capitalize on the higher glutathione (GSH) concentrations and more reducing cytoplasmic environment of malignant cells with respect to the surrounding tissue. 19…”

“…1a) remain stable in blood serum but are reduced intracellularly to release the corresponding formazans. 18 These metal-binding compounds in turn coordinate Fe( ii ) and disrupt intracellular iron homeostasis. The antiproliferative activities of these prochelators, however, are moderate, remaining in the 12–30 μM range in several cancer cell lines.…”

“…The activation of 2b and 4b was found to be incomplete after 48-hour incubations and likely limited their efficacy. 18 We reasoned that a more rapid and effective reduction would lead to a larger fraction of released chelators and thus to enhanced iron sequestration. As such, we sought to incorporate quinoline, isoquinoline, or quinoxaline into the new constructs with the intent to (i) maintain the same coordination mode of the pyridyl systems, and (ii) shift anodically the new reduction potentials due to the more electron-withdrawing, π-extended heterocycles.…”