Methemoglobin Formation by Triapine, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and Other Anticancer Thiosemicarbazones: Identification of Novel Thiosemicarbazones and Therapeutics That Prevent This Effect

Abstract

Thiosemicarbazones are a group of compounds that have received comprehensive investigation as anticancer agents. The antitumor activity of the thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone (3-AP; triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces methemoglobin (metHb) formation and hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed thiosemicarbazones, including di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified oxyhemoglobin, and a mouse model. The chelation of cellular labile iron with the formation of a redox-active thiosemicarbazone-iron complex was found to be crucial for oxyhemoglobin oxidation. This observation was substantiated using a thiosemicarbazone that cannot ligate iron and also by using the chelator, desferrioxamine, that forms a redox-inactive iron complex. Of significance, cellular copper chelation was not important for metHb generation in contrast to its role in preventing tumor cell proliferation. Administration of Dp44mT to mice catalyzed metHb and cardiac metmyoglobin formation. However, ascorbic acid administered together with the drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective thiosemicarbazone treatment regimens.

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

“…The PPY-based iron chelators are similar to previously studied HIV-1-inhibitory benzoylpyridine thiosemicarbazone compounds that also chelate cellular labile iron similarly to SIH (40). Only one aromatic ring was shown to be required to coordinate the binding of iron (31). Therefore, the benzyl moiety of PPYbased compounds can be utilized further for optimization of other properties, such as adjustment of the ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) profile.…”

“…The BpT-based iron chelators showed more antineoplastic activity than their DpT homologs in vitro (6). Recently, Dp44mT showed a significant amount of methemoglobin (metHb) formation in intact red blood cells and in mice (31). A modified analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone, did not generate production of metHb and thus was proposed to be suitable for further optimization (31).…”

“…Recently, Dp44mT showed a significant amount of methemoglobin (metHb) formation in intact red blood cells and in mice (31). A modified analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone, did not generate production of metHb and thus was proposed to be suitable for further optimization (31). Also, BpT was shown to induce metHb, whereas its analog, the lipophilic t-BuBpT chelator, was a less potent inducer of metHb (41).…”

“…1A). All of these chelators were shown to be toxic in vivo (31). Since the diarylketone and thiosemicarbozone moieties are likely to adopt a planar conformation that could potentially increase their ability to intercalate DNA, we substituted a benzyl group for the phenyl group.…”

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms

“…1E). The negative control compound, Dp2mT, which has a similar structure to Dp44mT but is unable to bind metals (20,27,44,52), did not alter the punctate fluorescence pattern of LC3-II relative to control after 24 and 48 h (Fig. 1E).…”