Fe(II) Ion Release during Endocytotic Uptake of Iron Visualized by a Membrane-Anchoring Fe(II) Fluorescent Probe

Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe­(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed <i>N</i>-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the <i>N</i>-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe­(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe­(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe­(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe­(II) over the course of cellular iron-uptake processes

Cytotoxicity of Tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide)-Induced DNA Damage in Chicken DT40 Cells

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II–DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that <i>tdp1</i>^–/–, <i>tdp2</i>^–/–, <i>parp1</i>^–/–, and <i>aptx1</i>^–/– cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5′-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase–DNA complexes and unligatable DNA ends under hypoxic conditions

2-Nitroimidazole-Tricarbocyanine Conjugate as a Near-Infrared Fluorescent Probe for <i>in Vivo</i> Imaging of Tumor Hypoxia

We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for <i>in vivo</i> imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. <i>In vivo</i> fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. <i>Ex vivo</i> analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising <i>in vivo</i> optical imaging probe for tumor hypoxia

Synthesis and Discovery of <i>N</i>-Carbonylpyrrolidine- or <i>N</i>-Sulfonylpyrrolidine-Containing Uracil Derivatives as Potent Human Deoxyuridine Triphosphatase Inhibitors

Recently, deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure–activity relationship (SAR) studies of uracil derivatives. <i>N</i>-Carbonylpyrrolidine- and <i>N</i>-sulfonylpyrrolidine-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (<b>12k</b>) having excellent potencies (IC₅₀ = 0.15 μM). The X-ray structure of a complex of <b>16a</b> and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds <b>12a</b> and <b>16a</b> markedly enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC₅₀ = 0.27–0.30 μM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors

Discovery of Highly Potent Human Deoxyuridine Triphosphatase Inhibitors Based on the Conformation Restriction Strategy

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound <b>7</b>, we designed and synthesized two conformation restricted analogues, i.e., compounds <b>8</b> and <b>9</b>. These compounds exhibited increased in vitro potency compared with the parent compound <b>7</b>. Further structure–activity relationship (SAR) studies identified a compound <b>43</b> with the highest in vitro potency (IC₅₀ = 39 nM, EC₅₀ = 66 nM). Furthermore, compound <b>43</b> had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage

1,2,3-Triazole-Containing Uracil Derivatives with Excellent Pharmacokinetics as a Novel Class of Potent Human Deoxyuridine Triphosphatase Inhibitors

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing uracil derivatives. Compound <b>45a</b>, which possesses 1,5-disubstituted 1,2,3-triazole moiety that mimics the amide bond of <i>tert</i>-amide-containing inhibitor <b>6b</b> locked in a cis conformation showed potent inhibitory activity, and its structure–activity relationship studies led us to the discovery of highly potent inhibitors <b>48c</b> and <b>50c</b> (IC₅₀ = ∼0.029 μM). These derivatives dramatically enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC₅₀ = ∼0.05 μM). In addition, compound <b>50c</b> exhibited a markedly improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that <b>50c</b> is a promising candidate for combination cancer chemotherapies with TS inhibitors

Discovery of a Novel Class of Potent Human Deoxyuridine Triphosphatase Inhibitors Remarkably Enhancing the Antitumor Activity of Thymidylate Synthase Inhibitors

Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure–activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor <b>7</b> (IC₅₀ = 100 μM), we developed compound <b>26</b>, which is the most potent human dUTPase inhibitor (IC₅₀ = 0.021 μM) reported to date. Not only does compound <b>26</b> significantly enhance the growth inhibition activity of 5-fluoro-2′-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC₅₀ = 0.075 μM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound <b>26</b> is a promising candidate for clinical development