7 research outputs found
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 FeÂ(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 FeÂ(II) redox state-selectively.
An imaging study with Mem-RhoNox and its derivatives reveals that
labile FeÂ(II) is transiently generated during the major iron-uptake
pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox
is capable of monitoring endosomal FeÂ(II) in primary cultured neurons
during endocytotic uptake. This report is the first example that identifies
the generation of FeÂ(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><sup>–/–</sup>, <i>tdp2</i><sup>–/–</sup>, <i>parp1</i><sup>–/–</sup>, and <i>aptx1</i><sup>–/–</sup> 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 (IC<sub>50</sub> = 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 (EC<sub>50</sub> = 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 (IC<sub>50</sub> = 39 nM, EC<sub>50</sub> = 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> (IC<sub>50</sub> = ∼0.029
μM). These derivatives dramatically enhanced the growth inhibition
activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells
in vitro (EC<sub>50</sub> = ∼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> (IC<sub>50</sub> = 100 μM), we
developed compound <b>26</b>, which is the most potent human
dUTPase inhibitor (IC<sub>50</sub> = 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 (EC<sub>50</sub> = 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