39 research outputs found
Radiosensitivity in cells in early M phase is comparable to that in KPU-300–treated cells.
<p>(A) Fractions sorted by flow cytometry. Fr. 1, whole cell population; Fr. 2, cell fraction enriched in early M phase following the shake-off method; Fr. 3, cell fraction accumulated in early M phase following KPU-300 treatment for 24 h. (B) Radiosensitivity in each cell fraction following various treatments. Radiation dose was 4 Gy, and concentration of KPU-300 was 30 nM. A value of “-” in the “Fr.” row (i.e., lanes 1 and 4) indicates that cell sorting was not performed. The SF for Lane No. 9 was normalized by dividing the SF for Lane No. 8 by that for Lane No. 5. Data represent means ± S.E. of values obtained from three independent experiments. Error bars are not displayed when they would have been smaller than the circular symbol indicating the mean. <i>N</i>.<i>S</i>., not significant by either ANOVA or t-test.</p
Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.
<p>(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 vs. lower values for the same time points.</p
Confocal fluorescence imaging of spheroids after treatment with KPU-300.
<p>The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 ÎĽm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 ÎĽm.</p
Characterization of abnormal Fucci fluorescence following KPU-300 treatment.
<p>(A) Representative images of abnormal fluorescence after treatment with KPU-300. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm (B) Relationship between abnormal Fucci fluorescence and M phase following KPU-300 treatment. (a) Two-dimensional flow-cytometric analysis of Fucci fluorescence. The area within a quadrangle represents cells expressing abnormal Fucci fluorescence. (b) Two-dimensional flow-cytometric analysis of DNA content and phosphorylated histone H3 (pHH3). The area within a quadrangle represents cells in M phase. The acquired time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. (c) Quantitative analysis of cells with abnormal Fucci expression and those in M phase in Fig 3a and 3b. Data represent means ± S.E. of values obtained from three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01 vs. controls at time 0.</p
Characterization of cell cycle kinetics in HeLa-Fucci cells following KPU-300 treatment.
<p>(A) Chemical structure of KPU-300. (B) Immunostaining for β-tubulin. Exponentially growing HeLa-Fucci cells were fixed and prepared for immunostaining following treatment with 30 nM KPU-300 for 16 h. Blue, DAPI; pink, β-tubulin. Bar, 5 μm. (C) Time course of Fucci fluorescence and histogram of DNA content following KPU-300 treatment. Cells were treated with the indicated concentrations of KPU-300 and prepared for time-lapse imaging and flow-cytometric analysis. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm. (D) Time course of the percentages of green fluorescent cells (a), M-phase cells (b), and total cell number (c) following KPU-300 treatment. Green cells and M-phase cells were manually counted in merged fluorescence and phase contrast images. A total of 170–350 cells obtained from 8–11 visual fields were counted in one experiment. M phase cells adopt a round-shape, accompanied by disappearance of the nuclear envelope. Data represent means ± S.E. of values from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 vs. treatment with 10 nM for the same duration of time.</p
An Overview of Severe Acute Respiratory Syndrome–Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy
Severe
acute respiratory syndrome (SARS) is caused by a newly emerged
coronavirus that infected more than 8000 individuals and resulted
in more than 800 (10–15%) fatalities in 2003. The causative
agent of SARS has been identified as a novel human coronavirus (SARS-CoV),
and its viral protease, SARS-CoV 3CL<sup>pro</sup>, has been shown
to be essential for replication and has hence been recognized as a
potent drug target for SARS infection. Currently, there is no effective
treatment for this epidemic despite the intensive research that has
been undertaken since 2003 (over 3500 publications). This perspective
focuses on the status of various efficacious anti-SARS-CoV 3CL<sup>pro</sup> chemotherapies discovered during the last 12 years (2003–2015)
from all sources, including laboratory synthetic methods, natural
products, and virtual screening. We describe here mainly peptidomimetic
and small molecule inhibitors of SARS-CoV 3CL<sup>pro</sup>. Attempts
have been made to provide a complete description of the structural
features and binding modes of these inhibitors under many conditions
Structural Basis for the Effective Myostatin Inhibition of the Mouse Myostatin Prodomain-Derived Minimum Peptide
Myostatin inhibition is one of the
promising strategies for treating muscle atrophic disorders, including
muscular dystrophy. It is well-known that the myostatin prodomain
derived from the myostatin precursor acts as an inhibitor of mature
myostatin. In our previous study, myostatin inhibitory minimum peptide <b>1</b> (WRQNÂTRYÂSRIEÂAIKÂIQILÂSKLRL-amide)
was discovered from the mouse myostatin prodomain. In the present
study, alanine scanning of <b>1</b> demonstrated that the key
amino acid residues for the effective inhibitory activity are rodent-specific
Tyr and C-terminal aliphatic residues, in addition to N-terminal Trp
residue. Subsequently, we designed five Pro-substituted peptides and
examined the relationship between secondary structure and inhibitory
activity. As a result, we found that Pro-substitutions of Ala or Gln
residues around the center of <b>1</b> significantly decreased
both α-helicity and inhibitory activity. These results suggested
that an α-helical structure possessing hydrophobic faces formed
around the C-terminus is important for inhibitory activity
Novel Hybrid-Type Antimicrobial Agents Targeting the Switch Region of Bacterial RNA Polymerase
The bacterial RNA polymerase (RNAP) is an ideal target
for the
development of antimicrobial agents against drug-resistant bacteria.
Especially, the switch region within RNAP has been considered as an
attractive binding site for drug discovery. Here, we designed and
synthesized a series of novel hybrid-type inhibitors of bacterial
RNAP. The antimicrobial activities were evaluated using a paper disk
diffusion assay, and selected derivatives were tested to determine
their MIC values. The hybrid-type antimicrobial agent <b>29</b> showed inhibitory activity against <i>Escherichia coli</i> RNAP. The molecular docking study suggested that the RNAP switch
region would be the binding site of <b>29</b>
Development of a New Benzophenone–Diketopiperazine-Type Potent Antimicrotubule Agent Possessing a 2‑Pyridine Structure
A new benzophenone–diketopiperazine-type
potent antimicrotubule
agent was developed by modifying the structure of the clinical candidate
plinabulin (<b>1</b>). Although the right-hand imidazole ring
with a branched alkyl chain at the 5-position in <b>1</b> was
critical for the potency of the antimicrotubule activity, we successfully
substituted this moiety with a simpler 2-pyridyl structure by converting
the left-hand ring from a phenyl to a benzophenone structure without
decreasing the potency. The resultant compound <b>6b</b> (KPU-300)
exhibited a potent cytotoxicity, with an IC<sub>50</sub> value of
7.0 nM against HT-29 cells, by strongly binding to tubulin (<i>K</i><sub>d</sub> = 1.3 ÎĽM) and inducing microtubule depolymerization
Discovery of Potent Hexapeptide Agonists to Human Neuromedin U Receptor 1 and Identification of Their Serum Metabolites
Neuromedin
U (NMU) and S (NMS) display various physiological activities, including
an anorexigenic effect, and share a common C-terminal heptapeptide-amide
sequence that is necessary to activate two NMU receptors (NMUR1 and
NMUR2). On the basis of this knowledge, we recently developed hexapeptide
agonists <b>2</b> and <b>3</b>, which are highly selective
to human NMUR1 and NMUR2, respectively. However, the agonists are
still less potent than the endogenous ligand, hNMU. Therefore, we
performed an additional structure–activity relationship study,
which led to the identification of the more potent hexapeptide <b>5d</b> that exhibits similar NMUR1-agonistic activity as compared
to hNMU. Additionally, we studied the stability of synthesized agonists,
including <b>5d</b>, in rat serum, and identified two major
biodegradation sites: Phe<sup>2</sup>-Arg<sup>3</sup> and Arg<sup>5</sup>-Asn<sup>6</sup>. The latter was more predominantly cleaved
than the former. Moreover, substitution with 4-fluorophenylalanine,
as in <b>5d</b>, enhanced the metabolic stability at Phe<sup>2</sup>-Arg<sup>3</sup>. These results provide important information
to guide the development of practical hNMU agonists