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 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.</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^pro, 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^pro 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^pro. 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> (WRQN­TRY­SRIE­AIK­IQIL­SKLRL-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 IC₅₀ value of 7.0 nM against HT-29 cells, by strongly binding to tubulin (<i>K</i>_d = 1.3 μM) 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: Phe²-Arg³ and Arg⁵-Asn⁶. The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in <b>5d</b>, enhanced the metabolic stability at Phe²-Arg³. These results provide important information to guide the development of practical hNMU agonists