Synthesis-guided structure revision of the monoterpene alcohol isolated from Mentha haplocalyx

The monoterpene isolated from Mentha haplocalyx, 3,3,5-trimethyl-2-oxabicyclo[2.2.2]oct-5-en-4-ol, was synthesized according to its proposed structure. However, the NMR data of the synthetic sample were not in agreement with those reported for the natural product. After considerable efforts, the genuine structure was confirmed as (1R*,2R*)-4-(1′-hydroxy-1′-methylethyl)-1-methylycyclohex-3-ene-1,2-diol.     

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits,ENaC β and γ

The epithelial sodium channel (ENaC) plays a pivotal role in sodium homeostasis, and the development of drugs that modulate ENaC activity is of great potential therapeutic relevance. We screened 6100 chemicals for their ability to activate sodium permeability of ENaC. We used a two-step strategy: a high throughput cell-based assay and an electrophysiological assay. Five compounds were identified showing common structural features including an indole or benzothiophene ring. ENaC consists of three subunits: α, β, and γ. Changing the heteromeric combination of human and mouse ENaC αβγ subunits, we found that all five compounds activated the human β subunit but not the mouse subunit. However, four of them exhibited lower activity when the human γ subunit was substituted by the mouse γ subunit. Our findings provide a structural basis for designing human ENaC activity modulators.

Abbreviations: ENaC: Epithelial sodium channel; ΔRFU: delta relative fluorescence units; EC₅₀: Half-maximal effective concentration; E_max: maximum effect value.     

A new class of pentapeptide KISS1 receptor agonists with hypothalamic–pituitary–gonadal axis activation

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.     

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1’s active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.     

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.     

The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization

The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of beta-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.     

Central melanocortin signaling restores skeletal muscle AMP-activated protein kinase phosphorylation in mice fed a high-fat diet

Little is known about the role of the central melanocortin system in the control of fuel metabolism in peripheral tissues. Skeletal muscle AMP-activated protein kinase (AMPK) is activated by leptin and serves as a master regulator of fatty acid beta-oxidation. To elucidate an unidentified role of the central melanocortin system in muscle AMPK regulation, we treated conscious, unrestrained mice intracerebroventricularly with the melanocortin agonist MT-II or the antagonist SHU9119. MT-II augmented phosphorylation of AMPK and its target acetyl-CoA carboxylase (ACC) independent of caloric intake. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the coadministration of SHU9119 or in KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat-diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin but was markedly restored by MT-II. Our data provide evidence for the critical role of the central melanocortin system in the leptin-skeletal muscle AMPK axis and highlight the system as a therapeutic target in leptin resistance.     

A mass conserved reaction-diffusion system captures properties of cell polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5'-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction-diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction-diffusion system with diffusion-driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction-diffusion system with diffusion-driven instability is one of such principles of cell polarity.     

A Mass Conserved Reaction–Diffusion System Captures Properties of Cell Polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5′-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction–diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction–diffusion system with diffusion–driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction–diffusion system with diffusion-driven instability is one of such principles of cell polarity.