De Novo Single-Stranded RNA-Binding Peptides Discovered by Codon-Restricted mRNA Display.

RNA-binding proteins participate in diverse cellular processes, including DNA repair, post-transcriptional modification, and cancer progression through their interactions with RNAs, making them attractive for biotechnological applications. While nature provides an array of naturally occurring RNA-binding proteins, developing de novo RNA-binding peptides remains challenging. In particular, tailoring peptides to target single-stranded RNA with low complexity is difficult due to the inherent structural flexibility of RNA molecules. Here, we developed a codon-restricted mRNA display and identified multiple de novo peptides from a peptide library that bind to poly(C) and poly(A) RNA with KDs ranging from micromolar to submicromolar concentrations. One of the newly identified peptides is capable of binding to the cytosine-rich sequences of the oncogenic Cdk6 3'UTR RNA and MYU lncRNA, with affinity comparable to that of the endogenous binding protein. Hence, we present a novel platform for discovering de novo single-stranded RNA-binding peptides that offer promising avenues for regulating RNA functions.

A suicide enzyme catalyzes multiple reactions for biotin biosynthesis in cyanobacteria.

In biotin biosynthesis, the conversion of pimeloyl intermediates to biotin is catalyzed by a universal set of four enzymes: BioF, BioA, BioD and BioB. We found that the gene homologous to bioA, the product of which is involved in the conversion of 8-amino-7-oxononanoate (AON) to 7,8-diaminononanoate (DAN), is missing in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We provide structural and biochemical evidence showing that a novel dehydrogenase, BioU, is involved in biotin biosynthesis and functionally replaces BioA. This enzyme catalyzes three reactions: formation of covalent linkage with AON to yield a BioU-DAN conjugate at the ε-amino group of Lys124 of BioU using NAD(P)H, carboxylation of the conjugate to form BioU-DAN-carbamic acid, and release of DAN-carbamic acid using NAD(P)+. In this biosynthetic pathway, BioU is a suicide enzyme that loses the Lys124 amino group after a single round of reaction.

Concise enantioselective synthesis of wine lactone via intramolecular Diels-Alder reaction.

An enantioselective synthesis of (3S,3aS,7aR)-wine lactone, a major aroma component of white wine and citrus juices, was achieved starting from (S)-2-methyl-3-butenoic acid. An intramolecular Diels-Alder reaction was employed as a key step.

Unified synthesis and assessment of tumor cell migration inhibitory activity of optically active UTKO1, originally designed moverastin analog.

UTKO1 is a synthetic analog of a natural tumor cell migration inhibitor, moverastin, isolated from microbial extracts of Aspergillus sp. 7720. UTKO1 was initially developed as a mixture of the stereoisomers. In this study, a concise and unified synthesis of the 4 optically active stereoisomers of UTKO1 was achieved from a known optically pure dihydro-α-ionone through a 5-step sequence. The key transformation in the synthesis was a Nozaki-Hiyama-Kishi (NHK) reaction between an optically active enoltriflate and a known aldehyde to install the chiral allylic hydroxy group at C2'. Simple chromatographic separation of the 2 diastereomers with regard to the allylic hydroxy group was possible by the derivatization into the corresponding acetals with Nemoto's optical resolution reagent, (S)- or (R)-5-allyl-2-oxabicyclo[3.3.0]octene (ALBO). All 4 synthetic stereoisomers of UTKO1 exhibited comparable tumor cell migration inhibitory activity.

14-3-3εa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

Asymmetric synthesis of trans-p-menth-3-ene-1,2,8-triol, the monoterpene isolated from herbal plants.

The monoterpene, trans-p-menth-3-ene-1,2,8-triol, is a naturally occurring alcohol isolated from several herbal plants. In the present work, the asymmetric synthesis of both enantiomers of this natural product was achieved using Sharpless asymmetric dihydroxylation as the key step. A reversal of enantiofacial selectivity was observed in the asymmetric dihydroxylation.

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.

Concise synthesis of (±)-litseaones A and B.

A concise synthesis of litseaones A and B, which were isolated from the stem barks of Litsea rubescens and L. pedunculata, is described in this study. Litseaone A was synthesized in just three steps from a known phloroglucinol derivative. The direct conversion of litseaone A into litseaone B was also achieved.

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits, ENaC ß 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, ß, and γ. Changing the heteromeric combination of human and mouse ENaC αßγ subunits, we found that all five compounds activated the human ß 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; Emax: maximum effect value.

First synthesis of (±)-myristicyclin A.

The first synthesis of myristicyclin A, which was isolated from the Papua New Guinean plant Horsfieldia spicata, is described. The synthesis features acid-mediated hydroarylation reaction to form a dihydrocoumarin moiety, construction of the 2,8-dioxabicyclo[3.3.1]nonane skeleton under acidic conditions, and regioselective Friedel-Crafts acylation at a later stage.

Estimation of total prescription weights of active pharmaceutical ingredients in human medicines based on a public database for environmental risk assessment in Japan.

The distribution of active pharmaceutical ingredients (APIs) in prescription medicines for human consumption in Japan was estimated using the public database of the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB). From the latest NDB, 2058 APIs were identified, and the prescription weight exceeded 1 tonne/year for 711 APIs. Of these, 298 APIs were selected for further analysis after removing 413 APIs that were not covered by current environmental risk assessment (ERA) directives or were combination products. Among the 298 APIs, 43 were relatively newly branded APIs that have been available on the Japanese market since 2001 or later and have no generic drugs, and only 5 of the branded APIs are used by more than 1% of the population. When prescription data from the 47 prefectures in Japan were analyzed, prescription weights for 257 of the 298 APIs were the highest in Tokyo, probably because of its large population. Though it has both advantages and limitations, this novel method based on a non-profit public database can provide a transparent, unbiased and cost-effective solution for the estimation of the environmental exposure of generic and branded human medicines distributed with prescriptions in Japan.

Identification of a pheromone that increases anxiety in rats.

Chemical communication plays an important role in the social lives of various mammalian species. Some of these chemicals are called pheromones. Rats release a specific odor into the air when stressed. This stress-related odor increases the anxiety levels of other rats; therefore, it is possible that the anxiety-causing molecules are present in the stress-related odorants. Here, we have tried to identify the responsible molecules by using the acoustic startle reflex as a bioassay system to detect anxiogenic activity. After successive fractionation of the stress-related odor, we detected 4-methylpentanal and hexanal in the final fraction that still possessed anxiogenic properties. Using synthetic molecules, we found that minute amounts of the binary mixture, but not either molecule separately, increased anxiety in rats. Furthermore, we determined that the mixture increased a specific type of anxiety and evoked anxiety-related behavioral responses in an experimental model that was different from the acoustic startle reflex. Analyses of neural mechanisms proposed that the neural circuit related to anxiety was only activated when the two molecules were simultaneously perceived by two olfactory systems. We concluded that the mixture is a pheromone that increases anxiety in rats. To our knowledge, this is the first study identifying a rat pheromone. Our results could aid further research on rat pheromones, which would enhance our understanding of chemical communication in mammals.

Detection of diastereomer peptides as the intermediates generating D-amino acids during acid hydrolysis of peptides.

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Synthesis of (-)-Chamobtusin A from (+)-Dehydroabietylamine.

Chamobtusin A, a unique diterpene alkaloid isolated from Chamaecyparis obtusa cv. tetragon, is considered to be biosynthesized from an abietane diterpenoid. On the basis of this biosynthetic hypothesis, ferruginol (15) was synthesized from (+)-dehydroabietylamine and then biomimetically transformed into (-)-chamobtusin A in 6 steps (12 steps from (+)-dehydroabietylamine).

An unsaturated aliphatic alcohol as a natural ligand for a mouse odorant receptor.

We report the identification of a physiological receptor-volatile pair in the mouse olfactory system. By activity-guided fractionation of exocrine gland extracts and subsequent chemical analysis, (Z)-5-tetradecen-1-ol was identified as a natural ligand for a mouse odorant receptor. (Z)-5-tetradecen-1-ol is excreted into male mouse urine under androgen control and enhances urine attractiveness to female mice. This report is to our knowledge the first to describe natural product-based deorphanization of an odorant receptor.

Asymmetric Formal Synthesis of Azadirachtin.

An asymmetric formal synthesis of azadirachtin, a potent insect antifeedant, was accomplished in 30 steps to Ley's synthetic intermediate (longest linear sequence). The synthesis features: 1) rapid access to the optically active right-hand segment starting from the known 5-hydroxymethyl-2-cyclopentenone scaffold; 2) construction of the B and E rings by a key intramolecular tandem radical cyclization; 3) formation of the hemiacetal moiety in the C ring through the α-oxidation of the six-membered lactone followed by methanolysis.

Identification of olfactory alarm substances in zebrafish.

Escaping from danger is one of the most fundamental survival behaviors for animals. Most freshwater fishes display olfactory alarm reactions in which an injured fish releases putative alarm substances from the skin to notify its shoaling company about the presence of danger. Here, we identified two small compounds in zebrafish skin extract, designated as ostariopterin and daniol sulfate. Ostariopterin is a pterin derivative commonly produced in many freshwater fishes belonging to the Ostariophysi superorder. Daniol sulfate is a novel sulfated bile alcohol specifically present in the Danio species, including zebrafish. Ostariopterin and daniol sulfate activate distinct glomeruli in the olfactory bulb. Zebrafish display robust alarm reactions, composed of darting, freezing, and bottom dwelling, only when they are concomitantly stimulated with ostariopterin and daniol sulfate. These results demonstrate that the fish alarm reaction is driven through a coincidence detection mechanism of the two compounds along the olfactory neural circuitry.

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration.

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.

Dexmedetomidine and clonidine inhibit ventricular tachyarrhythmias in a rabbit model of acquired long QT syndrome.

BACKGROUND: Agents with α-2 adrenoreceptor (AR) agonistic action have reportedly suppressed tachyarrhythmias. METHODS AND RESULTS: We hypothesized that α-2 AR agonists would have an inhibitory effect on abnormal repolarization-related ventricular tachyarrhythmias (VTs). To test this hypothesis, the effects of 2 clinically available α-2 AR agonists (dexmedetomidine and clonidine) on the occurrence of VTs were assessed in a methoxamine-sensitized rabbit model of acquired long QT syndrome (Study 1: n=45). In control rabbits, administration of methoxamine and nifekalant almost invariably caused VTs (14/15). In contrast, incidence of VT significantly decreased during the treatment with dexmedetomidine (1µg·kg(-1)·min(-1): 5/12 [P<0.01 vs. control]) or with clonidine (33.3µg·kg(-1)·min(-1): 10/18 [P<0.01]). To verify that VTs in this animal model are triggered by early afterdepolarization (EAD), the monophasic action potential on the left ventricular surface was recorded in 28 open-chest rabbits (Study 2). EAD-like hump was less frequently detected during treatment with clonidine or dexmedetomidine (2/14) than in saline-treated rabbits (9/10, P<0.005). Presence of a hump was significantly related to the advent of VTs (P<0.05). CONCLUSIONS: Agents with α-2 AR agonistic action have an inhibitory effect on VTs in a rabbit model of long QT syndrome. Alpha-2 AR agonists, especially dexmedetomidine, may be a therapeutic choice for abnormal repolarization-related VTs that are resistant to conventional treatment.