Alotamide B (2), a new cyclic depsipeptide, along with alotamide A (1) was isolated from assemblies of marine cyanobacteria mainly consisting of Moorena sp. collected at Kakeroma Is. The structure of 2 was determined by spectral analysis. The absolute configuration of 1 and 2 was analyzed by chemical degradation and modification followed by HPLC analysis.

Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.

Histone lysine methylation is an epigenetic mark that can control gene expression. In particular, H3K9me3 contributes to transcriptional repression by regulating chromatin structure. Successful mitotic progression requires correct timing of chromatin structure changes, including epigenetic marks. However, spatiotemporal information on histone modifications in living cells remains limited. In this study, we created an FRET-based probe for live-cell imaging based on the HP1α chromodomain (HP1αCD), which binds to H3K9me3. The probe was incorporated into chromatin and the emission ratio decreased after treatment with histone methyltransferase inhibitors, indicating that it successfully traced dynamic changes in H3K9me3. Upon entry into mitosis, the probe's emission ratio transiently increased with a concomitant increase in H3K9me3, then exhibited a stepwise decrease, probably due to loss of HP1αCD binding caused by phosphorylation of H3S10 and demethylation of H3K9me3. This probe will be a useful tool for detecting dynamic changes in chromatin structure associated with HP1α.

Plasmodium falciparum, the most virulent human malaria parasite, causes serious diseases among the infected patients in the world and is particularly important in African regions. Although artemisinin combination therapy is recommended by the WHO for treatment of P. falciparum-malaria, the emergence of artemisinin-resistant parasites has become a serious issue which underscores the importance of sustained efforts to obtain novel chemotherapeutic agents against malaria. As a part of such efforts, thirty-nine herbal extracts from traditional Chinese medicine (TCM) were assayed for their anti-malarial activity using 3D7 strain of P. falciparum. Three herbal supplements appeared to possess higher specific anti-malarial activity than the others. One of them (D3) was separated by two sequential fractionations with reverse-phase (the first step) and normal-phase (the second step) liquid chromatography, in which some fractions resulted in higher specific activities than those of D3 or the previous fractions. Cell toxicity assay was performed with the fractions of the first fractionation and demonstrated no obvious cell toxicity. These results suggest that structure determination of the major compound for the anti-malarial activity in D3 may help the development of more potent chemicals in the future.

Plants in the genus Curcuma have been widely used as traditional medicines in Asian countries. These plants contain bioactive compounds with neuroprotective properties or activities that increase neural stem cells (NSCs) and neurons. However, bioactive components in Curcuma that promote the differentiation of NSCs into astrocytes have not yet been reported. Here, the effects of Curcuma extracts on the in vitro differentiation of embryonic stem-cell-derived NSCs were evaluated. The extract of the wild turmeric, Curcuma aromatica, strongly promoted the differentiation of NSCs into astrocytes. Bioassay-guided isolation yielded coronarins C (1) and D (2), as well as (E)-labda-8(17),12-diene-15,16-dial (3) as the bioactive compounds. Coronarin D (2) markedly promoted the differentiation of NSCs into astrocytes up to approximately 4 times (3.64 ± 0.48) and increased the expression level of GFAP at the mRNA and protein level, while compounds 1 and 3 exhibited only weak effects, suggesting that the 15-hydroxy-Δ12-γ-lactone moiety is important for bioactivity. Moreover, compound 2 increased the number of pSTAT3-positive cells, suggesting that compound 2 promoted astrocytic differentiation through JAK/STAT signaling pathway.

Marine organisms are a rich source of bioactive secondary metabolites. Although many marine natural products with bioactivities have been isolated, successful elucidation of their mechanisms of action remains limited. In this study, we prepared a probe molecule based on the marine cyclic peptide kapakahine A (1) by introducing a linker with an azide terminal group, which enables the introduction of fluorescent groups for the effective monitoring of subcellular localization, or coupling to affinity beads for the pull-down of target proteins. The results of LC/MS/MS measurements, ProteinPilot analysis, and Western blotting suggest that kapakahine A interacts with the mitochondrial inner membrane proteins PHB1, PHB2, and ANT2, which is consistent with the results of the subcellular localization analysis using a fluorescent probe.

Nitrifying chemoautotrophs support the growth of diverse concomitant heterotrophs in natural or engineered environments by supplying organic compounds. In this study, we aimed to investigate this microbial association, especially (i) to distinguish whether the relationship between nitrifying chemoautotrophs and heterotrophs is commensal or mutualistic, and (ii) to clarify how heterotrophs promote the growth of autotrophic nitrite-oxidizing bacteria (Nitrospira). Pure cultured Nitrospira (Nitrospira sp. ND1) was employed in this study. Heterotrophs growing with metabolic by-products of Nitrospira as a sole carbon source were isolated from several environmental samples and used to test the growth-promoting activity of Nitrospira. Furthermore, liquid chromatography-mass spectrometry analysis was conducted to evaluate how heterotrophs consumed chemical compounds produced by Nitrospira and newly produced during co-cultivation. Notably, Nitrospira growth was stimulated by co-cultivation with some heterotrophs and the addition of spent media of some strains, suggesting that not only heterotrophs but also Nitrospira received benefits from their mutual co-existence. Furthermore, the data suggested that some of the growth-promoting heterotrophs provided as-yet-unidentified growth-promoting factors to Nitrospira. Overall, Nitrospira and heterotrophs thus appear to exhibit a mutualistic relationship. Such mutualistic relationships between autotrophs and heterotrophs would contribute to the stability and diversity of microbial ecosystems.

Changes in eating habits are brought about by drastic changes in lifestyle and environment, and, it has been pointed out, are strongly involved in the increase in neurological diseases and onset of cancer in younger adult ages. There is a wide variety of chemical substances in food, and there is a need to analyze the effects of complex exposures on complex mechanisms of action and to develop methods for evaluating and predicting them. The power of molecular nutrition needs to create an integrated approach to human nutrition in line with the grand social challenges of diet-related illnesses. The current article aims to explore some of these areas where integration is appropriate. Therefore, in this symposium, we will introduce the contents of four performers who are conducting cutting-edge research. 1) Chemoprevention by vitamin A and its derivatives, 2) Toxicity prediction of natural compounds from a developing database of bioactive gradients from Kampo medicine, 3) Toxicity prediction of chemicals using pluripotent stem cells. 4) Detection of bioactive compounds in “Aji” or “Umami” in food. By detecting and predicting the biological activity and toxicity of chemical substances such as nutrients in foods, it will be possible to provide better molecular information on dietary components. In addition, we will introduce next-generation health and prevention methods.

Selective cell tagging (SeCT) therapy is a strategy for labeling a targeted cell with certain chemical moietiesviaa catalytic chemical transformation in order to elicit a therapeutic effect. Herein, we report a cancer therapy based on targeted cell surface tagging with proapoptotic peptides (Ac-GGKLFG-X; X = reactive group) that induce apoptosis when attached to the cell surface. Using either Au-catalyzed amidation or Ru-catalyzed alkylation, these proapoptotic peptides showed excellent therapeutic effects bothin vitroandin vivo. In particular, co-treatment with proapoptotic peptide and the carrier-Ru complex significantly and synergistically inhibited tumor growth and prolonged survival rate of tumor-bearing mice after only a single injection. This is the first report of Ru catalyst applicationin vivo, and this approach could be used in SeCT for cancer therapy.

Increasing evidence indicates that many insecticides produce significant epigenetic changes during embryogenesis, leading to developmental toxicities. However, the effects of insecticides on DNA methylation status during early development have not been well studied. We developed a novel nuclear phenotypic approach using mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation changes via high-content imaging analysis. Exposure to imidacloprid, carbaryl, and o,p'-DDT increased the fluorescent intensity of granules in the nuclei, indicating global DNA methylating effects. However, DNA methylation profiling in cell-cycle-related genes, such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, decreased in imidacloprid treatments, suggesting the potential influence of DNA methylation patterns on cell differentiation. We developed a rapid method for evaluating global DNA methylation and used this approach to show that insecticides pose risks of developmental toxicity through DNA methylation.

Marine sponges are the most prolific marine sources for discovery of novel bioactive compounds. Sponge secondary metabolites are sought-after for their potential in pharmaceutical applications, and in the past, they were also used as taxonomic markers alongside the difficult and homoplasy-prone sponge morphology for species delineation (chemotaxonomy). The understanding of phylogenetic distribution and distinctiveness of metabolites to sponge lineages is pivotal to reveal pathways and evolution of compound production in sponges. This benefits the discovery rate and yield of bioprospecting for novel marine natural products by identifying lineages with high potential of being new sources of valuable sponge compounds. In this review, we summarize the current biochemical data on sponges and compare the metabolite distribution against a sponge phylogeny. We assess compound specificity to lineages, potential convergences, and suitability as diagnostic phylogenetic markers. Our study finds compound distribution corroborating current (molecular) phylogenetic hypotheses, which include yet unaccepted polyphyly of several demosponge orders and families. Likewise, several compounds and compound groups display a high degree of lineage specificity, which suggests homologous biosynthetic pathways among their taxa, which identifies yet unstudied species of this lineage as promising bioprospecting targets.

CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA Polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.

This study presents the early framework of selective cell tagging (SeCT) therapy, which is the concept of preferentially labeling specific cells in vivo with chemical moieties that can elicit a therapeutic response. Using glycosylated artificial metalloenzyme (GArM)-based protein labeling, this study reports two separate functional strategies. In one approach, early tumor onset can be suppressed by tagging cancer cells in living mice with an integrin-blocking cyclic-Arg-Gly-Asp (cRGD) moiety, thereby disrupting cell adhesion onto the extracellular matrix. In another approach, tumor growth in mice can be reduced by tagging with a cytotoxic doxorubicin moiety. Subsequent cell death occurs following internalization and drug release. Overall, experiments have shown that mouse populations receiving the mixture of SeCT labeling reagents exhibited a significant delay/reduction in tumor onset and growth compared with controls. Highlighting its adaptability, this work represents a foundational step for further development of SeCT therapy and its potential therapeutic applications.

Malaria remains one of the most important infectious diseases in the world. In 2017 alone, approximately 219 million people were infected with malaria, and 435,000 people died of this disease. Plasmodium falciparum, which causes falciparum malaria, is becoming resistant to artemisinin (ART) in Southeast Asia; therefore, new antimalarial drugs are urgently needed. Some excellent antimalarial drugs, such as quinine and ART, were originally obtained from plants. Hence, we analyzed the antimalarial effects of marine natural products to find new antimalarial agents. We used a malaria growth inhibition assay to determine the antimalarial ability and half-maximal inhibitory concentration (IC50) values of the marine organism-derived compounds. Three compounds (kapakahine A, kapakahine B, and kulolide-1) showed antimalarial effects, and one (kapakahine F) showed selective antimalarial effects on the Dd2 clone. Although the IC50 values obtained for these compounds were greater than that of ART, their potency against P. falciparum is sufficient to warrant further investigation of these compounds as possible drug leads.

A number of bioactive marine natural products have been isolated so far, but it is still difficult to disclose their modes of action. In this study, we prepared fluorescently labeled chemical probes from the cytotoxic marine cyclic peptides kapakahines A (1) and F (2) to visualize their localization as the first step of the study of their modes of action. We used fluorescent dyes 3a or 3a/b (a 1:1 mixture of 3a and 3b) whose terminal N-hydroxysuccinimide (NHS) group can react with the free amino groups of kapakahines. The fluorescently labeled kapakahine A (Kap A-5-FL, 5a) stained P388 murine leukemia cells and HeLa human cervical cancer cells, while cells treated with fluorescently labeled kapakahine F (Kap F-5-FL, 6a) only weakly stained them. Further analysis of the confocal images of the stained cells with higher magnification (×100) indicated the localization of Kap A-5-FL (5a) in the cells. In this paper, we report the small-scale preparation and a new delivery method of fluorescent probes, as well as the application of these procedures to cell staining.

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method's efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).

<title>ABSTRACT</title>Post-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

Jerusalem artichokes contain high amounts of inulin, which is a prebiotic that supports digestive health, as well as a variety of insoluble fibers and caffeoylquinic acid. The individual impact of these components on gut microbiota is well known; however, the combinatorial effects are less clear. In this investigation, we fractionated Jerusalem artichokes into three parts (water-soluble extract, insoluble extract, and organic extract) and powdered them. Mice were fed a high-fat diet that included one or more of these extracts for 10 days, and then their cecal pH, cecal short-chain fatty acids (SCFAs), and fecal microbiota were evaluated. The combination of the water-soluble and organic extract decreased cecal pH and increased the concentration of SCFAs and led to dynamic changes in the composition of the gut microbiota. These results demonstrate that both the water-soluble and organic extracts in Jerusalem artichokes are bioactive substances that are capable of changing SCFA production and the composition of gut microbiota. Powdered Jerusalem artichokes, rather than inulin supplements, may be superior for promoting a healthy gut.

Kakeromamide A (1), the first marine cyclopeptide inducing neural stem cells differentiation into astrocytes, was synthesized in 12 longest linear steps and 14% overall yield. Using this synthetic approach, four analogs of kakeromamide A were prepared and evaluated for neural differentiation- modulating activity.

Natural products have been regarded as a pivotal source for biological active compounds. In addition, structural modification of natural products has been simultaneously received much attention because these modified natural products would show not only enhanced activity and selectivity, but possibly also an entirely distinct bioactivity from those of the parent natural products. We describe herein a series of study on structual modification of ageladine A for exploring new biological activity. Ageladine A was isolated from the marine sponge Agelas nakamurai as an antiangiogenic compound. Based on total syntheses of this alkalod, its structual modifications were caried out as a part of SAR study, which revealed that a pyridine derivative shows a more potent activity. More recently, we have developed an efficient bio-inspired cascade reaction for the synthesis of ageladine A The cascade reaction involves an aza-electrocyclization and a novel 2-aminoimidazole formation that is modeled after a post-translational modification of arginine residue in protein. Employing the cascade reaction, ageladine A and its N1-substituted analogues were efficiently synthesized in one-pot fashion from anilines or guanidines. In addition, it was found that some analogs showed significant activity on modulating neuronal differentiation. Namely, these analogs selectively activate or inhibit the differentiation of neural stem cells to neurons, while negligible in astrocyte differentiation. The series of study represent a successful case in altering native bioactivity of natural products by structural modification.

A convenient route to RIKEN click probes for glycoconjugation to the amino groups of lysines was established. N-Glycoclusters prepared with human serum albumin by the new method showed the same cell interaction profiles as those synthesized previously. The new route has thus significantly expanding the applicability to glycoconjugation and drug delivery systems.

Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

The original article can be found online at https://doi.org/10.1038/ja.2017.145 .

Main Text(Stem Cell Reports 8, 634-647; March 14, 2017) An investigation by Kyoto University has identified fabrication and falsification in all the six main figures (Figures 1N, 2D, 3A, 4A, 4C, 5E, 6A, and 6C) and five out of six supplementary figures (Figures S2, S3A, S3B, S4A, S5D, S6A, S6B, S6C, and S6D) by the first and corresponding author, Kohei Yamamizu, in the above article. Multiple data that were used to make these figures are either missing in the original data set or have been manipulated. As a result of these modifications, the manuscript does not support its main conclusion that the authors reconstituted the blood-brain barrier by using human iPS cells. All authors have agreed to retract the paper. The authors sincerely apologize to the scientific community for publishing this erroneous article and profoundly regret any inconvenience and confusion that it might have caused.

Two new analogs of halistanol sulfate (1) were isolated from a marine sponge Halichondria sp. collected at Hachijo-jima Island. Structures of these new halistanol sulfates I (2) and J (3) were elucidated by spectral analyses. Compounds 1-3 showed inhibitory activity against SIRT 1-3 with IC 50 ranges of 45.9-67.9, 18.9-21.1 and 21.8-37.5 μM, respectively. X-ray crystallography of the halistanol sulfate (1) and SIRT3 complex clearly indicates that 1 binds to the exosite of SIRT3 that we have discovered in this study.

Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a β-amino acid, was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spectral analysis and the modified Marfey's method. Compound 1 induced differentiation of neural stem cells into astrocytes at the concentration of 10 µM.

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three β-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey's method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Krüppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.

A new halogenated diterpene, dolabellol A (1), was isolated from the opisthobranch Dolabella auricularia. The planar structure of dolabellol A was elucidated by NMR spectroscopic analysis and chemical reactions. The absolute stereochemistry was determined by single-crystal X-ray diffraction studies.

Rapidly growing cancer cells have increased levels of intracellular polyamines compared to normal, healthy tissues. Based on the selective reactivity of glycine propargyl esters, probes were synthesized that show evidence for selective polyamine reactivity, which was then applied for selective cancer cell imaging studies.

EGR2 is a zinc finger transcription factor that regulates myelination in the peripheral nervous system and T cell anergy. The transcriptional activity of EGR2 is known to be regulated by its co-activators and/or co-repressors. Although the activity of transcription factors is generally regulated not only by interactions with co-regulators but also posttranslational modifications including acetylation, little is known about posttranslational modifications of EGR2. Here we show that EGR2 is a novel acetylated protein. Through immunoblotting analyses using an antibody that specifically recognizes the acetylated form of EGR2, CBP and p300 were identified as acetyltransferases, while HDAC6, 10 and SIRT1 were identified as deacety-lases of EGR2. Although the NuRD complex containing HDAC1 and HDAC2 is known to associate with EGR2, the present study suggests that acetylation of EGR2 is regulated independently of NuRD. (C) 2017 Elsevier Inc. All rights reserved.

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an a-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting a-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-Au-III complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.

The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

Malformin A(1), a cyclic pentapeptide, was isolated from the marine-derived fungus Aspergillus tubingensis IFM 63452. The identity of the compound was established based on TOFMS and H-1 NMR data. Malformin A(1) exhibited trypanocidal activity against Trypanosoma congolense (IC50: 15.08 ng/mL). Interestingly, the compound was selective for T. congolense rendering a selectivity index value that ranged from 3.33 to 4.67. It also demonstrated cytotoxicity against HeLa (IC50: 50.15 ng/mL) and P388 (IC50: 70.38 ng/mL) cell lines. To further identify the possible mechanism of its cytotoxic effect, immunofluorescence staining was conducted to follow the epigenetic changes induced by the compound in the amino acid lysine of histone H3 and H4 in HeLa. The compound induced repressive levels of H3K27me3, H3K27ac and H4K5ac, and enhanced levels of H3K9me2, H3K9me3 and H4K16ac supporting the compound's chemotherapeutic potential.

A bio-inspired cascade reaction has been developed for the construction of the marine natural product ageladineA and a de novo array of its N1-substituted derivatives. This cascade features a 2-aminoimidazole formation that is modeled after an arginine post-translational modification and an aza-electrocyclization. It can be effectively carried out in a one-pot procedure from simple anilines or guanidines, leading to structural analogues of ageladineA that had been otherwise synthetically inaccessible. We found that some compounds out of this structurally novel library show a significant activity in modulating the neural differentiation. Namely, these compounds selectively activate or inhibit the differentiation of neural stem cells to neurons, while being negligible in the differentiation to astrocytes. This study represents a successful case in which the native biofunction of a natural product could be altered by structural modifications.

The core structure of cristaxenicin A having trans-fused dihydropyran and nine membered ring has been synthesized and evaluated the antileishmanial activity. The dihydropyran ring was synthesized by [4+2] cycloaddition reaction between an unsaturated aldehyde and a beta-alkoxy-alpha,beta-unsaturated ketone. The nine membered ring possessing alpha,beta-unsaturated aldehyde was constructed by the intramolecular NHK reaction followed by the Mitsunobu rearrangement. The racemic core structure of cristaxenicin A was evaluated the anti-leishmanial activity with an IC50 value of 2.4 mu M. (C) 2016 Elsevier Ltd. All rights reserved.

Acetylation is a well-characterized histone modification, which plays important roles in controlling epigenetic gene expression, and its malfunction is tightly associated with cancer. By taking advantage of the specific binding of BRD4 to acetylated lysine residues, we developed a FRET-based probe for visualizing histone H3 acetylation in living cells. BRD4, a protein known to be involved in acute myeloid leukemia and nuclear protein in testis midline carcinoma, recognizes the acetylation of histone H3 via its bromodomains. The probe exhibited a significant change in FRET signaling that was dependent on histone H3 acetylation. Mutagenesis studies revealed that an increase in the emission ratio reflected the acetylation of either K9 or K14 of histone H3 within the probe. Since BRD4 has increasingly drawn attention as a new anticancer drug target, we demonstrated that the developed fluorescent probe will also serve as a powerful tool to evaluate BRD4 inhibitors in living cells.

Piceatannol (3,3', 4', 5-trans-tetrahydroxystilbene) is a polyphenolic compound abundant in the seeds of passion fruit (Passiflora edulis). Piceatannol is an analogue of resveratrol (3, 4', 5-trans-trihydroxystilbene) and shares the structural motif and biological activities such as activation of SIRT1. Several studies have shown that piceatannol is more potent than resveratrol. In this study, we examined the effects of piceatannol on neural stem cell differentiation into astrocytes compared with those of resveratrol. At a concentration of 2.5 mu M, piceatannol promoted astrocyte differentiation, while resveratrol had no effect at this concentration. Furthermore, we found that oral administration of piceatannol increased the number of astrocytes in the brains of adult mice, while resveratrol administration showed no effects. These results suggest that piceatannol has a superior effect to resveratrol in promoting astrocyte differentiation.

Nodal signaling plays critical roles during embryonic development. The Nodal gene is not expressed in adult tissues but is frequently activated in cancer cells, contributing to progression toward malignancy. Although several regulatory elements of the Nodal gene have been identified, the epigenetic mechanisms by which Nodal expression is regulated over the long term remain unclear. We found a region exhibiting dynamic changes in DNA methylation at approximately -3.0 kb to -0.4 kb upstream from the transcriptional start site (TSS) that we termed the epigenetic regulatory element (ERE). The ERE was unmethylated in mouse embryonic stem cells (mESCs) but became increasingly methylated in differentiated cells and tissues, concomitant with the downregulation of Nodal mRNA expression. In vitro reporter assays identified an Oct3/4 binding motif within the ERE, indicating that the ERE is responsible for the activation of Nodal in mESCs. Furthermore, the ERE was a target of differentiation-associated Polycomb silencing, and the chromatin condensed when mESCs differentiated to embryoid bodies (EBs). Pharmacological inhibition of PRC2 led to the reactivation of Nodal expression in EBs and mouse embryonic fibroblasts (MEFs). The ERE was also targeted by PRC2 in normal human cells. In NODAL-expressing human cancer cells, accumulation of EZH2 and trimethylation of H3K27 at the ERE were diminished. In conclusion, Nodal is epigenetically controlled through the ERE in the mouse embryo and human cells. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Guanylation proceeded efficiently upon treatment of the various amines with cyanamide in the presence of catalytic amounts of scandium(III) triflate under mild conditions. The method did not require the guanylation reagents to be preactivated, and the reaction proceeded efficiently in water. The method, therefore, has practical utility for substrates that dissolve only in aqueous solutions, for example, peptides or pharmacologically important compounds.

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner. (C) 2013 Elsevier Inc. All rights reserved.

Cinanthrenol A (1), a new steroid composed of a phenanthrene and a spiro[2,4]heptane system, was isolated from the marine sponge Cinachyrella sp. It is the first phenathrene-containing steroid with estrogen activity.

Pregnane-X-receptor (PXR) is a member of nuclear receptors superfamily that activates gene transcription by binding to responsive elements in the promoter of target genes. PXR is a master gene orchestrating the expression/activity of genes involved in the metabolism of endobiotics including bilirubin, bile acids, glucose and lipid. In addition PXR oversights the metabolism of the large majority of xenobiotics including a large amount of prescribing drugs. Thus, developing PXR ligands represents a great opportunity for a therapeutic intervention on human diseases including diabetes, obesity, dyslipidemias and liver disorders. To this end, natural compounds represent an arsenal of new chemical scaffolds useful for the identification of novel PXR ligands. Here, we report a series of 4-methylenesteroid derivatives isolated from Theonella marine sponges as novel PXR modulators. In addition, combining medicinal chemistry, pharmacological experiments and computational studies, we have investigated the effects of different modifications on ring A and on the side chain of 4-methylenesteroid derivatives toward PXR modulation. This study provides the molecular bases of ligand/PXR interaction useful for designing novel PXR modulators. (C) 2013 Elsevier Masson SAS. All rights reserved.

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate
that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers. © 2013 American Chemical Society.

We report the conformational analysis of kulokekahilide-2, a cytotoxic cyclic depsipeptide, and its analogues. We also evaluated their cytotoxicity against human cancer cells. Although both the cis and trans conformations are possible for the amide bond between MePhe and MeGly, only one conformation was observed in DMSO. We also reveal that the configuration at C-43 in kulokekahilide-2 controls intramolecular ester exchange between the 26- and 24-membered cyclic depsipeptides. Kulokekahilide-2 and its analogues were evaluated for their cell-growth inhibition profile and using COMPARE analysis, which suggested a mechanism of action different from that of standard anticancer drugs. (C) 2013 Elsevier Ltd. All rights reserved.

Kulokekahilide-2, a 26-membered cyclodepsipeptide, was isolated from Hawaiian marine mollusk and possessed potent cytotoxicity in mammalian tumor cells. In the present study, we synthesized kulokekahilide-2 and its derivatives and examined the structure-activity relationships of these peptides in human cancer cells (A549, K562, and MCF7 cells). This study demonstrated that the cyclization of depsipeptide and the chirality of the 21 position in Ala in kulokekahilide-2 were important for its cytotoxic property and that addition of halogen at the para position of phenyl group in the 24-D-MePhe in kulokekahilide-2 as well as some derivatives remarkably increased their cytotoxicity in human cancer cells. These results suggest that the modifications of 24-D-MePhe in kulokekahilide-2, preserving its cyclization and the chirality at the 21-position, are promising strategy for exploring new derivative of kulokekahilide-2 as anti-tumor drug. (c) 2012 Elsevier Ltd. All rights reserved.

A new xenicane diterpenoid, cristaxenicin A (1), has been isolated from the deep sea gorgonian Acanthoprimnoa cristata. The structure of 1 was elucidated on the basis of spectral analysis including NMR and MS. The absolute configuration of 1 was determined on the basis of quantum chemical calculation of CD spectra. Cristaxenicin A (1) showed antiprotozoal activities against Leishmania amazonensis and Trypanosoma congolense with IC50 values of 0.088 and 0.25 mu M, respectively.

Using theonellasterol as a novel FXR antagonist hit, we prepared a series of semi-synthetic derivatives in order to gain insight into the structural requirements for exhibiting antagonistic activity. These derivatives are characterized by modification at the exocyclic carbon-carbon double bond at C-4 and at the hydroxyl group at C-3 and were prepared from theonellasterol using simple reactions. Pharmacological investigation showed that the introduction of a hydroxyl group at C-4 as well as the oxidation at C-3 with or without concomitant modification at the exomethylene functionality preserve the ability of theonellasterol to inhibit FXR transactivation caused by CDCA. Docking analysis showed that the placement of these molecules in the FXR-LBD is well stabilized when on ring A functional groups, able to form hydrogen bonds and pi interactions, are present.

Marine invertebrates are the rich source of small molecules with unique chemical skeletons and potent bioactivities; however, the major biological activities tested for marine natural products have been mainly limited to the traditional phenotype-oriented bioactivities such as cytotoxicity or antimicrobial activities. Enzyme inhibition is the target-oriented bioactivity and small molecule inhibitors are quite useful for controlling biological processes and understanding complicated biological phenomena. Recently, it has been recognized that the natural products are superior source of small molecule probes useful for the chemical biology. Especially, marine-derived enzyme inhibitors still remain to be fully investigated in spite of their high potentials as a source of unique compounds. In this chapter, selected enzyme inhibitors from marine invertebrates are described.

We successfully completed the total synthesis of kulokekahilide-2, a cytotoxic depsipeptide from the Cephalaspiedean mollusk Phillinopsis speciosa. We have revised the absolute stereochemistry of kulokekahilide-2 to 21-L-Ala, 24-D-MePhe, 37-L-Ile, 43-D-Ala, 15-D-Hica, and 5S,6S,7S-Dtda. We also investigated the cause of the mis-assignment of the configuration in the originally proposed kulokekahilide-2 and concluded that methanolysis using MeONa caused partial racemization, which led to the mis-assignment. The structure activity relationships of kulokekahilide-2 and its analogs indicate the importance of an L-amino acid at position 21. (C) 2011 Elsevier Ltd. All rights reserved.

Two dimeric steroid derivatives, shishicrellastatin A (1) and B (2), have been isolated as cathepsin B inhibitors from the marine sponge Crella (Yvesia) spinulata. Their structures were determined by interpretation of spectroscopic data. Shishicrellastatins inhibit cathepsin B with an IC(50) value of 8 mu g/mL each. (C) 2011 Elsevier Ltd. All rights reserved.

Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding.

A one-pot synthesis of ageladine A and analogues is reported. The key Pictet - Spengler reaction between 2-aminohistamine and aryl aldehydes has been successfully utilized for the synthesis of the natural product and 14 analogues. These compounds were screened for their matrix metalloprotease (MMP) and kinase inhibition to develop the first structure-activity relationship of ageladine A analogues. One compound, which showed significant kinase activity but little MMP inhibitory activity, was found to be highly active in an antiangiogenic screen, suggesting that the angiogenic activity of ageladine A is not associated with MMP inhibition but rather kinase inhibitory activity. Cytotoxicity was excluded as a mode of action by the assay of ageladine A and an analogue against 60 human cell lines.

Three new antiprotozoan compounds, gracilioethers A-C (1-3), have been isolated from the marine sponge Agelas gracilis. Their structures were elucidated on the basis of spectroscopic and chemical methods. Gracilioethers A-C showed antimalarial activity against Plasmodium falciparum with IC(50) values of 0.5-10 mu g/mL, whereas gracilioether B (2) also showed antileishmanial activity.

A new oxylipin, 15-hydroxy-tetracosa-6,9,12,16,18-pentaenoic acid (15-HTPE; 1) was isolated as an inhibitor of tube-formation from the soft coral Sinularia numerosa. Its structure was elucidated by means of spectral analysis and chemical degradation. 15-HTPE inhibited tube formation of EA.hy926 cells at the concentration of 20-40 mu M. (C) 2008 Elsevier Ltd. All rights reserved.

Kulokekahilide-2 is a 26-membered cyclic depsipeptide that exhibits potent cytotoxicity against HeLa and P388 cells with IC(50) values of 3.2 and 16 ng/mL, respectively. We have achieved a total synthesis of natural kulokekahilide-2, the NMR spectra of which showed complex signals because of trans and cis conformers at the amide bond. Besides, the spectra revealed that a mixture of 26- and 24-membered cyclic depsipeptides had been produced due to intramolecular ester exchange. The isolated 24-membered compound was transformed into the 26-membered compound over a period of several days. The two isomers have been shown to be in equilibrium and to display almost the same cytotoxicity. (C) 2008 Elsevier Ltd. All rights reserved.

Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual amino acid residues. The unusual amino acids are N(delta)-carbamoylasparagine and 2-(3-amino-2-hydroxy-5-oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia cells and the human colon tumor (HCT-116) cell line with an IC(50) value of 0.45 and 7.5 mu g/mL, respectively.

Three sesterterpenoids, aplysinoplides A -C (1-3), were isolated from the marine sponge Aplysinopsis digitata. Their structures were determined on the basis of spectroscopic data. They exhibited cytotoxic activity against P388 mouse leukemia cells.

A new pteridine derivative, asteropterin (1), was isolated as a cathepsin B inhibitor from the marine sponge Asteropus simplex. The structure of asteropterin (1) was elucidated by the analysis of spectral data. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of antiangiogenic activity of marine sponge derived azumamides by the in vitro vascular organization model using mouse induced pluripotent stem (iPS) cells was carried out. Azumamide E (5) strongly inhibited in vitro angiogenesis from iPS cells at 1.9 mu M while azumamide A (1) showed only weak inhibition at 19 mu M. These results were well correlated with HDAC inhibitory activity of these compounds, revealing the prospect of azumamides as the probe molecules useful for stem cell chemical biology. (C) 2008 Published by Elsevier Ltd.

Three lipopeptides, ciliatamides A-C (1-3), were isolated from the deep-sea sponge Aaptos ciliata, and their structures were elucidated on the basis of spectroscopic and chemical methods. Ciliatamides A (1) and B (2) were found to be antileishmanial, while 2 also exhibited marginal cytotoxicity to HeLa cells.

Kulokekahilide-2 is a potent cytotoxic depsipeptide isolated from the Hawaiian marine mollusk Philinopsis speciosa. The structure of kutokekahilide-2 was reported to be composed of five amino acids (L-Ala, L-Ile, MeGly, L-MePhe, D-Ala) and two hydroxy acids (D-Hica, 5S,6S,7S-Dtda); however, following its total synthesis, the (1)H NMR spectrum of the synthetic compound was found to be different from that of the natural one, suggesting that the stereochemistry of the reported structure was incorrect. To determine the stereochemistry of the natural compound, arrays of analogues have been prepared using different sets of chiral amino acids, and the absolute stereochemistry of kulokekahilide-2 has been unambiguously confirmed to involve the combination 21-L-Ala, 24-D-MePhe, and 43-D-Ala. (C) 2007 Elsevier Ltd. All rights reserved.

Two new polyacetylene carboxylic acids, petroformynic acids B (3) and C (4), were isolated from a marine sponge Pertrosia sp. as cytotoxic constituents. Their structures were determined by interpretation of 2D NMR data and tandem FABMS data. Absolute stereochemistry of 3 was assigned by application of the modified Mosher analysis. Petroformynic acids exhibit moderate cytotoxic activity against P388 cells.

In the course of our screening program for regulators of a molecular chaperone GRP78 expression, we isolated a novel inhibitor of GRP78 expression, designated as prunustatin A, from Streptomyces violaceoniger 4521-SVS3. The planar structure of prunustatin A was determined to be an oxidized type of the neoantimycin family. Its absolute stereochemistry was established to be 2R, 4S, 6S, 7R, 9S, and 29S by analyzing chemically degraded components obtained from the derivative of prunustatin A.

Three new serinolipid derivatives, shishididemniols C (1), D (2), and E (3), were isolated as antibacterial constituents of a tunicate of the family Didemnidae. Their planar structures were elucidated by interpretation of NMR and MS data, whereas the absolute stereochemistry was determined by chemical conversions. Shishididemniols C (3), D (4), and E (5) exhibited antibacterial activity against the fish pathogenic bacterium Vibrio anguillarum. similar to 2007 Published by Elsevier Ltd.

A 12-step synthesis of the angiogenesis inhibitory marine metabolite ageladine A is reported. The key steps include a 6 pi-1-azatriene electrocyclization for formation of the pyridine ring and a Suzuki-Miyaura coupling of N-Boc-pyrrole-2-boronic acid with a chloroimidazopyridine. In addition, an assessment of the biological activity of a variety of synthetic analogues of ageladine A prepared during this synthesis is described.

A new dimeric oroidin derivative, carteramine A (1), was isolated as a neutrophil chemotaxis inhibitor from the marine sponge Stylissa carteri. The structure of 1 was elucidated by the analysis of spectral data. (c) 2007 Elsevier Ltd. All rights reserved.

The unprecedented diastereoselective Mannich reaction of a Z-allylsulfoximine was a key step in the total synthesis of the marine natural products azumamide A and E, and an unnatural analogue. Their relative potency as histone deacetylase inhibitors was evaluated and found to correlate with predicted zinc-binding affinity.

Two new serinolipid derivatives, shishididemniols A (1) and B (2), were isolated as antibacterial constituents of a tunicate of the family Didemnidae. The structure of 1 was elucidated by interpretation of spectral data and the application of the modified Mosher method to 1 and its suitable degradation products. Compound 2 was the chlorohydrin of 1. Compounds 1 and 2 exhibited antibacterial activity against fish pathogenic bacterium Vibrio anguillarum.

An attempt to carry out a total synthesis of kulokekahilide-2 (1) by macrolactonization of a seco acid prepared from a suitably protected hexapeptide and a dioxy acid moiety unexpectedly resulted in the formation of the 43-epimer (1a) of the cytotoxic depsipeptide, for which structure 1b has previously been proposed. A second attempt involving macrolactamization of the corresponding amino acid gave the target product, 1b, but the spectral data of the product did not match those of natural 1. Furthermore, neither 1a nor 1b showed any cytotoxicity, from which it is concluded that the structure of natural 1 is incorrect and should be re-examined. © 2007 Elsevier Ltd. All rights reserved.

Marine sponges contain structurally intriguing and biologically active peptides of nonribosomal peptide synthase origin, often containing amino acids with novel structures. Here we report the discovery of asteropine A (APA), a cystine knot to be isolated from marine sponges. The solution structure of APA as determined by NMR belongs to the four-loop class of cystine knots similar to those of some conotoxins and spider toxins. However, the highly negatively charged surface of APA is uncommon among other cystine knots. APA competitively inhibits bacterial sialidases, but not a viral sialidase. APA was inactive against all other enzymes tested and did not have any apparent antitumor activity. Our data suggest that APA and other knotting peptides may be important leads for antibacterial and even antiviral drug development. © 2006 Elsevier Ltd. All rights reserved.

A zoom in on azumamides (1): Azumamides A-E are five new cyclic tetrapeptides isolated from the marine sponge Mycale izuensis. The azumamides display inhibitory activity toward histone deacetylase with IC50 values of 0.045-1.3 μg mL-1. Their structures were determined on the basis of spectroscopic analysis and chemical degradation. (Chemical Equation Presented) © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Structure-activity relationship of 13-deoxytedanolide showed that the southern and northern hemispheres play independent roles to exhibit potent biological activity. To obtain information of structure-activity relationships (SARs) of 13-deoxytedanolide, its chemical transformation has been carried out, targeting on such functional groups as an epoxide, hydroxyls, ketones, and olefins. A total of 10 derivatives have been prepared and their cytotoxicity against P388 murine leukemia cells and inhibitory activity of polypeptide elongation in yeast cell lysate provided some important SARs; the southern hemisphere comprises the pharmacophore, while the epoxide-bearing side chain is essential for the activity. © 2004 Elsevier Ltd. All rights reserved.

Axinelloside A was isolated from the lipophilic extract of the Japanease marine sponge Axinella infundibula as a strong human telomerase inhibitor (IC50 2.0 μg/mL). It has the molecular weight of 4780.4 as the monoisotopic mass of the 19 sodium salt. The chemical structure was elucidated mainly by spectroscopic methods (2D NMR and MS). Axinelloside A consists of twelve sugars, e.g., a scyllo-inositol, a D-arabinose, 5 D-galactoses, and 5 L-fucoses, together with an (R)-3-hydroxy-octadecanoic acid, 3 (E)-2-hexadecenoic acids, and 19 sulfates. © 2005 American Chemical Society.

A new α-glucosidase inhibitor, penasulfate A, has been isolated from a marine sponge Penares sp. Its structure was elucidated by spectral and chemical methods to be a scalemic mixture of methyl pipecolates acylated with a novel sulfated fatty acid.

A cytotoxic depsipeptide, kulokekahilide-2 (1), was isolated from a cephalaspidean mollusk, Philinopsis speciosa. The structure elucidation of kulokekahilide-2 was carried out by spectroscopic analysis and chemical degradation. Kulokekahilide-2 showed potent cytotoxicity against several cell lines (P388, SK-OV-3, MDA-MB-435, and A-10 with IC50 values ranging from 4.2 to 59.1 nM) indicating cancer cell selectivity.

The newly developed assay system using recombinant Leishmania amazonensis expressing enhanced green fluorescent protein (La/egfp) has been applied to the screening of Japanese marine sponges for antileishmanial activity. Bioassay-guided fractionation of an active sponge Neopetrosia sp. afforded an active compound which was identified as renieramycin A by spectroscopic analysis. It inhibited La/egfp with an IC50 value of 0.2 µg/mL.

Three new α-glucosidase inhibitors, schulzeines A-C (1-3), were isolated from the marine sponge Penares schulzei. Their structures were elucidated by spectral analysis and chemical degradations to be the isoquinoline alkaloids, encompassing two amino acids, and C28 fatty acid, the last of which was sulfated. Absolute stereochemistry of schulzeines was determined by application of the modified Mosher analysis to fragments obtained by chemical degradation. Schulzeines A-C inhibit α-glucosidase with IC50 values of 48-170 nM.

α-Glucosidases not only process protein glycosylation, but also control oligosaccharide metabolism. Thus, inhibitors of α-glucosidases are potential therapeutics for the treatment of such diseases as viral diseases, cancer, and diabetes. In the course of our continuing search for potential drug leads from Japanese marine invertebrates, we found a potent α-glucosidase-inhibitory activity in the hydrophilic extract of the marine sponge Penares schulzei, whose bioassay-guided fractionation afforded three new tetrahydroisoquinoline alkaloids named schulzeines A-C. The structure of schulzeine A was elucidated by spectral analysis and chemical degradations to be the isoquinoline alkaloids, encompassing two amino acids and a C_<28> trisulfated fatty acid. The absolute stereochemistry of schulzeine A was determined by application of the advanced Mosher analysis to fragments obtained by chemical degradation. Schulzeine B was a desmethyl derivative of schulzeine A, with a stereogenic center in the tricyclic portion being epimerised. Schulzeine C was an epimer of schulzeine B. NMR data of the tricyclic part in schulzeine C was superimposable on that of schulzeine A, while the fatty acid portion was identical with that of schulzeine B. Schulzeines A-C inhibit α-glucosidase with IC_<50> values of 48-170nM. It should be noted that desulfated schulzeines also inhibited the enzyme albeit with diminished activity, thereby indicating that the activity of schulzeines was not thoroughly due to the presence of the three sulfate groups.

A new cytotoxic diterpene, (Z)-sarcodictyin A, was isolated from the Japanese soft coral Bellonella albiflora.1 Its structure was elucidated on the basis of 1D and 2D NMR analysis and chemical degradation. (Z)-Sarcodictyin A showed strong cytotoxicity against HeLa human cervix cells.

Callysponginol sulfate A (1) was isolated from the marine sponge Callyspongia truncata as a membrane type 1 matrix metalloproteinase (MT1-MMP) inhibitor. Its structure was elucidated by a combination of spectroscopic and chemical methods and found to be a new sulfated C24 acetylenic fatty acid. Compound 1 inhibited MT1-MMP with an IC50 of 15.0 μg/mL.

(Matrix presented) Three new kapakahines E-G (1-3) have been isolated from the marine sponge Cribrochalina olemda. Limited quantities of these compounds required not only NMR analysis but also FAB-MS/MS analysis for the structure elucidation. Kapakahine E showed cytotoxicity against P388 murine leukemia cells.

The cytotoxic depsipeptide kulokekahilide-1, which contains two unusual amino acids, 4-phenylvaline and 3-amino-2-methylhexanoic acid, was isolated from the cephalaspidean mollusk Philinopsis speciosa. Structure elucidation of kulokekahilide-1 was carried out by spectroscopic analysis and chemical degradation. The absolute stereochemistry was determined by Marfey analysis for amino acids and chiral HPLC analysis for hydroxy acids. All four stereoisomers of 4-phenylvaline and 3-amino-2-methylhexanoic acid, which were necessary for Marfey analysis, were synthesized by use of the Heck reaction and Evans's method, respectively. Kulokekahilide-1 showed cytotoxicity against P388 murine leukemia cells with an IC50 value of 2.1 μg/mL.

A new polyacetylenic acid, callyspongynic acid (1), was isolated as an α-glucosidase inhibitor from the marine sponge Callyspongia truncata. Its structure was determined by spectroscopic and chemical methods.

Sodium 1-(12-hydroxy)octadecanyl sulfate (1) was isolated from a marine tunicate as a matrix metalloproteinase 2 (MMP2) inhibitor.1 The structure was elucidated by a combination of spectroscopic and chemical methods. Compound 1 inhibited MMP2 with an IC50 value of 9.0 μg/mL.

A neuraminidase inhibitor, nobiloside (1), was isolated from the marine sponge Erylus nobilis Thiele, 1903. Its structure was determined as a penasterol trisaccharide. The absolute configurations were determined by NMR and chiral GC analysis. It inhibited neuraminidase from the bacterium Clostridium perfringens with an IC50 value of 0.46μg/mL.

(2-Hydroxyethyl) dimethylsulfoxonium chloride (1), the well-known causative agent of Dogger Bank Itch, has been isolated as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis. The structure of 1 was determined by spectral and X-ray crystallographic analyses. © 2001 Elsevier Science Inc.

Ancorinosides B-D were isolated from the marine sponge Penares sollasi Thiele as inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP).1 Their structures were elucidated to be tetramic acid glycosides related to ancorinoside A by spectroscopic and chemical methods. Ancorinosides B-D inhibited MT1-MMP with IC50 values of 180-500 μg/mL. © 2001 Elsevier Science Ltd.

Two inhibitors of membrane-type matrix metalloproteinase (MT1-MMP) were isolated from a marine sponge Cribrochalina sp. Their structures were elucidated as unusual phosphorylated sterol sulfates by spectroscopic and chemical methods. One of the sterol sulfates was found to be identical to haplosamate A, whose structure was previously proposed to be a steroidal sulfamate ester, which led to structure revisions of both haplosamates A and B. The absolute stereochemistry of haplosamate A was determined by chemical transformation and the modified Mosher's method. The sterol sulfates inhibited MT1-MMP with IC50 values of 150 and 160 μg/mL, respectively. © 2001 Elsevier Science Ltd.

Three new bromotyrosine-derived metabolites, tokaradines A (1), B (2), and C (3), were isolated from the marine sponge Pseudoceratina purpurea. Their structures were determined by spectral methods. Tokaradines were lethal to the crab Hemigrapsus sanguineus. © 2001 Elsevier Science Ltd. All rights reserved.

Three new sulfated ceramides, calyceramides A-C (1-3) were isolated as inhibitors of neuraminidase from the marine sponge Discodermia calyx. Their structures were determined by spectroscopic and chemical methods. The ceramides inhibited neuraminidase with IC50 values of 0.2-0.8 μg mL-1. © 2001 Elsevier Science Ltd.

Penarolide sulfates A1 (1) and A2 (2) have been isolated as α-glucosidase inhibitors from a marine sponge Penares sp.1 Their structures including absolute stereochemistry were determined to be unique 30- and 31-membered macrolides encompassing a proline residue and three sulfate groups by interpretation of spectral data and chemical degradation. Penarolide sulfates A1 (1) and A2 (2) inhibit α-glucosidase with IC50 values of 1.2 and 1.5 μg/mL, respectively. (C) 2000 Elsevier Science Ltd.

Six new peptides with serine protease inhibitory activity have been isolated from the marine sponge Theonella swinhoei. Their structures including absolute stereochemistry were unambiguously established by interpretation of spectral data and chemical degradation. Pseudotheonamides A1 (1), A2 (2), and B2 (3) are linear pentapeptides embracing the rare piperazinone and piperidinoiminoimidazolone ring systems. Pseudotheonamide C (4) contains v-Tyr (vinylogous tyrosine) instead of a piperazinone ring. Pseudotheonamide D (5) is a tetrapeptide which lacks a C-terminal k-Arg (α- ketohomoarginine) unit. Dihydrocyclotheonamide A (6) is a reduction product of the known cyclotheonamide A (7).

Koshikamide A1, a new cytotoxic linear peptide, was isolated from a marine sponge, Theonella sp. Its structure was elucidated by spectroscopic and Chemical methods.

A new cathepsin B inhibitor, tokaramide A (1) has been isolated from the marine sponge Theonella aff. mirabilis. Its structure was determined by spectroscopic and chemical methods. Tokaramide A inhibits cathepsin B with an IC50 value of 29.0 ng / mL.

In addition to five new depsipeptides related to kulolide-1 (1), further examination of the mollusk Philinopsis speciosa has yielded a linear peptide, pupukeamide (14), and an unprecedented macrolide, tolytoxin-23-acetate (7). Chemical makeup suggested that the compounds originate from blue-green algae and are transmitted via herbivorous mollusks to P. speciosa. This could be verified by laboratory experiment. The structures were determined largely by spectroscopic techniques and the stereochemistry by hydrolysis of the peptides followed by chiral chromatography.

Bioassay-guided isolation of serine protease inhibitors from a marine sponge, Coscinoderma mathewsi, has yielded 1-methylherbipoline salts of halisulfate-1 and of suvanine. Structures of these compounds were identified by spectroscopic analyses and literature data. Antithrombin and antitrypsin activity was determined.

Two new potent serine protease inhibitors, cyclotheonamides E2 (3) and E3 (4), have been isolated from a marine sponge of the genus Theonella. Their structures were determined by interpretation of spectral data and chemical degradation studies. They are closely related to the previously reported cyclotheonamide E, from which they differ in the N-acyl group of the alanyl side chain. Cyclotheonamides E, E2, and E3 were more active against thrombin than against trypsin.

The sacoglossan mollusk Elysia rufescens elaborates six cyclic depsipeptides, kahalalides A- F. Its green algal diet (Bryopsis sp.) contains kahalalide G, which is an acyclic analogue of kahalalide F. Further analysis of the molluskan extract revealed two new acyclic peptides, kahalalide H (1) and kahalalide J (2), which share only four amino acids (leucine, phenylalanine, serine, and valine) with kahalalide F. Both contain aspartic acid and 4-hydroxyproline residues, and kahalalide J (2) also contains lysine. They have in common a β-hydroxy fatty acid, 3-hydroxy-9- methyldecanoic, previously encountered in kahalalide E. For kahalalide H (1) we succeeded in determining the sequential positions of the antipodal D- and L-phenylalanine. In common with the acyclic constituent of the alga, kahalalide G, the new compounds lack significant cytotoxicity.

A new cyclic heptapeptide cupolamide A (1) was isolated from two samples of the sponge Theonella cupola collected in Indonesia and Okinawa. It contains two L-Val, one D-Leu, and one D-Ser, and three uncommon amino acid residues, D-homoarginine (Har), trans-4-hydroxy-L-proline (Hyp), and L-2,4-diaminobutanoic acid (Dba). The structure elucidation of 1 was performed by NMR spectroscopic techniques and the stereochemistry was determined by Marfey's method. Cupolamide A (1) was active against P388 murine leukemia cells with an IC50 = 7.5 μg/mL.

Isolation of an aminoalkylpyridine, naloamine (4), and a cytotoxic polypropionate, nalodionol (5), from Smaragdinella calyculata is described. The structures were determined by interpretation of spectral data.

From the sponge, Ircinia dendroides, collected in Indonesia we isolated a new cyclic hexapeptide, waiakeamide (1). Its structure, consisting of three proline residues, two methionine sulfoxides, and one thiazolylphenylalanine, was elucidated by spectral analysis and chemical degradation. Isolation and structural elucidation of waiakeamide is described.

Four cyclic peptides, kapakahines A-D, were isolated from the marine sponge Cribrochalina olemda. Their structures including complete stereochemistry were elucidated by spectral analysis and chemical degradation. The unique structural feature of these peptides is the lack of an amide linkage between two tryptophan residues. Instead the ring is closed by a bond from the indole nitrogen of Trp-1 to the β-carbon of Trp-2.

Kulolide, a cyclic depsipeptide, was isolated from a cephalaspidean mollusk, Philinopsis speciosa Pease, 1860. Kulolide is made up of five amino acid residues, one each of L-Ala, L-Pro, and N-Me-D-Val and two of L-Val, and two carboxylic acids, L-3-phenyllactic acid and the unprecedented (R)-3-hydroxy-2,2-dimethyl-7-octynoic acid. The kulolide structure was elucidated by spectral techniques and hydrolytic reactions. Kulolide is active against L-1210 leukemia cells and P388 murine leukemia cells at IC50 values of 0.7 and 2.l μg/mL, respectively. Kulolide caused morphological change against rat 3Y1 fibroblast cells at the concentration of 50 μM.

A linear peptide pupukeamide (1) was isolated from a cephalaspidean mollusk Philinopsis speciosa. Its structure was elucidated by spectroscopic and chemical methods. Copyright (C) 1996 Elsevier Science Ltd.

Three new thrombin and trypsin inhibitors, cyclotheonamides C (3), D (4), and E (8) were isolated from the marine sponge Theonella swinhoei. Their structures were determined by spectral and chemical methods. © 1995.

A nucleoside, kumusine (1) from an Indonesian sponge Theonella sp. is 2-chloro-9-(5′-deoxy-2′-methyl-β-D-ribofuranosyl)-adeni ne. © 1995.

A unique thrombin inhibitor, named toxadocial A (1) has been isolated from the marine sponge Toxadocia cylindrica, and its structure was determined by spectroscopic and chemical methods to be a persulfated 7, 17, 31, 41-tetrahydroxyheptatetracontane-23-carbaldehyde. © 1993.

Three new thrombin-inhibitory metabolites, toxadocial B (2), toxadocial C (3), and toxadocic acid A (4) have been isolated from the marine sponge Toxadocia cylindrica. The structures of these compounds were determined by spectroscopic and chemical methods. © 1993.

New cytotoxic compounds 1-7 were isolated along with onnamide A (9) from a marine sponge Theonella sp. collected at Hachijo Island, whereas another Theonella sponge yielded 8. The structures of these compounds were determined by interpretation of the NMR spectral data as well as by comparison of spectral data with those of 9. Six compounds (1-6) were highly cytotoxic against the P388 cell line. © 1992.

Nazumamide A, a thrombin-inhibiting linear @Tetrapeptide, was isolated from a marine sponge, Theonella sp. The structure of nazumamide A was determined by interpretation of 2D NMR data and by chemical degradation as 2,5-dihydroxybenzoyl-l-arginyl-l-prolyl-l-isoleucyl-l-α-aminobutyric acid. © 1991.