Semisynthesis of Antitrypanosomal p-Quinone Analog Possesing the Komaroviquinone Pharmacophore.

A p-quinone analog having the komaroviquinone pharmacophore fused with a more conformationally flexible cycloheptane ring, was semisynthesized from natural demethlsalvicanol isolated from Perovskia abrotanoides via four steps in 26% overall yield. The IC50 for the antitrypanosomal activity of the analog was 0.55 µM.

Trehangelins ameliorate inflammation-induced skin senescence by suppressing the epidermal YAP-CCN1 axis.

Trehangelins (THG) are newly identified trehalose compounds derived from broth cultures of an endophytic actinomycete, Polymorphospora rubra. THG are known to suppress Cellular Communication Network factor 1 (CCN1), which regulates collagen homeostasis in the dermis. Although the physical properties of THG suggest a high penetration of the stratum corneum, the effect of THG on the epidermis has not been reported. Here we describe a possible mechanism involved in skin aging focusing on the effect of THG on epidermal CCN1. This study shows that: (1) THG suppress epidermal CCN1 expression by inhibiting the translocation of Yes-Associated Protein (YAP) to nuclei. (2) Epidermal CCN1, localized at the basement membrane, regulates the balance between the growth and differentiation of keratinocytes. (3) Keratinocytes secrete more CCN1 than fibroblasts, which leads to disruption of the basement membrane and extracellular matrix components. (4) The secretion of CCN1 from keratinocytes is increased by ultraviolet B exposure, especially in aged keratinocytes, and deteriorates the elastic fiber structures in the underlying dermis. (5) Topical application of THG ameliorates the structure of the basement membrane in ex vivo human skin explants. Taken together, THG might be a promising treatment for aged skin by suppressing the aberrant YAP-CCN1 axis.

Aldsulfin, a novel unusual anti-mannheimiosis epithiodiketopiperazine antibiotic produced by Lasiodiplodia pseudotheobromae FKI-4499.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

Jietacins, azoxy natural products, as novel NF-κB inhibitors: Discovery, synthesis, biological activity, and mode of action.

Deregulation of NF-κB plays an important role in various diseases by controlling cell growth, inflammation, the immune response, and cytokine production. Although many NF-κB inhibitors have been developed, to the best of our knowledge, none of them have been successfully translated into clinical practice as medicines. To overcome this issue, we aimed to develop a new class of NF-κB inhibitors. Previous reports indicated that the N-terminal cysteine is a promising target for NF-κB. Based on this, we first selected 10 natural products or their derivatives from the natural product library that we developed and examined the effect on NF-κB and the viability of cancer cells with constitutively strong NF-κB activity. Among them, we found that an azoxy natural product, jietacin A, with a vinylazoxy group and an aliphatic side chain, reduced cell viability and inhibited nuclear translocation of free NF-κB. In addition, we performed design, synthesis, and biological evaluation of jietacin derivatives for development of a novel NF-κB inhibitor. Of these derivatives, a fully synthesized derivative 25 with vinylazoxy and ynone groups had a potent effect. We clarified the structure-activity relationship of this compound. Jietacin A and 25 also inhibited tumor necrosis factor-α-mediated induction of NF-κB. The NF-κB inhibitory effect depended on the N-terminal cysteine and the neighboring Arg-Ser-Ala-Gly-Ser-Ile (RSAGSI) domain of NF-κB. We also found that 25 inhibited the association between NF-κB and importin α, suggesting inhibition of NF-κB at an early step of nuclear translocation. Overall, this study indicated that the vinylazoxy motif may compose a new class of NF-κB inhibitors, providing further insight for rational drug design and rendering a unique mode of action.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

Peyer's patch-immunomodulating glucans from sugar cane enhance protective immunity through stimulation of the hemopoietic system.

The aim of the present study was chemical clarification of in vitro Peyer's patch-immunomodulating polysaccharides in sugar cane molasses, and evaluation of in vivo modulating activity on immune function of T lymphocytes in Peyer's patches and on microenvironment of hemopoietic system. Five kinds of glucans, comprising of dextranase-sensitive and activity-related d-glucosyl moieties, were purified as in vitro Peyer's patch-immunomodulating polysaccharides from the molasses. Oral administration of a glucan-enriched subfraction induced IL-2 and GM-CSF-producing T lymphocytes in Peyer's patches, resulting in enhancement of IL-6 production in a hemopoietic microenvironment to boost neutrophil numbers in the peripheral blood stream. Oral administration of purified glucan or glucan-enrich sub-fraction of sugar cane reduced the number of Plasmodium berghei- or P. yoelii-infected erythrocytes in a murine infection model, using polysaccharide alone or via co-administration with the antimalarial drug, artesunate. These results suggested that Peyer's patch-immunomodulating glucans enhanced protective immunity through axis of Peyer's patches-hemopoietic system.

An Application of Photoactivatable Substrate for the Evaluation of Epithelial-mesenchymal Transition Inhibitors.

Epithelial-mesenchymal transition (EMT), phenotypic changes in cell adhesion and migration, is involved in cancer invasion and metastasis, hence becoming a target for anti-cancer drugs. In this study, we report a method for the evaluation of EMT inhibitors by using a photoactivatable gold substrate, which changes from non-cell-adhesive to cell-adhesive in response to light. The method is based on the geometrical confinement of cell clusters and the subsequent migration induction by controlled photoirradiation of the substrate. As a proof-of-concept experiment, a known EMT inhibitor was successfully evaluated in terms of the changes in cluster area or leader cell appearance, in response to biochemically and mechanically induced EMT. Furthermore, an application of the present method for microbial secondary metabolites identified nanaomycin H as an EMT inhibitor, potentially killing EMTed cells in disseminated conditions. These results demonstrate the potential of the present method for screening new EMT inhibitors.

Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 µM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 µM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

Decatamariic acid, a new mitochondrial respiration inhibitor discovered by pesticidal screening using drug-sensitive Saccharomyces cerevisiae.

A new decalin, decatamariic acid, was isolated from a cultured broth of the fungus Aspergillus tamarii FKI-6817. Its absolute configuration was elucidated by NMR and electronic circular dichroism. Decatamariic acid (10 µM) elicited ~50% inhibition of the ATP production in mitochondria isolated from wild-type Saccharomyces cerevisiae without affecting the activities of respiratory enzymes. The action manner of this compound may be interesting as a possible seed for new pesticides.

Herquline A, produced by Penicillium herquei FKI-7215, exhibits anti-influenza virus properties.

In the course of screening for new anti-influenza virus antibiotics, we isolated herquline A from a culture broth of the fungus, Penicillium herquei FKI-7215. Herquline A inhibited replication of influenza virus A/PR/8/34 strain in a dose-dependent manner without exhibiting cytotoxicity against several human cell lines. It did not inhibit the viral neuraminidase.

Synthesis and stereochemical determination of an antiparasitic pseudo-aminal type monoterpene indole alkaloid.

5-Nor stemmadenine alkaloids, isolated from the genus Tabernaemontana, display a range of bioactivity. 16-Hydroxy-16,22-dihydroapparicine, the active component of an extract from the Tabernaemontana sp. (dichotoma, elegans, and divaricate), exhibited potent antimalarial activity, representing the first such report of the antimalarial property of 5-nor stemmadenine alkaloids. We, therefore, decided to attempt the total synthesis of the compound to explore its antimalarial activity and investigate structure and bioactivity relationships. As a result, we completed the first total synthesis of 16-hydroxy-16,22-dihydroapparicine, by combining a phosphine-mediated cascade reaction, diastereoselective nucleophilic addition of 2-acylindole or methylketone via a Felkin-Anh transition state, and chirality transferring intramolecular Michael addition. We also clarified the absolute stereochemistries of the compound. Furthermore, we evaluated the activity of the synthetic compound, as well as that of some intermediates, all of which showed weak activity against chloroquine-resistant Plasmodium falciparum (K1 strain) malaria parasites.

Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 µM), DHA (100 µM), or siamycin I (2.5 µM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis.

Combination cellulose plate (non-agar solid support) and agar plate method improves isolation of fungi from soil.

This is the first report describing the improved isolation of common filamentous fungi via a method combining cellulose plate and agar plate system. A cellulose plate is a porous plate made of nanofibrous crystaline cellulose. Isolating fungi from soils using these types of media separately resulted in the number of fungal colonies appearing on cellulose plates being lower than that on agar plates. However, the number of actual fungal species isolated using cellulose plates alone was more or less the same as that found using agar plates. Significantly, the diversity of isolates using a combination of the two media was greater than using each media individually. As a result, numerous new or rare fungal species with potential, including previously proposed new species, were isolated successfully in this way. All fungal colonies, including the Penicillium species, that appeared on the cellulose plate penetrated in potato dextrose were either white or yellow. Cultivation on cellulose plates with added copper ion overcomes the change in coloration, the colonies appearing as they do following cultivation on potato dextrose agar.

Semisynthesis of salviandulin E analogues and their antitrypanosomal activity.

A series of analogues of salviandulin E, a rearranged neoclerodane diterpene originally isolated from Salvia leucantha (Lamiaceae), were prepared and their in vitro activity against Trypanosoma brucei brucei was evaluated with currently used therapeutic drugs as positive controls. One of the 19 compounds prepared and assayed in the present study, butanoyl 3,4-dihydrosalviandulin E analogue was found to be a possible candidate for an antitrypanosomal drug with fairly strong antitrypanosomal activity and lower cytotoxicity.

In vitro antitrypanosomal activity of bis(bibenzyls)s and bibenzyls from liverworts against Trypanosoma brucei.

During the course of our screening program to discover new antitrypanosomal compounds, 17 known plant aromatic compounds [12 bis(bibenzyls)s and 5 bibenzyls] were evaluated for in vitro activity against Trypanosoma brucei brucei. Sixteen compounds were found to exhibit antitrypanosomal activity. In particular, three compounds, marchantin A (1), plagiochin A (5) and 2(R)-2-isopropenyl-6,7-dihydroxy-4-(2-phenylethyl)dihydrobenzofuran (16) demonstrated moderate selective and potent antitrypanosomal activities in vitro. We detail here the antitrypanosomal properties and cytotoxicities of the compounds in comparison with two commonly used therapeutic drugs, eflornithine and suramin. Our finding represents the first report of the promising trypanocidal activity of these compounds. The research also provides valuable insight into structure-activity relationships and the possible mode of action of the compounds.

In vitro antitrypanosomal activity of some phenolic compounds from propolis and lactones from Fijian Kawa (Piper methysticum).

During our search to discover new antitrypanosomal compounds, eight known plant compounds (three phenolic compounds and five kawa lactones) were evaluated for in vitro activity against Trypanosoma brucei brucei. Among them, we found two phenolic compounds and three kawa lactones possessing an α-pyrone influenced antitrypanosomal property. In particular, ß-phenethyl caffeate, farnesyl caffeate and dihydrokawain exhibited high or moderate selective and potent antitrypanosomal activity in vitro. We detail here the antitrypanosomal activity and cytotoxicities of the compounds, in comparison with two commonly used antitrypanosomal drugs (eflornithine and suramin). Our findings represent the first report of the promising trypanocidal activity of these compounds.

A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium.

The type III secretion system (T3SS) is highly conserved in many Gram-negative pathogenic bacteria and functions as an injector of bacterial proteins (effectors) into host cells. T3SSs are involved in establishing disease processes, but this machinery is not essential for bacterial growth or homeostasis. Thus, T3SS is expected to be a candidate therapeutic target, and inhibitors of T3SSs could potentially reduce virulence without causing bacterial death, thereby avoiding any subsequent development of resistance. We identified a linear polyketide compound, aurodox, as a specific T3SS inhibitor from the culture broth of Streptomyces sp. using a screening system for the T3SS-mediated hemolysis of enteropathogenic Escherichia coli (EPEC) established by our group. Aurodox strongly inhibited T3SS-mediated hemolysis with an IC(50) value of 1.5 µg ml(-1) without affecting bacterial growth in liquid media. We also demonstrated that aurodox specifically inhibits the secretion of type III-secreted proteins such as EspB, EspF and Map, without affecting the expression of the housekeeping protein GroEL. Furthermore, an in vivo infection study using mice clearly indicated that the administration of aurodox allowed the mice to survive a lethal dose of Citrobactor rodentium, a model bacterium for human pathogens such as EPEC. Thus, our in vivo study directly demonstrated for the first time that this putative T3SS inhibitor can be applied as a novel class of anti-infective agents.

Oral adjuvant activity for nasal influenza vaccines caused by combination of two trihydroxy fatty acid stereoisomers from the tuber of Pinellia ternata.

Pinellic acid from the tuber of Pinellia ternata was isolated as an effective oral adjuvant for nasal influenza vaccine, and identified 9S,12S,13S-trihydroxy-10E-octadecenoic acid (9S,12S,13S) by the enantioselective total synthesis [Nagai et al., Int. Immunopharmacol., 2, 1183-93 (2002); Shirahata et al., Tetrahedron, 62, 9483-96 (2006)]. However, present study showed that synthetic 9S,12S,13S that was nearly 100% pure was not effective as an oral adjuvant. HPLC analysis also showed that the adjuvant active pinellic acid fraction from tuber of P. ternata contained the 9S,12S,13S as the main component and at least two minor components. Therefore seven other chemically synthesized stereoisomers were tested in combination with the 9S,12S,13S for oral adjuvant activity. Only the 9S,12S,13S in combination with the 9S,12R,13R isomer in a weight% ratio of 90.4:9.6 (pinellic acid mixture, PAM) was a potent oral adjuvant and elicited both antiviral IgA antibody (Ab) in bronchoalveolar lavage fluids and nasal washes and antiviral IgG(1) Ab in mice sera. Oral administration of the PAM followed by nasal influenza vaccination and infection with A/PR/8/34 virus showed increases in survival rate (22%, control versus 78% test) in mice orally administered PAM as adjuvant. Histopathological examination of lung tissue of mice given oral PAM with vaccine followed by influenza virus infection showed attenuated infiltration of inflammatory cells with decreases in the alveolar spaces and increases in the alveolar septa. The result of this study refutes the our previous study and suggests that the combination of 9S,12S,13S and 9S,12R,13R isomers is necessary for effective oral adjuvant activity when used in conjunction with nasal influenza vaccine.