A new gentiopicroside derivative improves cognitive deficits of AD mice via activation of Wnt signaling pathway and regulation of gut microbiota homeostasis.

BACKGROUND: In our previous study, we found that gentiopicroside (GPS) isolated from Gentiana rigescens Franch has a significant antiaging activity via regulation of mitophagy and oxidative stress. In order to increase the anti-aging activity of GPS, several compounds based on the chemical structure of GPS were synthesized and evaluated for bioactivity with yeast replicative lifespan assay, and 2H-gentiopicroside (2H-GPS) as leading compound was selected for AD treatment. PURPOSE AND METHODS: To investigate whether 2H-GPS has anti- Alzheimer's disease effects, we used D-galactose (Dgal)-induced model mice to evaluate the effect of 2H-GPS on AD mice. Furthermore, we explored the action mechanism of this compound with RT-PCR, Western Blot, ELISA and 16S rRNA gene sequence analysis. RESULTS: Memory dysfunction and reduction in the number of neurons in the brain of mice were observed in Dgal treated group. These symptoms of AD mice were significantly relieved by administering 2H-GPS and donepezil (Done), respectively. In the Dgal only treated group, the protein levels of ß-catenin, REST and phosphorylated GSK-3ß, involved in the Wnt signaling pathway were significantly decreased, whereas the protein levels of GSK-3ß, Tau, phosphorylated Tau, P35 and PEN-2 were significantly increased. Importantly, treatment with 2H-GPS resulted in restoration of memory dysfunction and levels of these proteins. Furthermore, the composition of the gut microbiota after 2H-GPS administration was explored through 16S rRNA gene sequence analysis. Moreover, the mice, in which depleted gut microbiota with antibiotic cocktail (ABX), were used for evaluation of whether the gut microbiota is involved to the effect of 2H-GPS. Significant changes in gut microbiota composition were observed between AD and 2H-GPS-treated AD mice, and ABX partially eliminated the AD-restoring effect of 2H-GPS. CONCLUSION: 2H-GPS improves the symptoms of AD mice through combination of the regulation of Wnt signaling pathway and the microbiota-gut-brain axis, and the mechanism of action of 2H-GPS is distinct from that of Done.

The Insulin Receptor: A Potential Target of Amarogentin Isolated from Gentiana rigescens Franch That Induces Neurogenesis in PC12 Cells.

Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.

Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods.

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA-DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Peanut skin extract ameliorates the symptoms of type 2 diabetes mellitus in mice by alleviating inflammation and maintaining gut microbiota homeostasis.

In this study, mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet were used to investigate the antidiabetic effect and mechanism of action of peanut skin extract (PSE). Results revealed that the fasting blood glucose, body weight, and food intake of mice with T2DM significantly decreased after they were given PSE. The effects of 80 mg/kg PSE were similar to those of 140 mg/kg metformin (MET). The glucose tolerance and insulin sensitivity of the mice also improved. The composition of intestinal microflora in the mice significantly changed after PSE administration. In particular, no Actinobacteria was detected in the PSE-treated group, and the ratio of Firmicutes to Bacteroidetes was remarkably reduced. PSE also increased the abundance of gut microbiota involved in fatty acid biosynthesis, lipid biosynthesis, and sucrose metabolism. The abundance of gut microbiota related to aminoacyl-tRNA biosynthesis also decreased. Lipopolysaccharide, interleukin (IL)-6, IL-1ß and tumor necrosis factor-α in the blood, liver and adipose tissue were reduced by PSE. Similarly, the mRNA expression levels of IkappaB kinase and nuclear factor kappaB in the hypothalamus were reduced by PSE. These results suggested that PSE and MET elicited significant antidiabetic effects by maintaining gut microbiota and inhibiting inflammation.

YO-001A, a new antifungal agent produced by Streptomyces sp. YO15-A001.

A new antifungal compound YO-001A was found from the culture broth of Streptomyces sp. YO15-A001, which was isolated from a soil sample collected in Toyama Prefecture. YO-001A was identified through morphological changes-based screening of the rice blast fungus, Pyricularia oryzae (P. oryzae). YO-001A is a new 26-membered macrolide of the oligomycin family, which exhibits potent antifungal activity against P. oryzae with an IC50 of 0.012 µM by disrupting mitochondrial respiration via inhibition of the FOF1-ATPase activity.

Structure Characterization and Action Mechanism of an Antiaging New Compound from Gastrodia elata Blume.

A new compound, bis(4-hydroxybenzyl)ether mono-ß-L-galactopyranoside (1), was isolated from the rhizome of Gastrodia elata Blume. Its structure was elucidated using extensive spectroscopic analysis, including 1D and 2D NMR, HR-ESI-TOF-MS, and chemical derivatization. Compound 1 extended the replicative lifespan of K6001 and the chronological lifespan of YOM36 yeast strains. To understand the mechanism of action, oxidative stress assessment, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, catalase (CAT) and total glutathione peroxidase (GPx) activity assays, and replicative lifespan assay of sod1, sod2, uth1, and skn7 yeast mutant strains were performed. Results indicated the significant increase in the survival rate of yeast under oxidative stress after treatment with 1. ROS and MDA levels were reduced significantly. Meanwhile, the activity of CAT and GPx was significantly increased. The lifespan of sod1, sod2, uth1, and skn7 mutants of K6001 was not affected by 1. Furthermore, we investigated the gene expression related to longevity after administrating 1. The significant increase of Sir2 and reduction of Uth1 gene expression in the 1-treated group were observed. These results indicated that antioxidative stress played an important role in the antiaging effect of 1; Sir2 and Uth1 genes were involved in antiaging effects of 1.

3ß,23,28-Trihydroxy-12-oleanene 3ß-Caffeate from Desmodium sambuense-Induced Neurogenesis in PC12 Cells Mediated by ER Stress and BDNF-TrkB Signaling Pathways.

3ß,23,28-Trihydroxy-12-oleanene 3ß-caffeate (compound 1) is a neuritogenic pentacyclic triterpenoid, which was isolated from Desmodium sambuense based on a PC12 cell bioassay system. Compound 1 induced neurite outgrowth dose-dependently in PC12 cells and primary cortical neurons at doses of 0.1, 0.3, and 1 µM. The potential target of compound 1 was predicted by ChemProteoBase profiling, and the mechanism of action was investigated using specific inhibitors, Western blot analysis, and PC12 [rasN17] and PC12 [mtGAP] mutants. Compound 1 activates endoplasmic reticulum (ER) as an ER stress inducer, and the maker of ER stress GRP78 protein significantly increased after treatment with compound 1. The inhibitors of tyrosine kinase B (TrkB), insulin-like growth factor 1 receptor (IGF-1R), mitogen-activated protein kinase (MEK), and phosphatidylinositol 3 kinase (PI3K) significantly decreased the neurite outgrowth induced by compound 1. Furthermore, the increases of phosphorylation of TrkB, IGF-1R, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT) were observed in the compound 1-treated group, and the phosphorylation of these proteins was diminished by corresponding inhibitors. Thus, the compound-1-induced neuritogenic activity depended on the activation of slight ER stress and associated BDNF-TrkB/Ras/Raf/ERK and IGF-1R/PI3K/AKT signaling pathways in PC12 cells.

A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell-accelerated fibroblast migration.

Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened ∼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity.

Construction of a potato fraction library for the investigation of functional secondary metabolites.

A potato fraction library was constructed to investigate functional secondary metabolites from 8 cultivars: Kitahime, Pilka, Sakurafubuki, Atlantic, Toyoshiro, Snowden, Kitamurasaki, and Northern Ruby, which were divided into flower, leaf, stem, roots, tuber peel, and tuber. Each fraction was a semi-purified extract and about 800 fractions were prepared for the library. They were analyzed by DAD-LC/MS to obtain structural information and were evaluated for various biological activities. LC/MS data showed that each part had a specific characteristic for their constituents supported by principal component analysis (PCA). Approximately 40% of fractions showed significant biological activities at 30 µg/mL, especially the flower fractions showed strong cytotoxicity. PCAs based on the activity and LC/MS data suggested that the strong cytotoxicity of flowers was derived from a complex mixture of potato glycoalkaloids. In addition, tuber peel fractions showed strong antimalarial activity, which had not been reported before. Also, some fractions showed significant antibacterial activities.

Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms.

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.

Bioactivity-guided isolation of neuritogenic triterpenoids from the leaves of Ilex latifolia Thunb.

Ilex latifolia Thunb is a traditional Chinese tea and herbal medicine. In this study, one new triterpene saponin (1) and six known triterpenoids (2-7) were isolated from the methanol extract of I. latifolia using a PC12 cell bioassay system. The structures and stereochemistry of these compounds were elucidated using spectroscopic methods and chemical derivatization. This new triterpene saponin (1) was characterized as an ursolic type acid with a 19α-hydroxyl and a trisaccharide moiety at C-3. Compound 1 significantly promoted the neurite outgrowth in PC12 cells by 52% at 10 µM, whereas compounds 2-7 showed less neuritogenic activity. Structure activity relationship studies indicated that introducing a trisaccharide moiety at C-3 is important for the neuritogenic activity, but the sugar group at C-28 decreased this activity. In addition, compound 1 increased the neurite outgrowth length in primary cortical neuron cells of mice and also exhibited a neuronal protection effect on H2O2-damaged PC12 cells at optimum concentrations.

Functional annotation of chemical libraries across diverse biological processes.

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells in the presence of a compound, revealing chemical-genetic interactions that can elucidate a compound's mode of action. We developed a highly parallel, unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized diagnostic mutant collection that is predictive for all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode-sequencing protocol, enabling the assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened seven different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway.

BACKGROUND: Neurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood-brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases. PURPOSE: The present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance. METHODS: Extract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments. RESULTS: The ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). Moreover, tyrosine kinase A (TrKA) and phosphatidylinositol 3 kinase (PI3K) were also involved in the signaling pathway. CONCLUSIONS: Two new cucurbitane triterpenoids, linderside A and lindersin B, were isolated from Lindernia crustacean. Neurite outgrowth induced by lindersin B in PC12 cells depends on activation of TrkA/PI3K/ERK signaling pathway.

Phenotypic screening meets natural products in drug discovery.

The Nobel Prize in Physiology or Medicine 2015 was awarded for discoveries related to the control of parasitic diseases using natural products of microbial and plant origin. In current drug discovery programs, synthesized compounds are widely used as a screening source; however, this award reminds us of the importance of natural products. Here, we introduce our phenotypic screening methods based on changes in cell morphology and discuss their effectiveness and impact for natural products in drug discovery.

Two new triterpenoids from the roots of Pinus densiflora.

Chemical investigation of the roots of Pinus densiflora led to the isolation of two new triterpenoids, (24S)-3ß-methoxy-24,25-epoxy-lanost-9(11)-ene (1) and 29-acetoxy-3α-methoxyserrat-14-en-21α-ol (2), together with three known serratene-type triterpenoids (3-5) and four known diterpenoids (6-9). Their structures were determined by spectroscopic analyses.

Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/ß and p38γ/δ.

Mammalian p38 mitogen activated protein kinases (MAPKs) are responsive to a variety of cellular stresses. The development of specific pyridinyl imidazole inhibitors has permitted the characterization of the p38 MAPK isoform p38α, which is expressed in most cell types, whereas the physiological roles of p38γ and p38δ are poorly understood. In this study, we report an approach for identifying selective inhibitors against p38γ and p38δ by focusing on the difference in gatekeeper residues between p38α/ß and p38γ/δ. Using GST-fused p38α wild type and T106M mutant constructs, wherein the p38α gatekeeper residue (Thr-106) was substituted by the p38γ/δ-type (Met), we performed comparative chemical array screening to identify specific binders of the mutant and identified SU-002 bound to p38αT106M specifically. SU-002 was found to inhibit p38αT106M but not p38α kinase activity in in vitro kinase assays. SU-005, the analog of SU-002, had inhibitory effects against the kinase activity of p38γ and p38δ in vitro but not p38α. In addition, SU-005 inhibited both p38γ and p38δ auto-phosphorylation in HeLa and HEK293T cells. These results demonstrate that the comparative chemical array screening approach is a powerful technique to explore specific inhibitors for mutant proteins with even single amino-acid substitutions in a high-throughput manner.

Screening of a virtual mirror-image library of natural products.

We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds.

Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening.

Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation.

Structures and biological activities of azaphilones produced by Penicillium sp. KCB11A109 from a ginseng field.

Twelve metabolites, including five highly oxygenated azaphilones, geumsanols A-E, along with seven known analogues were isolated from Penicillium sp. KCB11A109, a fungus derived from a ginseng field. Their structures were assigned by spectroscopic means (NMR and MS), and stereochemistries were determined by extensive spectroscopic analyses ((1)H-(1)H coupling constants, NOESY, and HETLOC) and chemical derivatizations (modified Mosher's method and acetonide formation). The isolates were evaluated for their anticancer, antimicrobial, antimalarial activities, and phenotypic effects in zebrafish development. Of these compounds possessing no pyranoquinone core, only geumsanol E exhibited cytotoxic activities and toxic effects on zebrafish embryos, suggesting that a double bond at C-11 and C-12 is important for biological activity.

Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups.

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies.