Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

A Diversifiable Synthetic Platform for the Discovery of New Carbasugar SGLT2 Inhibitors Using Azide-Alkyne Click Chemistry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are clinically available to control blood glucose levels in diabetic patients via an insulin-independent mechanism. It was found that some carbasugar analogs of known SGLT2 inhibitors exert a high inhibiting ability toward SGLT2 and have a prolonged blood glucose lowering effect. In this study, we designed new candidates of carbasugar SGLT2 inhibitor that can be synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) into an aromatic ring, which is a part of the pharmacophore at the final stage in the synthetic protocol for the easier discovery of superior SGLT2 inhibitors. Based on the results of molecular docking studies, some selected compounds have been synthesized. Evaluation of these compounds using a cell-based assay revealed that the majority of these compounds had SGLT2 inhibitory activity in a dose-dependent manner. The SGLT2 inhibitory activity of 7b and 7c was almost equal to that of SGLT2 inhibitors in current use. Furthermore, molecular dynamics simulations also revealed that 7c is a promising novel SGLT2 inhibitor.

Formation of Supramolecular Nanostructures through in Situ Self-Assembly and Post-Assembly Modification of a Biocatalytically Constructed Dipeptide Hydrazide.

Aqueous self-assembly of short peptides has attracted growing attention for the construction of supramolecular materials for various bioapplications. Herein, we describe how the thermolysin-assisted biocatalytic construction of a dipeptide hydrazide from an N-protected amino acid and an amino acid hydrazide leads to the formation of thermally stable supramolecular hydrogels. In addition, we demonstrate the post-assembly modification of the supramolecular architectures constructed in situ tethering hydrazide groups as a chemical handle by means of fluorescence imaging.

A platform for the discovery of new macrolide antibiotics.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

Betuletol, a Propolis Component, Suppresses IL-33 Gene Expression and Effective against Eosinophilia.

Propolis, a resinous substance produced by honeybees, has been used in folk medicine since ancient times due to its many biological benefits such as antitumor, antioxidant, antimicrobial, anti-inflammatory, and immunomodulatory effects. Propolis contains flavonoids, terpenoids, aromatic aldehydes, and alcohols, which vary with different climate and environmental conditions. In our study, we examined the antiallergic activity of Brazilian green propolis (BGP) and isolated the active compound that can suppress an allergy-sensitive gene, IL-33, expression and eosinophilia. Ethanolic extract of BGP freeze-dried powder was fractionated with several solvent systems, and the active fractions were collected based on activity measurement. The single active compound was found by thin-layer chromatography. Using column chromatography and NMR, the active compound was isolated and identified as 3,5,7-trihydroxy-6,4'-dimethoxyflavone, also known as betuletol. Further, the antiallergic activity of that has been examined in PMA-induced up-regulation of IL-33 gene expression in Swiss 3T3 cells. Our data showed the IL-33 gene suppression both by BGP and the isolated active compound, betuletol. We also found that betuletol suppressed ERK phosphorylation, suggesting it could be effective in suppressing IL-33 mediated eosinophilic chronic inflammation and will provide new insights to develop potent therapeutics against allergic inflammations.

Signaling Pathway of Histamine H1 Receptor-Mediated Histamine H1 Receptor Gene Upregulation Induced by Histamine in U-373 MG Cells.

Histamine H1 receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCß selective inhibitor Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.

Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway.

The 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway is an innate immune system that protects hosts against pathogenic viruses and bacteria through cleavage of exogenous single-stranded RNA; however, this system's selective targeting mechanism remains unclear. Here, we identified an mRNA quality control factor Dom34 as a novel restriction factor for a positive-sense single-stranded RNA virus. Downregulation of Dom34 and RNase L increases viral replication, as well as half-life of the viral RNA. Dom34 directly binds RNase L to form a surveillance complex to recognize and eliminate the exogenous RNA in a manner dependent on translation. Interestingly, the feature detected by the surveillance complex is not the specific sequence of the viral RNA but the 'exogenous nature' of the RNA. We propose the following model for the selective targeting of exogenous RNA; OAS3 activated by the exogenous RNA releases 2'-5'-oligoadenylates (2-5A), which in turn converts latent RNase L to an active dimer. This accelerates formation of the Dom34-RNase L surveillance complex, and its selective localization to the ribosome on the exogenous RNA, thereby promoting degradation of the RNA. Our findings reveal that the selective targeting of exogenous RNA in antiviral defense occurs via a mechanism similar to that in the degradation of aberrant transcripts in RNA quality control.

Chemical stimulus-responsive supramolecular hydrogel formation and shrinkage of a hydrazone-containing short peptide derivative.

Artificial supramolecular nanostructures showing transient properties have attracted significant attention in recent years. New discoveries in this area may provide insights into a better understanding of the sophisticated organization of complex biomolecular systems. Nevertheless, research concerning such materials is still limited. Better knowledge of the chemical reactivity and corresponding molecular transformations of self-assembling molecules, which guide their assembly/disassembly, may provide an opportunity to construct transient supramolecular nanostructures capable of showing chemical stimulus responsiveness. Herein, we report a short peptide derivative containing a hydrazone bond, which shows transient hydrogel formation (no only sol-to-gel but also gel-to-shrunken gel phase transition) accompanied by continuous transformation and growth of supramolecular nanostructures triggered by hydrazone-oxime exchange reaction in response to hydroxylamine. Such controlled shrinkage behavior of supramolecular hydrogels in response to specific chemical stimuli has rarely been explored compared with conventional polymer hydrogel systems.

Sulfonamide antibiotics inhibit RNAi by binding to human Argonaute protein 2 PAZ.

We found that sulfisomidine, a sulfonamide antibiotic, potently binds to the Piwi/Argonaute/Zwille (PAZ) domain of human Argonaute protein 2 and inhibits RNA interference (RNAi). To elucidate the effect on RNAi of strong affinity of the 3'-ends in small interfering RNA (siRNA) to the PAZ domain, chemically modified siRNAs bearing sulfisomidine at the 3'-end were synthesized.

Hybrid Soft Nanomaterials Composed of DNA Microspheres and Supramolecular Nanostructures of Semi-artificial Glycopeptides.

Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano- and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self-assembly under aqueous conditions. However, orthogonal self-assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single-stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi-artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli-responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions.

Regulation of microRNA expression and abundance during lymphopoiesis.

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity's microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.

GH30 Glucuronoxylan-Specific Xylanase from Streptomyces turgidiscabies C56.

Endoxylanases are important enzymes in bioenergy research because they specifically hydrolyze xylan, the predominant polysaccharide in the hemicellulose fraction of lignocellulosic biomass. For effective biomass utilization, it is important to understand the mechanism of substrate recognition by these enzymes. Recent studies have shown that the substrate specificities of bacterial and fungal endoxylanases classified into glycoside hydrolase family 30 (GH30) were quite different. While the functional differences have been described, the mechanism of substrate recognition is still unknown. Therefore, a gene encoding a putative GH30 endoxylanase was cloned from Streptomyces turgidiscabies C56, and the recombinant enzyme was purified and characterized. GH30 glucuronoxylan-specific xylanase A of Streptomyces turgidiscabies (StXyn30A) showed hydrolytic activity with xylans containing both glucuronic acid and the more common 4-O-methyl-glucuronic acid side-chain substitutions but not on linear xylooligosaccharides, suggesting that this enzyme requires the recognition of glucuronic acid side chains for hydrolysis. The StXyn30A limit product structure was analyzed following a secondary ß-xylosidase treatment by thin-layer chromatography and mass spectrometry analysis. The hydrolysis products from both glucuronoxylan and 4-O-methylglucuronoxylan by StXyn30A have these main-chain substitutions on the second xylopyranosyl residue from the reducing end. Because previous structural studies of bacterial GH30 enzymes and molecular modeling of StXyn30A suggested that a conserved arginine residue (Arg296) interacts with the glucuronic acid side-chain carboxyl group, we focused on this residue, which is conserved at subsite -2 of bacterial but not fungal GH30 endoxylanases. To help gain an understanding of the mechanism of how StXyn30A recognizes glucuronic acid substitutions, Arg296 mutant enzymes were studied. The glucuronoxylan hydrolytic activities of Arg296 mutants were significantly reduced in comparison to those of the wild-type enzyme. Furthermore, limit products other than aldotriouronic acid were observed for these Arg296 mutants upon secondary ß-xylosidase treatment. These results indicate that a disruption of the highly conserved Arg296 interaction leads to a decrease of functional specificity in StXyn30A, as indicated by the detection of alternative hydrolysis products. Our studies allow a better understanding of the mechanism of glucuronoxylan recognition and enzyme specificity by bacterial GH30 endoxylanases and provide further definition of these unique enzymes for their potential application in industry.IMPORTANCE Hemicellulases are important enzymes that hydrolyze hemicellulosic polysaccharides to smaller sugars for eventual microbial assimilation and metabolism. These hemicellulases include endoxylanases that cleave the ß-1,4-xylose main chain of xylan, the predominant form of hemicellulose in lignocellulosic biomass. Endoxylanases play an important role in the utilization of plant biomass because in addition to their general utility in xylan degradation, they can also be used to create defined compositions of xylooligosaccharides. For this, it is important to understand the mechanism of substrate recognition. Recent studies have shown that the substrate specificities of bacterial and fungal endoxylanases that are classified into glycoside hydrolase family 30 (GH30) were distinct, but the difference in the mechanisms of substrate recognition is still unknown. We performed characterization and mutagenesis analyses of a new bacterial GH30 endoxylanase for comparison with previously reported fungal GH30 endoxylanases. Our study results in a better understanding of the mechanism of substrate specificity and recognition for bacterial GH30 endoxylanases. The experimental approach and resulting data support the conclusions and provide further definition of the structure and function of GH30 endoxylanases for their application in bioenergy research.

Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides.

Glucosamino nucleic acids (GANAs) bearing a ß-N-glycoside bond between carbon 1 of the glucosamine and the nucleobase nitrogen were synthesized and incorporated into oligonucleotides (4',6'-GANA and 3',6'-GANA). The thermal stability of oligonucleotide duplexes containing the GANA zwitterionic nucleotides was also investigated.

Cyclic arylopeptoid oligomers: synthesis and conformational propensities of peptide-mimetic aromatic macrocycles.

Macrocyclic peptide-mimetic molecules are attracting renewed attention and have found widespread application in research fields ranging from biochemical science to materials science. Herein, we describe the synthesis and structural elucidation of cyclo[n]-p-arylopeptoids (classified into cyclic aromatic ε-amino acids) bearing various side chains, namely, C[n]pAP(Rn) (where n inside brackets denotes the number of main chain units and R inside parentheses represents side chains). We investigate the influence of n and R on the macrocyclization efficiency of linear p-arylopeptoid oligomers (n = 3, 4, 5) under high-dilution conditions with or without slow addition. The structures of the cyclo[4]-p-arylopeptoids (C[4]pAP(Rn)) and their conformational dynamics are disclosed on the basis of single-crystal X-ray analyses, viable-temperature (VT) 1H NMR studies, and density functional theory (DFT) calculations. We found two representative conformations (open and closed) of cyclo[4]-p-arylopeptoids (C[4]pAP(Rn)) in the solid state and whose preference in the solution state was most likely dependent on solvent polarity. We believe that this simple but dynamic macrocyclic peptide-mimetic molecular scaffold would be attractive for developing new functional molecular tools based on rational molecular design as well as molecular library screening strategies.

Effects of irradiation with narrowband-ultraviolet B on up-regulation of histamine H1 receptor mRNA and induction of apoptosis in HeLa cells and nasal mucosa of rats.

Narrowband-ultraviolet B (NB-UVB) phototherapy is used for the treatment of atopic dermatitis. Previously, we reported that irradiation with 200 mJ/cm2 of 310 nm NB-UVB suppressed phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) gene expression without induction of apoptosis in HeLa cells. However, the effect of NB-UVB irradiation on nasal symptoms is still unclear. Here, we show that low dose irradiation with 310 nm NB-UVB alleviates nasal symptoms in toluene 2,4-diisocyanate (TDI)-sensitized allergy model rats. Irradiation with 310 nm NB-UVB suppressed PMA-induced H1R mRNA up-regulation in HeLa cells dose-dependently at doses of 75-200 mJ/cm2 and reversibly at a dose of 150 mJ/cm2 without induction of apoptosis. While, at doses of more than 200 mJ/cm2, irradiation with 310 nm NB-UVB induced apoptosis. Western blot analysis showed that the suppressive effect of NB-UVB irradiation on H1R gene expression was through the inhibition of ERK phosphorylation. In TDI-sensitized rat, intranasal irradiation with 310 nm NB-UVB at an estimated dose of 100 mJ/cm2 once a day for three days suppressed TDI-induced sneezes and up-regulation of H1R mRNA in nasal mucosa without induction of apoptosis. These findings suggest that repeated intranasal irradiation with low dose of NB-UVB could be clinically used as phototherapy of AR.

Effect of Royal Jelly and Brazilian Green Propolis on the Signaling for Histamine H1 Receptor and Interleukin-9 Gene Expressions Responsible for the Pathogenesis of the Allergic Rhinitis.

The significant correlation between nasal symptom scores and level of histamine H1 receptor (H1R) mRNA in nasal mucosa was observed in patients with pollinosis, suggesting that H1R gene is an allergic disease sensitive gene. We demonstrated that H1R and interleukin (IL)-9 gene are the allergic rhinitis (AR)-sensitive genes and protein kinase Cδ (PKCδ) signaling and nuclear factor of activated T-cells (NFAT) signaling are involved in their expressions, respectively. Honey bee products have been used to treat allergic diseases. However, their pathological mechanism remains to be elucidated. In the present study, we investigated the mechanism of the anti-allergic effect of royal jelly (RJ) and Brazilian green propolis (BGPP). Treatment with RJ and BGPP decreased in the number of sneezing on toluene 2,4-diissocyanate (TDI)-stimulated rats. The remarkable suppression of H1R mRNA in nasal mucosa was observed. RJ and BGPP also suppressed the expression of IL-9 gene. RJ and BGPP suppressed phorbol-12-myristate-13-acetate-induced Tyr311 phosphorylation of PKCδ in HeLa cells. In RBL-2H3 cells, RJ and BGPP also suppressed NFAT-mediated IL-9 gene expression. These results suggest that RJ and BGPP improve allergic symptoms by suppressing PKCδ and NFAT signaling pathways, two important signal pathways for the AR pathogenesis, and suggest that RJ and BGPP could be good therapeutics against AR.

Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures.

The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-ß-d-ribofuranose (RE) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures.

Introduction of 2-O-benzyl abasic nucleosides to the 3'-overhang regions of siRNAs greatly improves nuclease resistance.

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (RHOBn) in their 3'-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3'-end.

Histamine H1 Receptor Gene Expression and Drug Action of Antihistamines.

The upregulation mechanism of histamine H1 receptor through the activation of protein kinase C-δ (PKCδ) and the receptor gene expression was discovered. Levels of histamine H1 receptor mRNA and IL-4 mRNA in nasal mucosa were elevated by the provocation of nasal hypersensitivity model rats. Pretreatment with antihistamines suppressed the elevation of mRNA levels. Scores of nasal symptoms were correlatively alleviated to the suppression level of mRNAs above. A correlation between scores of nasal symptoms and levels of histamine H1 receptor mRNA in the nasal mucosa was observed in patients with pollinosis. Both scores of nasal symptoms and the level of histamine H1 receptor mRNA were improved by prophylactic treatment of antihistamines. Similar to the antihistamines, pretreatment with antiallergic natural medicines showed alleviation of nasal symptoms with correlative suppression of gene expression in nasal hypersensitivity model rats through the suppression of PKCδ. Similar effects of antihistamines and antiallergic natural medicines support that histamine H1 receptor-mediated activation of histamine H1 receptor gene expression is an important signaling pathway for the symptoms of allergic diseases. Antihistamines with inverse agonist activity showed the suppression of constitutive histamine H1 receptor gene expression, suggesting the advantage of therapeutic effect.

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (-)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells.

The histamine H1 receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (-)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (-)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (-)-maackiain. A pull-down assay revealed that (-)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr(311) on PKCδ. These data suggest that (-)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (-)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.