Anti-inflammatory effect of covalent PPARγ ligands that have a hybrid structure of GW9662 and a food-derived cinnamic acid derivative.

Peroxisome proliferator-activated receptor γ (PPARγ) belongs to nuclear receptor superfamily and is involved in inflammatory process. Previously, we synthesized the ligands of PPARγ which possess the hybrid structure of a food-derived cinnamic acid derivative (CA) and GW9662, an irreversible PPARγ antagonist. These ligands activate the transcription of PPARγ through the covalent bond formation with the Cys285 residue of PPARγ, whereas their anti-inflammatory effect has not been examined yet. Here, we show the anti-inflammatory effect of the covalent PPARγ ligands in RAW264 cells, murine macrophage-like cells. GW9662 suppressed the production of nitric oxide (NO) stimulated by lipopolysaccharide and exerted a synergistic effect in combination with CA. The compounds bearing their hybrid structure dramatically inhibited NO production and transcription of proinflammatory cytokines. A comparison study suggested that 2-chloro-5-nitrobenzoyl group of the ligands is important for anti-inflammation. Furthermore, we synthesized an alkyne-tagged analogue which become an activity-based probe for future mechanistic study.

Insights into Dynamic Mechanism of Ligand Binding to Peroxisome Proliferator-Activated Receptor γ toward Potential Pharmacological Applications.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily, which regulates the transcription of a variety of genes involved in lipid and glucose metabolism, inflammation, and cell proliferation. These functions correlate with the onset of type-2 diabetes, obesity, and immune disorders, which makes PPARγ a promising target for drug development. The majority of PPARγ functions are regulated by binding of small molecule ligands, which cause conformational changes of PPARγ followed by coregulator recruitment. The ligand-binding domain (LBD) of PPARγ contains a large Y-shaped cavity that can be occupied by various classes of compounds such as full agonists, partial agonists, natural lipids, and in some cases, a combination of multiple molecules. Several crystal structure studies have revealed the binding modes of these compounds in the LBD and insight into the resulting conformational changes. Notably, the apo form of the PPARγ LBD contains a highly mobile region that can be stabilized by ligand binding. Furthermore, recent biophysical investigations have shed light on the dynamic mechanism of how ligands induce conformational changes in PPARγ and result in functional output. This information may be useful for the design of new and repurposed structures of ligands that serve a different function from original compounds and more potent pharmacological effects with less undesirable clinical outcomes. This review provides an overview of the peculiar characteristics of the PPARγ LBD by examining a series of structural studies focused on the dynamic mechanism of binding and the potential applications of strategies for ligand screening and chemical labeling.

Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1.

We examined whether the acetylenic fatty acids 6-octadecynoic acid (6-ODA) and 9-octadecynoic acid (9-ODA) perform as ligands for free fatty acid receptors of medium- and long-chain fatty acids FFAR1 and FFAR4, previously called GPR40 and GPR120, respectively. Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 was increased through FFAR1 but not through FFAR4 expressed in HEK 293 cells, suggesting that 6-ODA and 9-ODA function as an FFAR1 ligand, but not as an FFAR4 ligand. Activation of ERK in FFAR1-expressing HEK293 cells by 6-ODA and 9-ODA peaked at 10 min after stimulation followed by a slow decrease, similar to ERK activation by rosiglitazone, which peaked at 10 min after stimulation and lasted longer. Glucose-dependent production of insulin from MIN6 insulinoma cells was induced by 6-ODA and 9-ODA in an FFAR1-dependent manner. In this process, 6-ODA and 9-ODA stimulated the production of insulin not in the first phase that occurred within 10 min after stimulation but in the second phase. F-actin-remodeling that reflects insulin granule recruiting to the plasma membrane in the second phase of insulin secretion by 6-ODA and 9-ODA suggested that they have an FFAR1-dependent function in insulin secretion from MIN6 cells.

Importance of the Proximity and Orientation of Ligand-Linkage to the Design of Cinnamate-GW9662 Hybrid Compounds as Covalent PPARγ Agonists.

Covalent agonists of PPARγ cause unique receptor conformational changes and behave as selective PPARγ modulators, whereas there are few covalent agonists other than endogenous unsaturated fatty acids metabolites. Previously, we established a cell-based strategy to identify new PPARγ ligands and synthesized a new-type of covalent agonist that possesses the hybrid structure of a plant-derived cinnamic acid derivative and GW9662, a covalent antagonist. Herein, we report six analogues that differ in how the two fragments are linked together. Compounds with a simplified linker showed potent agonistic activity with improved EC50 values (less than 5 nM), indicating that close proximity between the two fragments improves binding affinity. When the position of cinnamic acid moiety was placed at 4' carbon of aniline ring, PPARγ agonist activity was completely abolished. Docking studies suggested that the activation profile likely depends on interaction with the cavity around helix 3, ß-sheet, and Ω-loop region in the ligand-binding domain. Furthermore, a cell-based assay revealed that agonist-type compounds activate PPARγ transcription in a manner dependent on covalent linkage with the Cys285 residue leading to prolonged transactivation. This activation feature reflects pharmacological benefits of covalent drugs, suggesting that these hybrid compounds may serve as potential leads for a new-class of covalent PPARγ ligands.

The isoflavone fraction from soybean presents mRNA maturation inhibition activity.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line.

Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation.

Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.

The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.

Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist.

6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.

Induction of hepatocyte growth factor production in human dermal fibroblasts by caffeic acid derivatives.

Hepatocyte growth factor (HGF) has mitogenic, motogenic, and morphogenic activities in epithelial cells. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. In this study, we examined the effects of caffeic acid derivatives including 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) on HGF production in Neonatal Normal Human Dermal Fibroblasts (NHDF). Both 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) significantly induced HGF production dose-dependent manner. To know the important substructure for HGF production activity, we next investigated the effect of the partial structure of these caffeic acid derivatives. From the results, caffeic acid (3) showed strong activity on the promotion of HGF production, while hydroxytyrosol (4) and quinic acid (5) didn't show any activity. Our findings suggest that the caffeoyl moiety of caffeic acid derivatives is essential for accelerated production of HGF. The compound which has the caffeoyl moiety may be useful for the treatment of some intractable organ disease.

Inhibition of amyloid ß aggregation by acteoside, a phenylethanoid glycoside.

We examined the effects of acteoside (1a), which was isolated from Orobanche minor, and its derivatives on the aggregation of a 42-mer amyloid ß protein (Aß42) in our search for anti-amyloidogenic compounds for Alzheimer's disease (AD) therapy. Acteoside (1a) strongly inhibited the aggregation of Aß42 in a dose-dependent manner. The structure-activity relationship for acteoside (1a) and related compounds suggests the catechol moiety of phenylethanoid glycosides to be essential for this inhibitory activity.

A strategy for orthogonal deubiquitination using a bump-and-hole approach.

We have successfully applied a bump-and-hole approach to establish orthogonal deubiquitination in which a ubiquitin substrate variant is specifically targeted by an engineered deubiquitinating enzyme (DUB). This makes it possibe to selectively observe and measure a single type of DUB activity in living cells.

Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid ß-protein.

Alzheimer's disease (AD), a neurodegenerative disorder, is characterized by aggregation of 42-mer amyloid ß-protein (Aß42). Aß42 aggregates through ß-sheet formation and induces cytotoxicity against neuronal cells. Aß42 oligomer, an intermediate of the aggregates, causes memory loss and synaptotoxicity in AD. Inhibition of Aß42 aggregation by small molecules is thus a promising strategy for the treatment of AD. Caffeoylquinic acid (CQA), a phenylpropanoid found widely in natural sources including foods, shows various biological activities such as anti-oxidative ability. Previously, our group reported that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA) rescued the cognitive impairment in senescence-accelerated-prone mice 8. However, structure-activity relationship of CQA derivatives on the aggregation and neurotoxicity of Aß42 remains elusive. To evaluate the anti-amyloidogenic property of CQA-related compounds for AD therapy, we examined the effect of CQA and its derivatives on the aggregation and neurotoxicity of Aß42. In particular, 4,5-di-O-caffeoylquinic acid (4,5-di-CQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA) strongly inhibited the aggregation of Aß42 in a dose-dependent manner. Structure-activity relationship studies suggested that the caffeoyl group in CQA is essential for the inhibitory activity. These CQAs also suppressed the transformation into ß-sheet and cytotoxicity against human neuroblastoma cells of Aß42. Furthermore, 3,4,5-tri-CQA blocked the formation of Aß42 oligomer. These results indicate that 3,4,5-tri-CQA could be a potential agent for the prevention of AD.

Establishment of a monitoring system to detect inhibition of mRNA processing.

A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing.

[A Screening System for the Identification of Fragment Molecules toward the Creation of Nuclear Receptor Ligands].

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily, which plays an important role in glucose and lipid metabolism as well as inflammation. The transcriptional activity of PPARγ is regulated by the binding of its ligand and the accompanied conformational change followed by the recruitment of cofactors. The ligand-binding pocket (LBP) of PPARγ comprises multiple sub-pockets and includes a large, Y-shaped cavity. In some cases, more than two ligands simultaneously occupy the LBP and cooperatively activate PPARγ transcription. Inspired by this peculiar character, the author proposed a strategy to create new PPARγ ligands in two steps: first, identifying a combination of ligands that cooperatively activate PPARγ, and second, designing and synthesizing their hybrid structure. Cooperative activation can be detected by a conventional cell-based assay using a reporter gene, which may provide advantages over the existing fragment-based drug discovery approach. Using this strategy, a plant-derived cinnamic acid derivative was found to synergistically activate PPARγ in combination with GW9662, an irreversible antagonist. The designed hybrid structure was synthesized and found to behave as a covalent agonist, which partially activates PPARγ transcription. Structure-activity studies revealed the importance of proximity and orientation in the linkage of the two units. The strategy discussed in this article may contribute to the development of a highly potent PPARγ agonist.

Cyanidin 3-Glucoside Induces Hepatocyte Growth Factor in Normal Human Dermal Fibroblasts through the Activation of ß2-Adrenergic Receptor.

Hepatocyte growth factor (HGF) is expressed in various organs and involved in the fundamental cellular functions such as mitogenic, motogenic, and morphogenic activities. Induction of HGF may be therapeutically useful for controlling organ regeneration, wound healing, and embryogenesis. In this study, we examined the stimulation effect of cyanidin 3-glucoside (C3G), an anthocyanidin derivative, on HGF production in normal human dermal fibroblasts (NHDFs) and the underlying mechanisms. C3G induced HGF production at both mRNA and protein levels in NHDF cells and enhanced the phosphorylation of cAMP-response element-binding protein. We also observed that treatment with C3G increased intracellular cAMP level and promoter activity of cAMP-response element in HEK293 cells expressing ß2-adrenergic receptor (ß2AR). In contrast, cyanidin, an aglycon of C3G, did not show the activation of ß2AR signaling and HGF production. These results indicate that C3G behaves as an agonist for ß2AR signaling to activate the protein kinase A pathway and induce the production of HGF.

Structure-activity relationship of caffeoylquinic acids on the accelerating activity on ATP production.

Caffeoylquinic acid (CQA) is one of the phenylpropanoids which have various bioactivities such as antioxidant, antibacterial, anticancer, antihistamic, and other biological effects. We previously reported that 3,5-di-O-caffeoylquinic acid inhibited amyloid ß(1-42)-induced cellular toxicity on human neuroblastoma SH-SY5Y cells and increased the mRNA expression level of glycolytic enzymes and the intracellular ATP level. To investigate structure-activity relationship on the accelerating activity on ATP production, we synthesized 1,4,5-tri-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, 3,4,5-tri-O-caffeoylquinic acid, and other derivatives. Additionally, we evaluated intracellular ATP level in SH-SY5Y treated with each CQA derivative. As a result, 3,4,5-tri-O-caffeoylquinic acid showed the highest accelerating activity on ATP production among tested compounds. It was suggested that caffeoyl groups bound to quinic acid are important for activity and the more caffeoyl groups are bound to quinic acid, the higher accelerating activity on ATP production exhibits.

Pycnalin, a new α-glucosidase inhibitor from Acer pycnanthum.

A new compound, pycnalin (1), together with four known compounds, ginnalins A (2), B (3), C (4), and 3,6-di-O-galloyl-1,5-anhydro-D-glucitol (3,6-di-GAG) (5), were isolated from Acer pycnanthum. The structure of 1 was determined on the basis of 2D-NMR spectral data and synthesis of 1. Pycnalin (1) is the first 1,5-anhydro-D-mannitol linked to a gallic acid, while compounds 2-5 were 1,5-anhydro-D-glucitol linked to gallic acids. All compounds were tested in vitro for α-glucosidase inhibitory and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. Pycnalin (1) exhibited moderate α-glucosidase inhibitory activity as well as free radical scavenging activity. Ginnalin A (2) and 3,6-di-GAG (5), which have two galloyl groups, exhibited potent α-glucosidase inhibition, compared to those of other compounds 1, 3, and 4 containing a galloyl group. These results suggest that α-glucosidase inhibition is influenced by the number of galloyl groups.