Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis.

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.

Synthesis, Stability, and Antidiabetic Activity Evaluation of (-)-Epigallocatechin Gallate (EGCG) Palmitate Derived from Natural Tea Polyphenols.

This work describes a novel approach for the synthesis of (-)-epigallocatechin gallate (EGCG) palmitate by a chemical-synthesis method, where the elevated stability of the EGCG derivative is achieved. Various parameters affecting the acylation process, such as the base, solvent, as well as the molar ratio of palmitoyl chloride, have been studied to optimize the acylation procedure. The optimized reaction condition was set as follows: EGCG/palmitoyl chloride/sodium acetate was under a molar ratio of 1:2:2, with acetone as the solvent, and the reaction temperature was 40 °C. Under the optimized condition, the yield reached 90.6%. The EGCG palmitate (PEGCG) was isolated and identified as 4'-O-palmitoyl EGCG. Moreover, the stability of PEGCG under different conditions was proved significantly superior to EGCG. Finally, PEGCG showed better inhibition towards α-amylase and α-glucosidase, which was 4.5 and 52 times of EGCG, respectively. Molecular docking simulations confirmed the in vitro assay results. This study set a novel and practical synthetic approach for the derivatization of EGCG, and suggest that PEGCG may act as an antidiabetic agent.

Dibenzoate esters of cis-tetralin-2,3-diol as analogs of (-)-epigallocatechin gallate: synthesis and crystal structure of anticancer drug candidates.

(-)-Epigallocatechin gallate (EGCG), the main component of green tea extract, displays multiple biological activities. However, it cannot be used as a drug due to its low cellular absorption, instability and metabolic degradation. Therefore, there is a need to provide analogs that can overcome the limitations of EGCG. In this work, six synthetic analogs of EGCG sharing a common tetralindiol dibenzoate core were synthesized and fully characterized by 1H NMR, 13C NMR, HRMS and IR spectroscopies, and X-ray crystallography. These are (2R,3S)-1,2,3,4-tetrahydronaphthalene-2,3-diyl bis[3,4,5-tris(benzyloxy)benzoate], C66H56O10, and the analogous esters bis(3,4,5-trimethoxybenzoate), C30H32O10, bis(3,4,5-trifluorobenzoate), C24H14F6O4, bis[4-(benzyloxy)benzoate], C38H32O6, bis(4-methoxybenzoate), C26H24O6, and bis(2,4,6-trifluorobenzoate), C24H14F6O4. Structural analysis revealed that the molecular shapes of these dibenzoate esters of tetralindiol are significantly different from that of previously reported dimandelate esters or monobenzoate esters, as the acid moieties extend far from the bicyclic system without folding back over the tetralin fragment. Compounds with small fluorine substituents take a V-shape, whereas larger methoxy and benzyloxy groups determine the formation of an L-shape or a cavity. Intermolecular interactions are dominated by π-π stacking and C-H...π interactions involving the arene rings in the benzoate fragment and the arene ring in the tetrahydronaphthalene moiety. All six crystal structures are determined in centrosymmetric space groups (either P-1, P21/n, C2/c or I2/a).

Duo of (-)-epigallocatechin-3-gallate and doxorubicin loaded by polydopamine coating ZIF-8 in the regulation of autophagy for chemo-photothermal synergistic therapy.

To achieve highly systemic therapeutic efficacy, chemotherapy is combined with photothermal therapy for chemo-photothermal synergistic therapy; however, this strategy suffers from high toxicity and unsatisfactory sensitivity for cancer cells. Herein, we developed a pH- and photothermal-responsive zeolitic imidazolate framework (ZIF-8) compound for loading a dual-drug in the tumor site and improving their curative effects. Since autophagy always accompanies tumor progression and metastasis, there is an unmet need for an anticancer treatment related to the regulation of autophagy. Green tea polyphenols, namely, (-)-epigallocatechin-3-gallate (EGCG) and doxorubicin (DOX), both of which exhibit anticancer activity, were dual-loaded via polydopamine (PDA) coating ZIF-8 (EGCG@ZIF-PDA-PEG-DOX, EZPPD for short) through hierarchical self-assembly. PDA could transfer photothermal energy to increase the temperature under near-infrared (NIR) laser irradiation. Due to its pH-response, EZPPD released EGCG and DOX in the tumor microenvironment, wherein the temperature increased with the help of PDA and NIR laser irradiation. The duo of DOX and EGCG induced autophagic flux and accelerated the formation of autophagosomes. In a mouse HeLa tumor model, photothermal-chemotherapy could ablate the tumor with a significant synergistic effect and potentiate the anticancer efficacy. Thus, the results indicate that EZPPD renders the key traits of a clinically promising candidate to address the challenges associated with synergistic chemotherapy and photothermal utilization in antitumor therapy.

A semisynthetic approach for the simultaneous reaction of grape seed polymeric procyanidins with catechin and epicatechin to obtain oligomeric procyanidins in large scale.

Polymeric procyanidins (PPCs) were the major constituents of procyanidins, while they have poor bioactivity. To better utilize PPCs, a semisynthetic approach for converting PPCs to oligomeric procyanidins (OPCs) was proposed. Grape seed PPCs were simultaneously reacted with catechin (C) and epicatechin (EC) under acid condition. Combining response surface methodology (RSM) and single-factor experiments, an optimized semisynthetic condition was confirmed with the ratio of PPCs with C and EC of 1:1:1, temperature of 40 °C, reaction time of 20 min and 0.1 M methanolic HCl. High-speed counter-current chromatography (HSCCC) was adopted to obtain three fractions from semisynthetic products and preparative-HPLC was used to isolate individual procyanidins. Thirteen B-type procyanidins including monomers, dimers and trimers were got with high yield of 0.8-17.8 mg from 200 mg semisynthetic products and high purity over 91%. The developed semisynthesis combined with separation method was efficient to obtain individual OPCs in preparative scale.

Development and evaluation of resveratrol, Vitamin E, and epigallocatechin gallate loaded lipid nanoparticles for skin care applications.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been studied as potential carriers for both dermal and transdermal drug delivery. SLN contain lipid droplets that are fully crystallized and have a highly-ordered crystalline structure. NLC are modified SLN in which the lipid phase contains both solid and liquid lipids at room temperature. SLN and NLC are thought to combine the advantages of polymeric particles, liposomes and emulsions. Therefore they provide high encapsulation percentages, better protection for incorporated actives and allow for control of desired release profile. In this work, Resveratrol, Vitamin E (VE), and Epigallocatechin Gallate (EGCG) all potent antioxidants known to provide protection to the skin, were formulated into lipid nanoparticles. Several different formulations were successfully developed and demonstrated high uniformity and stability. Both resveratrol and VE lipid nanoparticles provided effective protection of actives against UV induced degradation. However, lipid nanoparticles did not show protection from UV degradation for EGCG in this work. An active release study exhibited a sustained release of resveratrol over 70% after 24h. Skin penetration studies showed that lipid nanoparticles directionally improved the penetration of resveratrol through the stratum corneum. Our findings suggest that lipid nanoparticles are promising viable carriers for the delivery of resveratrol and VE to provide longlasting antioxidant benefits to the skin.

Challenges and complexity of functionality evaluation of flavan-3-ol derivatives.

Flavan-3-ol derivatives are common plant-derived bioactive compounds. In particular, (-)-epigallocatechin-3-O-gallate shows various moderate biological activities without severe toxicity, and its health-promoting effects have been widely studied because it is a main ingredient in green tea and is commercially available at low cost. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly due to their relative unavailability. Here, I outline the major factors contributing to the complexity of functionality studies of flavan-3-ol derivatives, including proanthocyanidins and oligomeric flavan-3-ols. I emphasize the importance of conducting structure-activity relationship studies using synthesized flavan-3-ol derivatives that are difficult to obtain from plant extracts in pure form to overcome this challenge. Further discovery of these minor constituents showing strong biological activities is expected to produce useful information for the development of functional health foods.

A novel ECG analog 4-(S)-(2,4,6-trimethylthiobenzyl)-epigallocatechin gallate selectively induces apoptosis of B16-F10 melanoma via activation of autophagy and ROS.

Autophagy-induced cancer cell death has become a novel strategy for the development of cancer therapeutic drugs. Numerous studies have indicated that green tea polyphenols induce both autophagy and apoptosis in a variety of cancer cells. Here, we synthesized a series of green tea polyphenol analogues, among which JP8 was shown to potently activate autophagy. JP8 treatment had a stronger effect on apoptosis in B16-F10 melanoma cells than that in normal AML-12 hepatocytes. JP8 selectively resulted in reactive oxygen species (ROS) accumulation in B16-F10 cells, and this effect was associated with corresponding increases in key components of the ER stress-mediated apoptosis pathway. Pharmacological inhibition of ROS by N-acetyl-L-cysteine (NAC) attenuated JP8-induced autophagy and apoptosis, indicating an upstream role of ROS in JP8-induced autophagy. An in vivo study showed that JP8 had significant antitumor effects in a B16-F10 xenograft mouse model. Our results indicate that JP8 is a novel anticancer candidate with both autophagy and ROS induction activities.

Synthesis of Effective Food Constituents toward the Development of Chemical Biology Investigations.

This article describes the development of various probes and immunogens for chemical-biological investigations of food flavonoids. We accomplished a large (gram)-scale asymmetric synthesis of a key intermediate, 5-aminopentyl deoxy epigallocatechin-3-gallate (APDOEGCg; 3), an analogue of green tea polyphenol EGCg, in which the key step was cationic cyclization utilizing neighboring group participation of the gallate carbonyl group. The synthetic APDOEGCg (3) was efficiently converted to a fluorescent probe 18 and an immunogen 19 by utilizing the high reactivity of the amine functional group. We confirmed the usefulness of these probes for imaging studies and the generation of antibodies, respectively. We also describe the efficient synthesis of a positron emission tomography (PET) probe [11C]20 by incorporation of 11C into EGCg (1), for which synthetic 4″-Me-EGCg (20) was utilized as an authentic sample. Our synthetic strategy was also applied for the practical synthesis of nobiletin (21), a polymethoxylated flavone from citrus. Synthetic nobiletin was readily converted to various probes by selective demethylation and incorporation of fluorescein, biotin or 11C. These probes should be useful for a range of biological applications. Detailed examination of the mechanisms and further applications are in progress.

Targeted intracellular protein delivery based on hyaluronic acid-green tea catechin nanogels.

A novel ternary nanogel based on the self-assembly of hyaluronic acid-epigallocatechin gallate conjugates (HA-EGCG), linear polyethylenimine (PEI) and Granzyme B (GzmB) in an aqueous environment was developed for the targeted intracellular delivery of GzmB into cancer cells. Lysozyme-encapsulated HA-EGCG nanogels were first prepared and characterized. HA-EGCG nanogels exhibited smaller particle sizes and a more homogeneous size distribution than the HA counterpart. Fluorescence quenching and lysozyme activity studies revealed that EGCG moieties facilitated protein binding through physical interactions and led to the formation of stable nanogels. When CD44-overexpressing HCT-116 colon cancer cells were treated with GzmB-encapsulated HA-EGCG nanogels in vitro, a significant cytotoxic effect was observed. Caspase assays and intracellular trafficking studies confirmed that cell death was due to apoptosis triggered by the delivery of GzmB to the cytosol of those cells. In comparison, little cytotoxic effect was observed in CD44-deficient cells treated with GzmB-encapsulated HA-EGCG nanogels. This study highlights the potential utility of HA-EGCG as effective intracellular protein carriers for targeted cancer therapy. STATEMENT OF SIGNIFICANCE: Intracellularly activated cytotoxic proteins can be used to kill cancer cells but viable carriers for such proteins are lacking. In this work, we developed novel nanogels based on selfassembly of hyaluronic acid (HA)-(-)-epigallocatechin-3-gallate (EGCG) conjugates, linear polyethylenemine (PEI) and the cytotoxic protein Granzyme B (GzmB) for the intracellular delivery of GzmB for cancer therapy. HA was exploited for its ability to target CD44 which are overexpressed in many types of cancer cells, while EGCG, the main component of green tea catechins, was chosen for its ability to bind to proteins. Characterization studies showed that EGCG facilitated protein complexation through physical interactions and led to the formation of stable nanogels. HA-EGCG nanogels were able to achieve CD44 targeted killing of HCT-116 cancer cells by delivering GzmB into the cytosol of these cells. We believe that the applications of the HA-EGCG nanogels can be expanded to the intracellular delivery of other cytotoxic protein drugs for cancer therapy.

Synthesis of Arecatannin A1 from Dimeric Epicatechin Electrophile.

Synthesis of arrecatannin A1 (1) was accomplished from dimeric epicatechin electrophile, which was prepared by Zn(OTf)2 mediated self-condensation, and monomeric catechin nucleophile. The condensation was successfully worked using Yb(OTf)3 as a Lewis acid in good yield.

Synthesis and quantitative analysis of plasma-targeted metabolites of catechin and epicatechin.

Grape seed polyphenolic extract (GSPE) rich in the flavan-3-ols (+)-catechin and (-)-epicatechin beneficially modulates Alzheimer's Disease phenotypes in animal models. The parent molecules in the extract are converted to a series of methylated and glucuronidated derivatives. To fully characterize these metabolites and establish a robust quantitative assay of their levels in biological fluids, we have implemented a partial synthetic approach utilizing chemical methylation followed by enzymatic glucuronidation. Liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to assign unequivocal structures to the compounds. An analytical method using solid-phase extraction and LC-MS/MS in selective reaction monitoring mode (SRM) was validated for their quantitation in plasma. These studies provide a basis for improvements in future work on the bioavailability, metabolism, and mechanism of action of metabolites derived from dietary flavan-3-ols in a range of interventions.

Inhibitory activity of synthesized acetylated Procyanidin B1 analogs against HeLa S3 cells proliferation.

Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.

Factors influencing the antifolate activity of synthetic tea-derived catechins.

Novel tea catechin derivatives have been synthesized, and a structure-activity study, related to the capacity of these and other polyphenols to bind dihydrofolate reductase (DHFR), has been performed. The data showed an effective binding between all molecules and the free enzyme, and the dissociation constants of the synthetic compounds and of the natural analogues were on the same order. Polyphenols with a catechin configuration were better DHFR inhibitors than those with an epicatechin configuration. Antiproliferative activity was also studied in cultured tumour cells, and the data showed that the activity of the novel derivatives was higher in catechin isomers. Derivatives with a hydroxyl group para on the ester-bonded gallate moiety presented a high in vitro binding to DHFR, but exhibited transport problems in cell culture due to ionization at physiologic pHs. The impact of the binding of catechins to serum albumin on their biological activity was also evaluated. The information provided in this study could be important for the design of novel medicinal active compounds derived from tea catechins. The data suggest that changes in their structure to avoid serum albumin interactions and to facilitate plasmatic membrane transport are essential for the intracellular functions of catechins.

Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils.

Protein misfolding and/or aggregation has been implicated as the cause of several human diseases, such as Alzheimer's and Parkinson's diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, named after the cross-ß-sheet amyloid fibril aggregates or deposits common to these disorders. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work toward an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of Aß(1-40), IAPP(8-24), or Sup35NM(7-16). We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in Aß(1-40), IAPP(8-24), or Sup35NM(Ac7-16) Y→F amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail.

Chemical synthesis of tea polyphenols and related compounds.

Tea polyphenol "catechin", which is a major constituent of green tea extract, has received special attention for its important bioactivities. In this article, we review the recent various syntheses of catechins by classifying the mode of of flavan ring-construction. Additionally, we also take notice of the application of the modified catechins that were created to uncover the mechanism of bioactivities and discover new activities.

Anti-angiogenic activity and intracellular distribution of epigallocatechin-3-gallate analogs.

Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 µM. To elucidate antiangiogenic effect of EGCG, we used a 1 µM concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria.

Towards the synthesis of proanthocyanidins: half a century of innovation.

Results emanating from the synthesis of proanthocyanidins played a crucial role in defining the constitution, regiochemistry, and absolute configuration of this complex but fascinating group of plant secondary metabolites. The initial efforts, commencing in 1966, were focused on structure elucidation of, especially, the procyanidins, profisetinidins, and prorobinetinidins. However, over the past 12 years the emphasis has shifted to the synthesis of the bioactive procyanidins and some of their derivatives at a scale that would permit assessment of their pharmacological properties. With a few exceptions, the vast majority of these synthetic protocols involve the formation of the interflavanyl bond by acid/Lewis acid activation at C-4 of a flavan-3,4-diol or its equivalent, and subsequent trapping of the incipient C-4 carbocation by the nucleophilic centers of a flavan-3-ol (catechin). This review represents the first comprehensive chronicle depicting the development of the subject of proanthocyanidin synthesis.

Comparison of a pair of synthetic tea-catechin-derived epimers: synthesis, antifolate activity, and tyrosinase-mediated activation in melanoma.

Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin-derived compounds, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG) and 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro-apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non-melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.

Concise synthesis of catechin probes enabling analysis and imaging of EGCg.

A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for imaging studies and the generation of antibodies.