MKP-1 발현 증가를 통해 항염증 활성을 갖는 CMDD8299의 항동맥경화 기전 규명

학위논문 (박사)-- 서울대학교 대학원 자연과학대학 지구환경과학부, 2017. 8. 강헌중.Atherosclerosis is known as a chronic inflammatory disease and numerous attempts to improve atherosclerosis by anti-inflammatory molecules were conducted. Marine sponges are rich sources of anti-inflammatory natural products. To find the anti-atherosclerotic molecule, anti-inflammatory effects of marine natural products against cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β) were evaluated. I identified several marine natural products including phorbaketal A, which showed anti-inflammatory effects. Phorbaketal A is a marine sponge-derived bioactive sesterterpenoid, which isolated from Phorbas sp. and Monanchora sp. Byun MR et al. demonstrated that phorbaketal A stimulates osteogenic differentiation and inhibits adipogenic differentiation through activation of transcriptional coactivator with PDZ-binding motif (TAZ) and extracellular signal-regulated kinase (ERK) in C3H10T1/2 and human mesenchymal stem cell. In the present study, phorbaketal A significantly reduced IL-6, TNF-α and IL-1β mRNA expression in lipopolysaccharide (LPS)-induced raw 264.7 macrophages and inhibited IL-6 and TNF-α secretion. Phorbaketal A reduced nitric oxide (NO) secretion through inhibition of inducible nitric oxide synthase (iNOS) expression. In addition, pre-incubation of human umbilical vein endothelial cells (HUVECs) with phorbaketal A abrogated TNF-α-induced expression of VCAM-1 and MCP-1. To demonstrate anti-atherogenic effect of phorbaketal A in vivo, 5 mg/kg of phorbaketal A administered to three types of atherosclerosis in vivo models with atherogenic diet (21% fat, 1.25% cholesterol). The severity of atherosclerosis was analyzed by en face staining of aorta. Phorbaketal A treated mice had less atherosclerotic lesions than control group in progression models. MAPKs regulate numerous fundamental cellular processes especially inflammation and immune response. TLR agonists activate MAPKs signals in innate immune cells and contribute to pathogenesis of atherosclerosis. To identify the molecular mechanisms of phorbaketal A, effects of phorbaketal A on MAPK activation were estimated. Phorbaketal A inhibited LPS-induced phosphorylation of p38 MAPK but it has no effect on ERK or JNK phosphorylation in raw264.7 macrophage. In addition, phorbaketal A abrogated p38 phosphorylation in TNF-α stimulated HUVECs. The inhibitory effect of phorbaketal A recovered by MAPK phosphatase 1 (MKP-1) inhibitor, triptolide. MKP-1 is known to inhibit inadequate inflammatory response through negative feedback of MAPKs, especially p38 and JNK. Phorbaketal A induced MKP-1 mRNA and protein expression in concentration-dependent manners. Transcription factors which regulate MKP-1 expression, such as cAMP response element binding protein (CREB) and activating transcription factor 2 (ATF-2) were phosphorylated at early time (5 min) by phorbaketal A. The phosphorylation of ERK pathway signaling molecules were suppressed by U0126 (ERK inhibitor). These suggest that phorbaketal A induced MKP-1 through ERK-activated CREB and ATF-2 signals. In this study, I identified a novel mechanism of phorbaketal A, and demonstrated its anti-atherogenic effects on model of atherosclerosis in vitro and in vivo. These results suggest that phorbaketal A could be a drug candidate for treatment of inflammatory disease, such as atherosclerosis.Chapter 1. Introduction 1 1.1 Atherosclerosis 1 1.2 Inflammation in atherosclerosis 3 1.3 MAPK phosphatase 1 (MKP-1) 5 1.4 Aim of study 6 Chapter 2. Materials and Methods 7 2.1 Reagents and Antibodies 7 2.2 Cell Culture 7 2.3 Animal Experiment 8 2.4 En face Staining 9 2.5 Cell Viability 9 2.6 Total RNA Preparation and Quantitative Real-time PCR 10 2.7 miR101-PCR 12 2.8 Western Blotting 13 2.9 ELISA 13 2.10 Nitrite Assay 14 2.11 Statistical analysis 14 Chapter 3. Results 15 3.1 Phorbaketal A 15 3.2 Phorbaketal A inhibited nitric oxide production by suppression of inducible nitric oxide synthase expression in LPS-activated raw264.7 19 3.3 Phorbaketal A inhibited production of pro-inflammatory cytokines in LPS-activated raw264.7 21 3.4 Phorbaketal A differentially inhibited adhesion molecules expression in TNF-α stimulated HUVEC 24 3.5 Phorbaketal A abrogated MCP-1 expression in TNF-α induced HUVEC 26 3.6 Effects of phorbaketal A on apoE knockout mice lesion formation 28 3.6.1 Progression model-1 lesion analysis 28 3.6.2 Progression model-2 lesion analysis 31 3.6.3 Regression model lesion analysis 34 3.7 Effects of phorbaketal A on apoE knockout mice aortic inflammatory gene expression 37 3.8 Phorbaketal A activated MAPK phosphorylation 42 3.9 Phorbaketal A abrogated p38 phosphorylation in LPS-activated raw264.7 47 3.10 Phorbaketal A induced MKP-1 expression 49 3.11 MKP-1 stabilized by Phorbaketal A 52 3.12 Phorbaketal A suppressed p38 phosphorylation by MKP-1 54 3.13 Phorbaketal A upregulated MKP-1 expression through the ERK in raw264.7 58 3.14 Effects of phorbaketal A on MAPK in TNF-α stimulated HUVEC 65 Chapter 4. Discussion 70 References 81 국문 초록 101Docto

Synthetic Investigations into Alotane Derived Sesterterpenoid Marine Natural Products.

PhDPhorbaketal A (I) and phorone A (II) are members of a growing family of biologically active sesterterpenoid natural products, derived from marine sponges. These compounds possess potent biological activities and intriguing molecular architectures, which have inspired efforts towards their total synthesis. Early work in this thesis describes how new methodology is developed aimed at the synthesis of phorbaketal A (I). The synthesis and subsequent treatment of the model hydroxyphenol III with PhI(OAc)2 led to the development of a new method for spiroketal synthesis via cascade oxidative dearomatisation. This new spiroketalisation method was then further elaborated for the total synthesis of phorbaketal A (I). This required the construction of the noralotane carbon skeleton IV, employing the union of the aryl aldehyde V and the propargylated geraniol VI. Treatment of the noralotane IV with PhI(OAc)2 gave the spiroketal VIII as a complex mixture of labile diastereomers. This new method for spiroketalisation ultimately proved unsuitable for the total synthesis of these spiroketal natural products. Following this, attempts at expanding the substrate scope for this new oxidative dearomatisation method were explored for the formation of a range of saturated spiroketals. v The second part of this thesis describes efforts towards the total synthesis of the related sesterterpenoid natural product phorone A (II). Considerable synthetic effort led to synthesis of the novel (Z)-bromoalkene IX and the drimane aldehyde X, which were coupled to give the ansellane alcohol XI, in a total of 17 steps. This work represents the first ever synthesis of the ansellane skeleton. Efforts to oxidise and cyclise the alcohol XI were initiated; however difficulties with eliminative dehydration prevented its conversion to phorone A (2). The final work in this thesis describes alternate coupling methods aimed at circumventing these unforeseen issues.Engineering and Physical Sciences Research Council (EPSRC

Obesity: The metabolic disease, advances on drug discovery and natural product research

Obesity is a global health threat. OECD reported that more than half (52%) of the adult population in the European Union is overweight or obese. Obesity and obesity-related co-morbidities have deep negative effects on morbidity, mortality, professional and personal quality of life. Healthcare costs represent a negative impact of this disease, with an associated economic cost of 100 billion US$ per year in the United States. The most prescribed drugs for obesity treatment worldwide are orlistat, and phentermine/topiramate extended release, while the major prescribed drug for the same disease in the US are exenatide and dapagliflozin. The so far developed drugs, targeting weight loss, have a long history of malignant secondary effects. There is still a lack of efficient and safe drugs to treat obesity and related metabolic complications since in many cases cure cannot be reached by bariatric surgery or healthy lifestyle habits. Terrestrial and aquatic organisms are a promising source of valuable, bioactive compounds, often with interest for human health. Some of the natural compounds or organisms have been used for centuries by humans as traditional medicine foods. In this review, we give insights into the adipose tissue function and development, and the progress in traditional anti-obesity pharmacotherapy. A major focus is to highlight the state of the art of natural compounds with anti-obesity properties and their potential as candidates for drug development; an overview is given about natural compounds derived from different marine animal sources, cyanobacteria, marine phytoplankton, fungus or plants. © 2016 Bentham Science Publishers.This study was funded by the Project MARBIOTECH (reference NORTE-07-0124-FEDER-000047) within the SR&TD Integrated Program MARVALOR - Building research and innovation capacity for improved management and valorization of marine resources, supported by ON.2 Program and by the European Regional Development Fund (ERDF) through COMPETE - Operational Competitiveness Programme and NOVOMAR (reference 0687-NOVOMAR- 1-P), and national funds through FCT - Foundation for Science and Technology, through the project UID/Multi/04423/2013. Ralph Urbatzka was supported by grant SFRH/BPD/112287/2015 (FCT)

Biological Activity of Recently Discovered Halogenated Marine Natural Products

This review presents the biological activity—antibacterial, antifungal, anti-parasitic, antiviral, antitumor, antiinflammatory, antioxidant, and enzymatic activity—of halogenated marine natural products discovered in the past five years. Newly discovered examples that do not report biological activity are not included

Unified total syntheses of the antibiotic macrolides aldgamycin N and mycinamicin IV & de novo syntheses of their carbohydrate units

The present thesis provides a unified total synthesis approach to the 16-membered macrolide antibiotics aldgamycin N and mycinamicin IV. Both natural products represent two distinct series of macrolide targets, which are characterized by a highly conserved “eastern” acid half but subtle variations within their macrocyclic frameworks, as well as their specific glycosides. The unified total syntheses are enabled by the swift assembly of the macrocyclic frameworks by merging individual carbonyl and alkyne modules as the two main synthetic fragments and the subsequent ruthenium-catalyzed transformation of the C–C triple bonds into the key functionalities distinguishing the individual targets. The eastern carbonyl fragments are reached from a single common terminal alkene building block formed on multi-gram scale by an asymmetric vinylogous Mukaiyama-type aldol reaction. Wacker oxidation of the common alkene terminus provides a ketone as the anchor point towards aldgamycin N, while an unprecedented stereo- and branch-selective hydroformylation at this site controlled by a literature-reported rhodium complex furnishes an aldehyde to serve as the handle for the preparation of mycinamicin IV. After combination of these fragments with their respective alkyne counterparts by carbonyl addition and closure of the macrolactone rings by an unusual stannoxane-mediated transesterification, the individual target moieties are forged using ruthenium-catalyzed transformations of the propargylic alcohols obtained from the carbonyl additions. Specifically, these late-stage key steps comprise a regioselective hydrostannation/Chan-Lam-type oxygenative coupling sequence to unveil the acyloin of aldgamycin N, and a rare example of a rearrangement of a secondary propargylic alcohol into the unsaturated ketone of mycinamicin IV. Completion of the target syntheses by attachment of the carbohydrates proved challenging and required close attention to both the timing of the glycosidation events and the exact glycosylation conditions employed. Systematic screening of the glycosylation conditions showcases the crucial distinction in reactivity between different silyl triflates for the activation of trichloroacetimidate donors. The rare branched-chain octose D aldgarose and the basic amino sugar D desosamine, required as the 4,6 dideoxy carbohydrates at the C5 position of both natural products, are also reached by a unified approach. The employed common building block is synthesized using an enantioselective hetero-Diels Alder reaction, and intermediates of both de novo syntheses may serve the practical preparation of other naturally occurring 4,6 dideoxy sugars as well as derivatives thereof

Design and Synthesis of Nickel N-Heterocyclic Carbene Catalyst Systems and Their Application in the Cross-Coupling of Silyloxyarenes

The invention of transition-metal catalyzed cross-coupling reactions has fundamentally changed how chemists approach the synthesis of small molecules. Moreover, the ability to perform reactions catalytically while employing Earth abundant first row transition-metals has had positive impacts regarding sustainability. In addition to environmental considerations, first row transition-metals, like nickel, have been found to possess reactivity that is complementary to metals like palladium, which has implications for how the metal performs in the various elementary steps of catalytic processes. These insights have arisen from the study, design, and development of nickel catalyst systems, which have propelled the invention of catalytic strategies for the formation of a wide array of C‒C and C‒heteroatom bond forming reactions. The nature of reaction development and catalyst design are intimately related as achieving the development of novel bond forming reactions relies upon innovative advances that reduce the limitations associated with existing catalyst systems to provide enhanced performance, or to make the catalysts, themselves, more accessible. An account of these efforts in the context of C‒O bond functionalization and air-stable, discrete nickel(0) (Ni(0)) n-heterocyclic carbene (NHC) catalysts are described, herein. Chapter 1 is largely oriented towards the utility of phenol derivatives as electrophilic coupling partners with low-reactivity C‒O bonds being of main interest. The challenges regarding the activation of these low-reactivity C‒O bonds is discussed, as well as current strategies to overcome their inherently low-reactivity. Although low-reactivity C‒O bonds are challenging to activate, their functionalization is of import as possessing electrophilic coupling partners that range in reactivity enables highly selective sequential couplings, resulting from leveraging the orthogonal reactivity of each electrophile. Chapter 2 chronicles the utility of aryl boronic acids in organic synthesis, with particular attention paid to their role in cross-coupling reactions. The synthesis of these compounds spanning from traditional approaches to modern techniques, which includes the state of the art in the borylation of low-reactivity C‒O bonds. The limitations of these strategies are covered and the development of a borylation of silyloxyarenes is described along with synthetic demonstrations that highlight the ability of silyloxyarenes to be used orthogonally to other electrophiles. Chapter 3 entails the advantages of catalysis, and the transition metal catalysts we rely upon to mediate these processes, with special attention to nickel, have evolved. For the purposes of this dissertation, nickel catalysts have been categorized as 1) Ni(II) salts, 2) discrete Ni(II) pre-catalysts, 3) Ni(0) pre-catalysts, 4) air-stable Ni(0) pre-catalysts, and 5) discrete air-stable Ni(0) pre-catalysts. The advantages and disadvantages, activation strategies, and synthesis of each category are described in detail. Preliminary work regarding progress towards the synthesis of an air-stable, discrete Ni(0) NHC complex from nickel (II) (Ni(II)) precursors and NHC salts is described.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169764/1/pein_1.pd

Human and plant proteins as reservoirs of host defence peptides

Antimicrobial peptides (AMPs), known also as host defense peptides, are fundamental evolutionarily conserved components of innate immunity. Constitutively or inducibly expressed in response to invasion by pathogens, they operate synergistically with other defence molecules to combat infections. Despite differences in their size and sequence, many of them share a net positive charge at neutral pH, and fold into amphipathic structures, often after contact with bacterial surfaces. HDPs are attractive alternative candidates for antibiotic treatment, because they offer several advantages over the currently used drugs. They combat pathogens by targeting bacterial membranes, thus impairing essential membrane-related functions, and, in some cases, also target intracellular components. Due to their peculiar mechanism, the resistance towards these peptides would be difficult for the bacteria to develop. Several proteins, including proteins apparently not involved in immunity, can behave as sources of HDPs hidden in their primary structures and released by the action of host and/or bacterial proteases. Recently it has been developed a bioinformatic tool allowing to identify such “cryptic HDPs”. Analyzing a library of 4000 proteins, we have identified and studied several novel cryptic HDPs from human and plant. Among these, three human peptides (GVF27, ApoBS and ApoBL) show pharmacologically relevant properties like significant antimicrobial activity on a broad spectrum of bacteria (including some clinical isolates), very promising antibiofilm properties (both on pre-formed and attached biofilm), strong affinity for endotoxins as LPS and LTA and immunomodulatory properties on LPS induced murine macrophages, while two peptides from plant (IKY31 and IKY23) are strong anti-biofilm agents and not toxic against eukaryotic cells. Overall our data suggest that these new cryptic HDPs, could serve as leads for the design of innovative antimicrobials with immunostimulating and immunomodulatory properties

Crytic antimicrobial peptides hidden in protein precursors: identification of novel bioactive molecules

Antibiotics are the mainstay in treatment of bacterial infections. However, resistance to antibacterial treatments has been rising since the 1970s, causing serious problems in the treatment and control of infectious diseases. Antibiotic resistance is now considered as one of the major global health threats of the 21st century in that the worldwide use of antibiotics is predicted to increase by more than 65% in the coming decades due to the increasing demand for meat and shift in agriculture practices in developing countries. New antibacterial drugs are urgently needed, but only three antibacterial drugs have been brought to the market since 1999 and very few new antibiotics are currently in development. This has prompted the search for alternatives to conventional antibiotics. Multiple alternative anti-infective strategies are being investigated, including vaccines, probiotics and phage therapy. Other promising alternatives to antibiotics are host defense peptides (HDPs), an important component of the first line of defense against infection, found in all multicellular organisms. HDPs are seen as true multifunctional peptides with activities as diverse as chemotaxis, inhibition of LPS-induced inflammation, modulation of leukocyte differentiation and promotion of wound healing. Interestingly, novel functions of these peptides are still being described to date. Many human proteins with functions not necessarily related to host defense behave as sources of HDPs. Some examples are lactoferrin, lysozyme and thrombin. Since these peptides are hidden in large proteins, they can be defined as “cryptic”. In order to identify by a rational approach further human proteins carrying cryptic HDPs, we recently developed an in-silico screening method to localize antimicrobial regions hidden inside the primary structure of precursor proteins. A wide list of potential new antimicrobial peptides was obtained by applying this method to about 4,000 human extracellular proteins. The main aim of this PhD project was the identification of interesting potential HDPs, to develop novel bioactive peptides. Firstly, we developed a novel and cost-effective method to produce recombinant HDPs, based on the use a cheap and efficient medium to be employed in an auto-inducing fermentation process. Furthermore, to avoid HDPs toxicity towards bacterial host, a novel fusion system based on a carrier protein (derived from a Rana pipiens ribonuclease) was used. Once optimized the production system, a broad characterization of two novel recombinant peptides, previously identified in human Apolipoprotein B (ApoB), was performed. We demonstrated that both peptides are endowed with a significant antimicrobial activity towards Gram-negative and Gram-positive strains, and are able to prevent biofilm formation in several strains at concentrations lower than those required to directly kill planktonic bacterial cells. Moreover, ApoB-derived peptides were found to be endowed with anti-inflammatory properties as well as the ability to promote wound healing in keratinocytes. In addition, two further cryptic HDPs have been structurally and functionally characterized. One of these HDPs has been identified in human 11-hydroxysteroid dehydrogenase-1 β-like, the other represents the first HDP from an archaeal protein, the transcription factor Stf76 encoded by the hybrid plasmid-virus pSSVx from Sulfolobus islandicus. By means of a multidisciplinary approach including biochemical, cellular biology and spectroscopic techniques, the action mechanism of both peptides has been elucidated, and intriguing results have been obtained by testing their immunomodulatory and anti-cancer activities. Hence, the in silico-derived panel of potential HDPs is a rich source of peptides with pharmacologically relevant properties

Totalsynthese von Limaol

Limaol ist ein aus dem benthischen Dinoflagellaten Prorocentrum lima isoliertes C40-Polyketid mit auffälligen Strukturmerkmalen wie vier nicht-konjugierten exo-Methylengruppen im nördlichen Teil und einem chiralen Spiroketalkern. Diese Dissertation beschreibt die Synthese von Limaol. Aufgrund der Größe des Zielmoleküls wurde ein fragmentbasierter Ansatz gewählt. Retrosynthetisch wurde das Molekül in drei Bausteine von ungefähr gleicher Komplexität aufgeteilt, was eine konvergente Synthese ermöglichte. Das zentrale und das südliche Fragment wurden durch eine asymmetrische Allylierung vereinigt, welche entgegen der erwarteten Cram-Chelat-Selektivität verlief und eine Inversion der Stereochemie an C27 nötig machte. Das nördliche Trien-Fragment wurde durch eine Allyl-Alkenyl-Kreuzkupplung mit dem Rest des Zielmoleküls verbunden. Insgesamt wurden auf diese Weise 3,3 mg Limaol hergestellt. In dem Bestreben, die Materialausbeute für biologische Tests zu erhöhen, wurde eine Synthese der zweiten Generation entwickelt. Die größten Engpässe des ersten Ansatzes waren auf die ungünstige Sterik des Zentralfragments zurückzuführen. Ein Wechsel der Schutzgruppen verbesserte die Effizienz der neuen Route erheblich. Die Gesamtausbeute wurde gegenüber dem Ansatz der ersten Generation vervierfacht. Darüber hinaus erwies sich die überarbeitete Route als skalierbar und lieferte 277 mg Limaol in einem Durchgang