Rozprawy doktorskie na temat „Novel glycolipids”

The glycolipid α-galactosyl ceramide, α-GalCer, has been shown to stimulate the proliferation of murine spleen cells and activate the immune system. Stimulation occurs through binding of the glycolipid to the protein CD1d. Subsequent presentation of the CD1d−glycolipid complex to invariant Natural Killer T cells (iNKT cells) initiates the proliferation of a host of cytokines leading to an immune response The therapeutic potential of α-GalCer is currently being explored; however the induction of both Th1 and Th2 cytokines by this agent is likely to limit its therapeutic application. Significantly, analogues of α-GalCer have been shown to induce iNKT cell-derived cytokines more selectively through a skewed Th1-Th2 response. To date, very few alterations around the amide bond have been explored. To investigate its importance in iNKT cell stimulation, a range of α-GalCer and threitol ceramide (ThrCer) analogues has been synthesised in which the amide functionality in these two leads has been replaced with different carbonyl functional groups. These compounds have been tested for iNKT cell induction and in particular their Th1/Th2 response, which determined their therapeutic potential. Labelled derivatives of α-GalCer and ThrCer have also been designed and synthesised to find application in lipid trafficking studies.

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagonists on HEK cells and the capacity to inhibit LPS-induced septic shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the panel as hits for anti-sepsis drug development. In addition in the studies presented in this PhD thesis new classes of natural compounds are tested for their ability to interact with TLR4 receptor complex.

NKT cells are an important part of human immune system and recognize a specific set of antigens called glycolipids. Only a handful of "natural" NKT cell antigens are known till date. Although NKT cells play a protective role against pathogenic organisms, imbalances in NKT cell functions are implicated in many diseases including asthma. Allergic asthma, a Th2 driven inflammation of airways, is primarily caused by inhalation of environmental allergens. In the last decade, inhaled allergen Aspergillus fumigatus has been under scrutiny for the presence of NKT cell antigens that might trigger asthma. We successfully isolated, characterized and synthesized a "natural" antigenic glycolipid which activates NKT cells in CD1d dependent manner. When this glycolipid is administered intranasally to mice, WT but not CD1d-/- mice developed airway hyperreactivity (AHR), which is a cardinal feature of asthma. Our results indicate that this glycolipid also triggers the production of key cytokines responsible for development of airway hyperreactivity, namely IL-4 and IL-13. Widespread use of antibiotics has convoluted the problem of antimicrobial resistance. Our research group has developed a novel class of antimicrobial peptide mimics called Ceragennins. These cholic acid based antimicrobial compounds have many desirable properties including low MICs, effectiveness against biofilms, and relatively low manufacturing cost. In order to advance the clinical development of Ceragennins, we developed analytical methods for qualitative and quantitative determination of these compounds in complex biological matrices. These methods were also used for carrying out the stability studies of Ceragenins under varying pH and temperatures

Invariant NKT (iNKT) cells are a subset of T lymphocytes that express an invariant \(\alpha\) \(\beta\) T cell receptor (TCR) as well as an NK1.1 marker. They play an important role in autoimmune diseases, such as type I diabetes and lupus. In contrast to conventional CD4+ and CD8+ T lymphocytes that recognise foreign peptides bound to the major histocompatibility complex (MHC) class I or MHC class II, iNKT cells recognise a range of foreign lipids and glycolipids bound to CD1d proteins. \(\alpha\) \(\beta\)-Galactosyl Ceramide (\(\alpha\) \(\beta\)GalCer), originally isolated from a marine sponge, is a powerful agonist of CD1d capable of triggering an immune response that results in the proliferation of a range of regulatory cytokines, including IFN-\(_y\) (Th1), as well as IL-4 (Th2). This mixed cytokine response (i.e. Th1 and Th2), combined with the “unresponsive state” of iNKT cells after activation with \(\alpha\)GalCer, limits the therapeutic potential of this agonist. To address some of these issues, we have also synthesised an \(\alpha\)GalCer analogue, namely Threitol Ceramide (ThrCer), that exhibits attenuated activity relative to \(\alpha\)GalCer. ThrCer is a truncated analogue of \(\alpha\)GalCer that conserves the stereochemistry of the hydroxyl functions present in \(\alpha\)GalCer and that exhibit a much stronger ether bond under acidic hydrolysis linking the sugar moiety with ceramide than the glycosidic bond in \(\alpha\)GalCer. We have labelled ThrCer with a biotin residue and with a 14C radiolabel, and our collaborators have used these derivatives to show that ThrCer behaves similarly to \(\alpha\)GalCer in endogenous lipid trafficking and its tissue distribution in vivo. We have also made advances towards the stereoselective synthesis of recently discovered natural agonists of iNKT cells from pathogenic origin, namely \(\alpha\)-galactosyl diacylglycerol (\(\alpha\)GalDAG).

The development of novel medicinal agents is becoming increasingly important in solving several serious healthcare issues. Tuberculosis (TB) is among one of the world’s most serious health threats, especially in developing countries, and although treatment is available, current methods take 6 – 24 months. Prolonged treatment times often result in noncompliance and subsequently result in selection for drug resistance. As there are several multidrug resistant strains of Mycobacterium tuberculosis current treatment methods are proving inadequate, and it is becoming more and more essential to develop novel drugs to control the incidence and spread of TB; alongside other pathogenic bacterial species of clinical importance. Three major approaches towards the creation of novel antitubercular and antimicrobial agents and treatment regimens through the use of carbohydrate-based chemistry were explored within this project; (i) structure-based drug design and synthesis of novel competitive inhibitors targeting mycolic acid synthesis of M. tuberculosis (Chapter 1 & 2); (ii) modification of aminoglycosides (AGs) through amphiphilic substitution, aimed to restore activity towards resistant bacteria or create agents with novel mechanisms of action (Chapter 3 & 4); (iii) the development of novel liposomal drug carriers to selectively deliver therapeutics towards severe intracellular respiratory infections (Chapter 5 & 6). Mycolic acids are essential for the growth and survival of M. tuberculosis, making mycolic acid inhibition a highly promising drug target for treating TB. Recently, a novel pathway of fatty acid synthesis has been identified as a promising drug target. FadD32, a fatty acyl-AMP ligase (FAAL) involved in the progression of TB represents a promising target for novel drug design. A family of sulfonylacetamide compounds have shown promising preliminary results in treating TB, however these failed within in vivo experiments due to their poor pharmacokinetic properties. Further development of these lead compounds with a rational structure-based drug design approach, led to the design and synthesis of compounds directly targeting the FadD32 biosynthetic pathway. This synthesised library of compounds were evaluated for their antitubercular inhibitory effect and several lead structures were identified. The most potent derivatives synthesised within this study displayed an MIC90 of 13 – 16 μg/mL towards M. tuberculosis (H37Rv) within a normoxic resazurin reduction microplate assay. Structural modification of the AG scaffold has historically remained a successful approach towards creating novel antibiotics. This approach led to the discovery of amikacin, a potent AG that is used for the treatment of TB today. Recently, a new class of antibiotics derived from the AG scaffold has produced several promising lead compounds that have revived antimicrobial activities against drug resistant bacterial species of clinical importance. This new class of antimicrobial agents are derived from conjugation of hydrophobic groups to AGs, creating semi-synthetic amphiphilic aminoglycosides (AAGs). AAGs have shown promising preliminary results against several drug resistant bacterial strains, including mycobacteria by evading common mechanisms of resistance and exerting novel mechanisms of action. A variety of novel and structurally diverse AAGs have been synthesised within this study providing lead structures with promising activity for the development of antimicrobial agents. A series of AAGs synthesised from the scaffolds of amikacin, kanamycin and neomycin were synthesised by conjugation of several hydrophobic groups to the AG scaffold. Derivatives were prepared by utilizing either the active functionality from the sulfonylacetamides conjugated via amide or triazole linkers; or by an alternative prodrug-based strategy utilizing long alkyl chains conjugated via ester linkages. Due to AGs broad-spectrum antibacterial activities, these compounds were evaluated for their antimicrobial inhibitory effect towards M. tuberculosis and S. aureus. Several structures were identified to display potent antitubercular activities. One of the kanamycin-based prodrugs was found to exert an increased antitubercular activity in comparison to the parent scaffold (MIC90 3 μg/mL), while amikacin derivatives maintained significant inhibitory effect (MIC90 3 – 6 μg/mL). The most active AAG utilizing the active functionality of the sulfonylacetamides was an amide-linked conjugate of amikacin (MIC90 22 μg/mL). Intracellular infections from bacterial pathogens such as M. tuberculosis are the causative agents of several serious diseases worldwide. Although the mononuclear phagocyte system primarily consists of macrophages, and constitutes the primary line of defence against infections, several bacterial pathogens have evolved the ability to survive and reproduce within intracellular host organelles. Intracellular infections are difficult to eradicate through traditional chemotherapeutic approaches due to the poor intracellular penetration of antibiotics. Moreover, the resulting low intracellular concentration of antibiotics is often ineffective against intracellular pathogens and furthermore promotes the development of drug resistance. Consequently, novel approaches to improve therapeutic effectiveness must be considered to advance treatment towards persistent intracellular bacterial infections. Nanoparticle-based drug delivery systems are one such approach; enabling improved antibiotic bioavailability and targeted site-specific drug delivery, offering the ability to overcome the less than desirable effects most antibiotics display towards intracellular pathogens. Liposomal drug delivery vectors are the most established and investigated nanocarriers, offering nontoxic biological delivery systems that can enhance the antimicrobial efficiency and therapeutic index of various chemotherapeutics. Liposomes provide unique delivery systems for antibiotics, such as AGs, as they can enhance efficiency whilst decreasing toxicity. Liposomal encapsulated AGs have demonstrated enhanced bactericidal activity towards intracellular infections in vitro and in vivo. Liposomal encapsulated amikacin has demonstrated its effectiveness in clinical trials and is currently FDA approved for the treatment of Mycobacterium avium complex. Within this study, amphiphilic-based prodrug derivatives of amikacin and mannosylated targeting ligands were utilized to formulate novel liposomal preparations for the treatment of pulmonary infections. Amphiphilic derivatization of amikacin allowed passive drug loading of the active therapeutic within the lipid bilayer, providing improved drug loading capacities compared to conventional approaches of loading amikacin within the aqueous core. Additionally, incorporation of the therapeutic AAG within the bilayer resulted in vesicles with improved membrane stability and more desirable physiochemical properties compared to conventional formulations. To test these liposomal formulations as a delivery system against intracellular pathogens in vitro, a murine macrophage cell line expressing mannose receptor (RAW264.7) was infected with S. aureus and bacterial inhibition was determined. All formulations displayed concentration-dependent killing efficiencies, while some were capable of complete intracellular bacterial inhibition at higher concentrations. The lipid derivatized amikacin-based prodrug delivery systems provide a novel liposomal delivery platform for intracellular infections, whilst offering the potential to overcome the shortcomings of current liposomal formulations such as poor stability and drug leakage. Once realized, this application could improve treatment towards serious intracellular pulmonary infections such as M. tuberculosis.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
Full Text

Les liquides biologiques ont ete etudies par spectroscopie de resonance magnetique nucleaire. La premiere partie concerne la mise au point de methodologies de rmn adaptees a l'etude des liquides biologiques. Une nouvelle methode de quantification des metabolites plasmatiques, basee sur l'utilisation du formate comme reference, a ete developpee. Les resultats des dosages par rmn sont en bon accord avec les donnees obtenues par les methodes de dosages biochimiques. Les sequences multi-impulsionnelles (echo de spin, cosy, j-resolue) ont ete adoptees pour la simplification des spectres et l'attribution des resonances. L'elimination complete du signal de l'eau a ete obtenue grace a l'addition aux echantillons de chlorure d'ammonium. Dans une deuxieme partie nous avons aborde les perturbations du metabolisme lipidique et glycoproteique chez les sujets cancereux. Le rapport des protons methyle/methylene ainsi que les residus glycosyles sont augmentes. Leur taux varie avec le stade et la nature de la tumeur. L'etude des extraits lipidiques par la rmn du proton et du phosphore-31 a montre l'augmentation du rapport triglycerides/phospholipides en parallele avec la diminution du taux des phospholipides. Enfin, la rmn-2d a permis de montrer que les glycolipides fucosyles ne constituent qu'une fraction mineure dans le plasma des sujets cancereux. La puissance analytique de la rmn des liquides biologiques et son contenu informationnel sont de nature a faciliter le developpement de cette technique dans le domaine de la biologie clinique

Background: Vascular diseases represent a significant burden on healthcare systems in the developed world, heavily contributing to mortality and morbidity. TLR4 is an innate immune receptor integral to host defence against pathogens. However, overactivation of this response can be detrimental and is associated with development and progression of chronic inflammatory based conditions such as vascular diseases. Modulation of TLR4 activity therefore represents a potential avenue for development of pharmacological interventions. Objective: Professor Peri (University of Milano) has developed a series of synthetic glycolipid small molecule modulators of TLR4. The current body of work aimed to screen and assess the potential of several predicted agonist and antagonists to affect TLR4 signalling in a cellular system, investigate the mechanism of action of these compounds and to determine their validity as modulators of haematopoietic TLR4 signalling in human and mouse cell lines. Methods: Human and mouse monocyte and macrophages were used as a model. MTT was used to determine cytotoxic effects of ligands and small molecule antagonists and agonists. TLR4 activation was stimulated by exposure of cells to bacterial endotoxin or sterile oxidised lipids and effects. FP compounds were tested in the presence or absence of TLR4 ligands. Two readouts were used: activation of signalling mediators and endpoint proinflammatory protein production. These were measured using Western blot, ELISA, and a proinflammatory protein antibody array. Results: Screening revealed two antagonists and one agonist which were selected for further mechanistic studies. FP7 and FP12 were shown to negatively regulate both human and mouse MyD88-dependent signalling and human TRIF-dependent pathways. FP11 and FP18 were more effective at inducing MyD88-dependent signalling than TRIF-dependent but may still activate signalling via IFNAR. Conclusions: Results from the current body of work provide novel evidence towards the effects and mechanism of action of FP antagonists and agonists as modulators of TLR4-dependent proinflammatory signalling. Together, this helps to validate two antagonist and two agonist molecules as potential novel therapeutics for further preclinical investigations for treatment of inflammatory based disorders.

碩士
國立臺灣大學
生化科學研究所
96
According to previous studies, glycolipids are the major components present in the cell surface of thermophilic bacteria. The high proportion of glycolipids in the cell membranes could possibly to contribute to the ability of the bacteria to grow at high temperature, however, the molecular basis of this phenomenon is still unclear. The structure of glycolipid of thermophilic bacteria has long been a topic and has been proved to exhibit great structural diversity due to its high temperature growth condition. This study focuses on the structural analyses of glycolipids isolated from this new indigenous of Taiwan, NTU-1233. Lipid crude extracts from two type strains, Truepera radiovitrix and Deinococcus murrayi; and five indigenous strains, NTU-484, NTU-1233, NTU-1085, NTU-1089, NTU-407 were subjected to TNF-α induction assay and found that lipid crude extracts of NTU-1233 exhibits stronger immunostimulation activity. The structures of three major glycolipids purified from lipid crude extracts of NTU-1233 were determined using TLC, NMR spectroscopy (TOCSY, COSY, HSQC, HMBC), GC-MS, MALDI-MS and ESI-MS, and chemical analyses. According to the spectroscopic and chemical analyses, the structures of three glycolipids are elucidated to be two sulfolipids sulfoquinovosyl diacylglycerol [SQDG] and its acylated derivative, 2’-O-acyl-sulfoquinovosyl diacylglycerol [ASQD], and a novel sphingolipid, glucosaminyl(1-3)-β-galactosyl-(1-1’)-dihydroceramide [GlcNAcα3Galβ- Cer]. The acyl chain of fatty acid moiety of these three glycolipids are saturated according NMR analysis. It is the first time for ASQD to be found in nonphotosynthetic organism, and for GlcNAcα3GalβCer, it is a novel compound that has never been reported before.

碩士
國立臺北科技大學
生物科技研究所
97
Glycolipids exhibit great structural diversity and play important roles in biology system. Previous studies indicated that the structures of glycolipids were the major components of thermophilic bacteria cell membrane. Here, we aim to discover the novel glycolipids which have bioactivity of immunomodulation from radiation-resistant thermophilic bacteria found in Taiwan. Our study focus on the immunoactivity and structural analyses of Genus Rubrobacter glycolipids isolated from hot springs indigenous to Taiwan. The glycolipid crude extracts from strains NTU-520, NTU-806, NTU-808, NTU-829, NTU-1102, NTU-1106, NTU-1110, NTU-1119 and NTU-1129 of Genus Rubrobacter were subjected to TNF-α induction assay. Result shows glycolipid crude extracts of NTU-806 possesses highest immunostimulation activity. Structure composition analysis of NTU-806 by Column Chromatography, and were determined NMR spectroscopy, Mass spectrometer, indicated this strain contain two major sulfolipids; (6-(2-butyramido- 3-hydroxyheptyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methanesulfonate (SQC) and (6-(2-(butyryloxy)-3-propoxypropoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl) methanesulfonate (SQMG). It is the very first time for SQC to be found in linkage between sulfoquinovose and ceramide which is a novel compound that has never been reported before.

The thesis entitled “Novel Redox Responsive Cationic Lipids, Lipopolymers, Glycolipids and Phospholipid-Cationic Lipid Mixtures: Syntheses, Aggregation and Gene Transfection Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel cholesterol based cationic lipids with ferrocene as the redox moiety, polyethylenimine based ferrocenylated lipopolymers and cholesterol based non-ionic glycolipids. The thesis also discusses the cationic phospholipid-cationic lipid mixtures as superior gene transfection agents. The work has been divided into six chapters. Chapter 1. Introduction Part A. Various Cholesterol based Systems for Applications as Biomaterials Liposomes composed of cationic lipids have become popular gene delivery vehicles. A great deal of research is being pursued to make efficient vectors by varying their molecular architecture. Cholesterol being ubiquitous component in most of the animal cell membranes is increasingly being used as a hydrophobic segment of synthetic cationic lipids. In this chapter we describe various cholesterol based cationic lipids and focus on the effect of modifying various structural segments like linker and the headgroup of the cationic lipids on gene transfection efficiency with a special emphasis on the importance of ether linkage between cholesteryl backbone and the polar headgroup. Interaction of cationic cholesteryl lipids with dipalmitylphosphatidycholine membranes is also discussed here. Apart from cholesterol being an attractive scaffold in the drug/gene delivery vehicles, certain cholesteryl derivatives have also been shown to be attractive room temperature liquid-crystalline materials. Part B. Diverse Applications of Ferrocene Derivatives This chapter gives a brief overview of ferrocene chemistry followed by description of major applications of ferrocenyl derivatives in a variety of fields like catalysis, materials chemistry, electrochemical sensors, medicinal chemistry etc. We discuss the use of ferrocene as an electrochemical and redox active switch to achieve control over supramolecular aggregation. It also reviews ferrocene based amphiphiles including surfactants, lipids and polymers with an emphasis on the role of ferrocene over aggregate formation and their utilization in biological applications. Chapter 2: Optimization of Redox Active Alkyl-Ferrocene Modified Polyethylenimines for Efficacious Gene Delivery in Serum 1a-c, n = 6, P8-C6-F1, P8-C6-F2, P8-C6-F3 2a-c, n = 11, P8-C11-F1 P8-C11-F2, P8-C11-F3 % ferrocene grafting, F1 = 15%, F2 = 25% and F3 = 50% Figure 1. Structure of the alkyl-ferrocene modified 800 Da Branched Polyethylenimine. In this chapter we present six new lipopolymers based on low molecular weight polyethylenimines (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and redox state of ferrocene in the lipopolymer on the self assembly properties were investigated in detail by transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements. The assemblies displayed a redox induced increase in the size of the aggregates. The coliposomes comprising of the lipopolymer and a helper lipid 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) showed excellent gene delivery capability in serum containing environment in two cancer cell lines (HeLa, U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI 25 KDa and even better than commercial Lipofectamine 2000 as evidenced from luciferase activity and EGFP expression analysis. Oxidation of ferrocene in lipopolymers led to reduced levels of gene transfection which was also followed by cellular internalization of fluorescently labeled pDNA using confocal microscopy. Cytotoxicity assay revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels. Overall, we have exploited the redox activity of ferrocene in PEI based polymeric gene carriers for trenchant control over gene transfection potential. RLU/mg protein HeLa Cells Figure 2. Maximum transfection efficacies of optimized redox lipopolymer/DOPE formulations by (A) Luciferase Assay and (B) Flow cytometry (GFP expression). Chapter 3. Membranes derived from Redox-active Cholesterol based Cationic Lipids and their Interactions with DNA and Phospholipid Membranes Figure 3. Molecular structures of the electroactive cholesterol based monomeric and gemini lipids. This chapter describes the synthesis and aggregation properties of two series of redox-active ferrocene containing monomeric and gemini cationic lipids with cholesterol as a hydrophobic domain. These cationic lipids are modified at their headgroup region using ferrocene terminated alkyl chains of differing length. All the four cationic lipids formed stable suspensions in water. Aggregation behavior of these cationic lipids in aqueous suspensions in their unoxidized and oxidized state was studied using TEM, DLS, zeta potential measurements and XRD studies. Cationic lipids with ferrocene in natural, reduced state were found form bigger sized vesicles which upon oxidation became smaller aggregates with increased zeta potential. XRD results indicate the existence of nice lamellar arrangements of the lipid bilayers. Thermotropic phase transition behavior of DPPC membranes incorporated with cationic ferrocene lipids was also studied using differential scanning calorimetry. Finally, we assayed pDNA (plasmid DNA) binding ability of all the four cationic lipids using ethidium bromide intercalation assay where all the cationic lipid formulations showed excellent DNA binding capability. In the experiments involving SDS-induced release of DNA, we observed that redox-active monomeric lipids (3a-b) were found to be more efficient in facilitating the release of DNA from the liposome-DNA complex in the presence of negatively charged SDS micelles than their gemini counterparts (4a-b). Chapter 4. Redox-responsive Gene Delivery by Ferrocene containing Cationic Cholesteryl Lipids in Serum This chapter describes the transfection efficacy of redox-active monomeric and gemini cationic lipids with cholesterol backbone. The transfection efficiency of all the lipids could be tuned by changing the oxidation state of the ferrocene moiety. Gene transfection capability was assayed in terms of EGFP expression using pEGFP-C3 plasmid DNA in three cancer cell lines of different origin, namely Caco-2, HEK293T and HeLa in the presence of serum. Figure 4. Effect of oxidation state of ferrocene on maximum transfection efficacies of monomeric and gemini lipids in three different cell lines (Caco-2, HEK 293T and HeLa). Cationic liposomal formulations with ferrocene in its reduced state were observed to be potent transfectants reaching the EGFP expression levels even better than commercial lipofectamine 2000 in the presence of serum as evidenced by flow cytometry. EGFP expression was further substantiated using fluorescence microscopy studies. All liposomal formulations containing oxidized ferrocene displayed diminished levels of gene expression and interestingly, these results were consistent for each formulation in all the three cell lines. Assessment of EGFP expression mediated by both reduced and oxidized ferrocene containing formulations was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Lipoplexes derived from different liposomal formulations with reduced and oxidized ferrocene were characterised using TEM, AFM, zeta potential and DLS measurements. Overall, we demonstrate here controlled gene transfection levels using redox driven, transfection efficient cationic monomeric and gemini lipids. Chapter 5: Synthesis of ‘Click Chemistry’ Mediated Glycolipids: Their Aggregation Properties and Interaction with DPPC Membranes This chapter describes the synthesis and aggregation properties of cholesterol based glycolipids along with their interaction with a model phosphatidylcholine membranes. Three series of non-ionic glycolipids with hydrophobic cholesterol backbone and various monosaccharide and disaccharide sugars as the hydrophilic polar domain have been synthesized. These were conjugated to the cholesteryl backbone via oligooxyethylene spacers of different lengths (n = 1, 3 and 4) using Cu (I) catalyzed Huisgen [3+2] cycloaddition, which is popularly known as „Click Chemistry‟. All the synthetic glycolipids (5a-d, 6a-d and 7a-d) formed vesicular aggregates in aqueous medium as confirmed by TEM and DLS. XRD studies with the cast films of lipids revealed that the bilayer width increased with increase in the length of oligoethylene spacer unit that has been incorporated between the hydrophobic and hydrophilic domains. Also, within the same series containing a particular oligoethylene unit, bilayer widths were found to be more for the lipids containing disaccharides as their headgroup than monosaccharides. Figure 5. Molecular structures of various cholesterol-based glycolipids. Calorimetry studies of the coaggregates containing naturally occurring 1, 2-dipalmitoylphosphatidylcholine (DPPC) and various mol-% of each of the glycolipids revealed that more than 30 mol-% of glycolipids are required to completely abolish the phase transition of DPPC membranes. These results were further supported by fluorescence anisotropy measurements of the co-aggregates using 1, 6-diphenylhexatriene (DPH) as a probe. Fluorescence anisotropy of the neat vesicles revealed that 9a and 9c were more rigid than DPPC vesicles in the solid-like gel phase, while the glycolipids with longer oxyethylene spacers (n = 3 and 4) were less rigid than the DPPC vesicles. Chapter 6. Hydrophobic Moiety Decides the Synergistic Increase in Transfection Efficiency in Cationic Phospholipid/Cationic Lipid mixtures This chapter describes the effect of inclusion of cationic lipid/cationic gemini lipids into the membranes of a cationic phospholipid on the gene delivery efficiency across HeLa and HEK293T cell lines. Although all the three cationic lipids have the same quaternary ammonium moiety as their headgroup, they differ from each other in terms of their hydrophobic moiety and in the number of cationic headgroups. Chol-N is a cholesterol based monocationic lipid, while 2C14-N and 2C14N-5-N2C14N are monomeric and gemini cationic lipids respectively with pseudoglycerol backbone consisting of tetradecyl (n-C14H29) chains. Each of the three cationic lipids under the current investigation, namely, Chol-N, 2C14-N and 2C14N-5-N2C14N were added in different ratios to EtDMoPC and the resultant mixed membranes were studied for the biophysical characterization and gene delivery efficacies. Figure 6. Molecular structures of cationic lipids used in this study. All the formulations were characterized using dynamic light scattering and zeta potential measurements to obtain their hydrodynamic diameters and surface charge properties respectively. Their DNA binding ability was also studied by measuring changes in zeta potential and gel electrophoresis of the lipoplexes formed by the coliposomal formulations and pDNA at different Lipid/DNA weight ratios. The gene delivery efficacies of various formulations were studied in terms of EGFP expression using pEGFP-C3 plasmid DNA in two different cell lines, namely HeLa and HEK293T. In the absence of serum we found that the formulation (EtDMoPC+2C14N-5-N2C14N) showed better transfection efficiency than the individual lipids. However, in the case of others, i.e., (EtDMoPC+Chol-N) and (EtDMoPC+2C14-N) formulations, there was a slight decrease in transfection efficiency compared to the individual lipids. In the presence of serum, the formulations (EtDMoPC+2C14-N) and (EtDMoPC+2C14N-5-N2C14N) showed significantly higher transfection efficacies compared to their individual lipids. Fusion assay using labelled cationic lipid formulations and unlabelled anionic liposomes revealed that lipoplexes prepared from EtDMoPC+ 2C14-N and EtDMoPC+ 2C14N-5-N2C14 exhibited much higher fusogenicity as compared to the lipoplexes prepared using EtDMoPC+Chol-N as well as the individual lipids. Thus, the liposome formulations which showed better transfection activity fused more readily with the anionic liposomes than did the formulations with poorer activity. Overall, we found that the hydrophobic domain of the cationic lipid/cationic gemini lipid that is added to cationic phospholipid has an important role on the transfection efficiency of the mixed formulations. Additionally the cytotoxicity studies revealed that each of these formulations was not significantly toxic making them viable for applications in vivo. (For structural formula pl see the abstract pdf file)

Mycobacteria are atypical bacteria possessing unusual cell envelopes comprised of an outer membrane, covalently linked to an arabinogalacatan-peptidoglycan structure via waxy mycolic acids, in addition to the conventional inner membrane. This thick and highly impermeable cell envelope is a major deterrent to antibiotic treatment of clinically relevant mycobacterial pathogens, including Mycobacterium tuberculosis (Mtb), which infects a third of the world’s population and kills millions each year. Thus, the regulation of mycobacterial cell envelope biosynthesis is of great interest for the development of more effective therapeutics for treating Mtb infections. Using the model organism Mycobacterium smegmatis (M. smegmatis), we identified a novel protein, Spe2, with an unknown role in the biosynthesis of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM). Based on the observation that Δspe2 mutants produce truncated LM/LAM, I speculated Spe2 might enhance the elongation of these products. Here, I use biochemical assays to show Spe2 is localized to the periplasm where it can directly interact with the LM/LAM biosynthetic pathway. I further utilize a genetic approach to demonstrate that Spe2 acts at the stage in which the mannosyltransferase MptA mediates periplasmic LM elongation. Moreover, native polyacrylamide gel electrophoresis (PAGE) and co-immunoprecipitation techniques failed to reveal Spe2 protein binding partners. Together, these data suggest Spe2 is a periplasmic protein involved in regulating LM/LAM biosynthesis, perhaps through direct interactions with LM intermediates.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

碩士
國立臺灣大學
生化科學研究所
96
Aberrant glycosylation is often a hallmark during tumor progression and correlates with poor prognosis. Diverse tumor associated antigens existed in the form of glycolipids or glycoproteins have been characterized. Globo H (Fucα1→2Galβ1→3 GalNAcβ1→3Galα1→4Galβ1→4Glu) hexasaccharide was first isolated from metastatic breast cancer cells by Hakomori in 1984 and initially synthesized by Danishefsky using glycal assembly strategy in 1995. Globo H was present on most cancers of epithelial origin, but only minimal expression on normal secretory tissue which is not readily accessible to immune system. Taking advantage of the exclusive expression signature, Globo H has been an attractive target for immunotherapy against prostate, breast, colon and ovarian cancers. Although, numerous vaccines are undergoing clinical evaluation and some shows improved survival rate in patients, the ultimate goal is to prevent the tumor recurrence. Therefore, we aim to develop a more effective vaccine and adjuvant against a variety of cancers. Here we reported the promising carbohydrate based vaccine enhanced antibody production and delayed tumorigenesis in xenograft studies. Overall, systematic optimization with respect to formulation, adjuvant, dosage, glycan/protein ratio and timing of immunization regimen may further improve the protection against tumors. Finally, conjugation of diverse carbohydrate antigens to various carriers or dendrimer will be planned in the near further.

The search for new pharmacologically active agents by screening natural sources has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases. The goal of my thesis was to discover structurally novel type III secretion inhibitors. Active extracts from marine sponge P. johnstonia were identified via high throughput bioassays for the EPEC type III secretion inhibitors. Guided by this novel bioassay model, a new glycolipid family, the pachymosides, was fractionated and identified. To determine the structure of the pachymosides, chemical degradation was performed and the glycolipids were divided into aglycon and monosaccharide parts. Three aglycons were isolated and identified as 6, 8 and 9. [diagrams not included] Two monosaccharide residues were identified by NMR as glucose and galactose. Further chiral GC analysis determined their absolute configuration as D-glucose and D-galactose. Acetylation was performed on the pachymosides followed by 13C labelling. The structure of pachymoside A (16), a member of a novel bioactive glycolipids family, was figured out. This is the second identified structure for the type III secretion inhibitor bioassay. [diagrams not included]