Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

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Heparanase (Hpa1) is an enzyme overexpressed in nearly all cancers, typically at the tumour growth front. It cleaves proteoglycan heparan sulfate (HS) chains to release growth factors necessary for tumour growth. While some carbohydrate-based mimetic inhibitors have progressed to advanced clinical trials, new inhibitors and tools to further investigate heparanase are of continued interest. This thesis proposes a HS mimetic trisaccharide sequence that can bind Hpa1 and is suitable both for biological evaluation and inhibitor development. Synthetic work was then undertaken toward the progression of this moiety. Exploring building blocks applicable to the trisaccharide, conformationally-locked glucose derivatives were developed. This included the introduction of a conformational switch that resulted in the isolation of constrained half-chair conformers. The synthetic work toward trisaccharide formation also evaluated the utility of 1,2-cyclohexane-diacetal as a protecting group with glucuronic acid. The disarming qualities of these moieties were assessed, leading to the development of alternate routes. A more linear approach resulted in the formation of important disaccharide building blocks that contribute toward the synthesis of the core trisaccharide, including isolated 1,2-orthoesters. Further development of the chemistry established herein should allow for the formation of the desired core trisaccharide, while contributions have additionally been made toward its tool functionalisation and use in multivalent schemes.

Syndecan-1 is a cell surface proteoglycan which participates in cell adhesion, differentiation, motility, morphogenesis and intracellular signaling. The two glycosaminoglycans heparan sulfate and chondroitin sulfate are covalently attached to the ectodomain of syndecan-1 via a tetra saccharide linkage sequence. However, the ectodomain can be modified having only one or neither of the glycosaminglycans attached. The glycosaminoglycans are capable of binding ligands such as fibroblast growth factors (FGFs) and support activation of receptors. The ectodomain is proteolytically cleaved from the cell surface by metalloproteinases in a process known as shedding. Shedding turns the ectodomain into a soluble effector which can stimulate other cells in the surroundings by delivering growth factors and also translocate into cells through endocytosis. In this study the aim was to find out if a modified ectodomain, which only contains chondroitin sulfate, could support intracellular signaling in the absence of heparan sulfate. The aim was also to find out whether a modified ectodomain could translocate into the cell. The methods used were cell culturing, isolation and purification of syndecan-1 ectodomain, cell signaling and immunohistochemistry. It was found that modified shed syndecan-1 ectodomain was able to support intracellular signaling almost to the same degree as wild type syndecan-1 ectodomain. This may suggest that heparan sulfate does not have to be present on the ectodomain to support intracellular signaling, although the signal is slightly higher when present. When trying to detect translocation of the ectodomain the results were too uncertain and further research is required.

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

Polynuclear platinum compounds represent a new class of potential platinum anticancer therapeutics. Derived from the most widely used platinum anticancer drug, cisplatin, these novel compounds are distinct in their interactions with bio-molecules. The effectiveness of platinum anticancer agents is influenced by three pharmacological factors: (i) their resistance to deactivating sulfur nucleophiles, (ii) the ability to gain cellular entry and efficient cellular uptake, and (iii) the ability to form stable and specific complexes with DNA. BBR 3464, the first multinuclear platinum compound to reach phase II clinical trials, has created a new approach to cancer drug design. Large, highly charged platinum compounds have been shown to form favorable covalent and noncovalent interactions with bio-molecular structures. Compounds such as BBR 3464, form an immediate pre-association with anionic structures on biomolecules before covalent attachment. To better characterize these interactions, a new set of compounds was designed that exclusively interacts via electrostatic associations and hydrogen bonding. The investigation of noncovalent complexes between DNA, proteins, and peptides with a variety of synthetic and biological relevant structures has become increasingly more common with the coupling of electrospray ionization and mass spectrometry (ESI-MS). Mass spectrometry has been useful to the drug design community by allowing the rapid and accurate characterization of drug binding sites. In the first project we have explored the use of collision induced dissociation (CID) to map the potential binding sites of noncovalent polynuclear platinum compounds of varying size and charge with an antisense oligonucleotide of the Bcl-2 sequence. In the second project, the gas-phase dissociation and stabilizing effects of these polynuclear platinum compounds on duplex DNA were determined. Correlations between the size and charge of associating platinum compounds were determined by comparing the change in gas phase stability under CID conditions. Additionally, the association of these new types of noncovalently binding polynuclear platinum compounds was investigated with model cell surface structures such as anionic heparan sulfate and phospholipids.

The work discussed here is divided into two projects. The first project involves the interactions between antidepressants and the platinum based chemotherapeutics while the second project begins to investigate possible implications of a recently discovered uptake mechanism for positively charged platinum drugs. Gaining understanding of the interactions between antidepressants and platinum-based chemotherapeutics is important due to the frequency with which they are prescribed together. Although using a combination regimen of antineoplastics is beneficial to the patient, not all drug interactions are. For instance, many of the serotonin reuptake inhibitors have been shown to decrease the efficacy of tamoxifen. Desipramine, a tricyclic antidepressant used to treat neuropathic pain, has been shown to increase the cytotoxicity of cisplatin, oxaliplatin and carboplatin in the human colon carcinoma cell line, HCT116 wt. To study this interaction, the cell line specificity as well as the drug specificity with regard to both the platinum-based chemotherapeutic and the antidepressant were investigated. The data show that the effect is both cell line specific as well as drug specific with respect to both types of drugs. To elucidate the mechanism behind the alteration in cytotoxicity of the platinum drugs, the effect of p53 status was investigated. A reduction of the effect is observed in the absence of p53, suggesting that there is a p53 dependent mechanism as well as a p53 independent mechanism. The tricyclic antidepressants and fluoxetine are known to be calmodulin inhibitors. Calmodulin inhibition mirrored some of the effects seen with the antidepressants suggesting that calmodulin inhibition might also play a role in the mechanism. The second project is based on the discovery that heparan sulfate proteoglycans mediate the uptake of positively charged platinum complexes. Heparan sulfate proteoglycans are important in cell-cell as well as cell-extracellular matrix adhesion. In cancer, heparanase, the enzyme that cleaves heparan sulfate, is over expressed creating a pro-angiogenic and pro-metastatic state. This work demonstrates that the positively charged platinum complexes can inhibit heparanase activity by binding to the substrate (heparan sulfate proteoglycans). This suggests that this class of drugs may have the capacity to be anti-angiogenic and anti-metastatic as well as cytotoxic.

Heparan sulfate (HS) is a cell-surface sulfated polysaccharide that binds to multiple proteins and has been implicated in cancer, viral infection and Alzheimer's disease. Due to the heterogeneity of HS, the structural requirements for protein binding are ill- defined. Chemical synthesis of structurally-defined HS oligosaccharides, which are tunable in terms of length, order of monosaccharides and sulfation pattern, is required for the investigation of HS-protein binding. Single molecule methods have been utilised in biophysics to study dynamic processes and can allow observation of rare events which would be 'averaged out' in ensemble measurements. Access to fluorescently labelled HS oligosaccharides should allow investigation of interactions with proteins at the single molecule level using methods such as single molecule FRET, providing a method complementary to NMR studies (ensemble) and X-ray crystallography (non-dynamic).This thesis presents the development of a method for the fluorescent labelling of a chemically synthesised HS disaccharide utilising a reducing-end amine tag. Analysis of the fluorescence properties of the labelled disaccharide at ensemble and single molecule level indicated no perturbation of the fluorophore when attached to the sugar. Fluorescence correlation spectroscopy measurements of the fluorescent HS disaccharide with the protein FGF-1 showed no binding, which is attributed to the low concentration (1 nM) of disaccharide required in the experiment. Additional work is presented in this thesis on the development of a method for atom-specific 13C labelling of HS oligosaccharides, which has been initiated by synthesis of a 13C labelled L-iduronate monosaccharide and a 13C labelled disaccharide. New strategies for the synthesis of HS oligosaccharides based on orthogonal thioglycoside-based glycosylations employing S-benzoxazolyl and S-thiazolyl donors have been investigated. Development of a chemoselective glycosylation strategy for HS oligosaccharide synthesis utilising a 'super-disarmed' [2.2.2] L-iduronic lactone is presented.

Proteoglycans containing Heparan Sulfate (HS), a sulfated glycosaminoglycan (GAG), play a major role in the cell signaling process, interacting with many different proteins. HS is over expressed on the surface of many cancer cells. Enzymatic cleavage of HS-GAGs by heparanase causes release of angiogenic growth factors leading to tumor cell migration. Heparanase is also over-expressed in tumors with significant correlation between metastatic potential and heparanase activity. Proteoglycans and their associated enzymes are thus significant drug targets of high biological relevance. A functional consequence of strong PPC-HS binding has been shown in proof-of-concept studies confirming inhibition of the model pentasaccharide, Fondaparinux, by bacterial Heparinase. Such metalloshielding by PPCs may also protect HS from enzymatic cleavage by the mammalian heparanase; preventing growth factors from binding to HS and/or preventing release of bound growth factors and thus inhibiting the metastatic response in the cancer cells. HS-GAGs are also receptors for cellular accumulation of cationic Polynuclear Platinum Complexes (PPCs) through high-affinity binding to the highly anionic HS. PPCs competitively inhibit uptake of TAMRA-R9, a fluorescent nona-arginine derivative, in CHO cells. The previously reported series of Pt(II) complexes were investigated as DNA binders, initiating the apoptotic cascade. The result of PPC-DNA binding produces long range inter and intra-strand cross-links, that produce structural and conformational changes. Hydrogen bonding between phosphate oxygens and square planar Pt(II) nitrogen results in bidentate complexes by either backbone tracking or groove spanning of DNA. This complex forms a clamp like structure, called a phosphate clamp, similar to that of the arginine fork. Understanding this clamp allows us to investigate the structurally similar sulfate binding between metal complexes and target HSPG. HSPGs may allow significant research into both a novel cellular internalization of principal metals and “metalloshielding” of heparin by these compounds. Previous studies have shown that a wide range of metal ions have high affinity to heparin. The trend of metal/heparin affinity is believed to be dependent on parameters consisting of the metal’s overall size, spatial orientation of the ligands attached to each metal, the net charge and oxidation state of these metals, and number of binding sites. Studies have shown relative affinities of sulfate and carboxylate groups for the metal ions. These metal cations play an important role in the affinity, specificity, and stability of many protein/heparin interactions. The study of simple coordination compounds, like Pt, Mn, V, Ru and Co, will allow preliminary results which will extend into the PPCs mode of binding. This thesis focuses on the concept of metalloglycomics and reviews the interactions of various metal complexes with heparin. The covalent and non-covalent interactions of metal complexes with heparin resulting in strong bonding are explained through spectroscopy and calorimetry. The cleavage inhibition of heparanase by metal complexes is also described. Sulfate cluster anchoring shields the sulfates from loss as seen in mass spectrometry. The study of metalloglycomics offers potential understanding into the relevance of metal-heparin interactions and possibilities into the development of new compounds as therapeutic agents.

<p>Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS.</p><p>The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.</p>