Doctor of Philosophy
Nicholas P. Farrell
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.
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Available for download on Saturday, December 07, 2019