Synthesis of Heterogeneous Heparan Sulfate Oligosaccharides to Investigate Structure- Activity Relationship (SAR)

Abstract

Understanding the relationship between the chemical structure of a molecule and its biological activity holds tremendous potential for drug candidates’ development in several pathophysiological conditions. Carbohydrates, the most diverse biomolecules present on cell surfaces or extracellular matrix spaces, remains the least explored molecules in this scenario due to their structural complexity and bioavailability. Heparan sulfate (HS), a carbohydrate polymer accompanied by a broad range of biological activities, is undisputedly the most complex polysaccharide present in living systems. Despite decades of research investigating HS’s structure-activity relationship, our current understanding is limited by its extensive microheterogeneity. Identification of the structural epitopes of HS that are responsible for orchestrating various cellular processes could enable us to fine-tune the interactions among those processes that modulate biological activities. Inspired by this, we have synthesized a combinatorial library of HS tetrasaccharides and investigated their structural relationships with chemokines, growth factors, P-selectin, and Tau protein. Chapter 1 briefly introduces cell-surface glycans and discusses the structural details of glycosaminoglycans, one of the major classes of surface glycans, with a focus on HS heterogeneity. Also, with the help of a literature review, the chapter introduces the aim of the thesis by reviewing the previously known structure-activity relationship (SAR) details of HS with heparan sulfate binding proteins (HSBPs). Chapter 2 summarizes the synthesis of the novel heparin tetrasaccharides (HT) library used to investigate the structure-activity relationship. To discover the new binding epitopes responsible for selectively activating chemokines, comprehensive microarray and surface plasmon resonance (SPR) analyses were conducted using a broad spectrum of chemokines. Surprisingly, the rare N-unsubstituted (NU) domain carrying the HT ligands HT-2,6S-NH and HT-3,6S-NH was found to modulate the activities of CCL2 and CCL5. Furthermore, HT-3,6S-NH was successfully used to inhibit CCL2-mediated cancer cell proliferation, migration, and invasion. We believe that CCL2 and CCL5 are only the fourth and fifth proteins that have been found to recognize HS’s NU domain. Chapter 3 describes the systematic investigation method used to identify high-affinity HS ligands that modulate angiogenesis. The HS library described in Chapter 2 was used to determine their binding affinities with the growth factors, namely BMP-2, amphiregulin, HB-EGF, VEGF165, and FGF-2. Glycan microarray and SPR analysis identified the wide binding preferences of HS ligands with growth factors. However, the unexpectedly high binding of HT-6S-NAc and HT-2,6S-NAc with the growth factor VEGF165 was observed, which suggested that HT ligands can be used to target the cellular signaling mediated by VEGF165. To substantiate the binding profiles of VEGF165 and the HT ligands 6S-NAc and 2,6S-NAc, in vitro experiments with human umbilical vein endothelial cells (HUVECs) were conducted. The successful inhibition of VEGF165-mediated HUVEC cell proliferation, migration, and endothelial tube formation in the presence of synthetic HT ligands revealed the hidden structural features of HS that could be crucial for the development of antiangiogenic agents. Chapter 4 addresses a new aspect to study structure-activity relationship of HT ligands with HSBPs wherein the multivalent fluorescent glyco-gold nanovehicle is constructed to target the epidermal growth factor receptors (EGFR) present on triple-negative breast cancer cells (TNBCs). A series of confocal images using HT-6S-NAc- and HT-6P-modified nanovehicles showed that HT-6S-NAc-capped gold nanoparticles in the two-dimensional cell culture model had uptake that was preferential compared with that of HT-6P. Also, this preferential uptake was in good agreement with the cell-surface EFGR expression on the TNBCs, with MDA-MB-468 (which had the highest EGFR expression) showing remarkably high uptake compared to that of SK-Br-3 (which had the lowest EGFR expression). Furthermore, the results were validated by selectively targeting cancer cells in the tumor microenvironment using the three-dimensional cell culture technique. Overall, the results support the use of HT ligands in targeted cancer therapy. Finally, Chapter 5 summarizes HT ligands’ high-throughput screening profiles with P-selectin and Tau protein acquired by glycan microarray. We have identified a structurally well-defined N- unsubstituted HT ligand (HT-2S-NH) with exceptionally high P-selectin binding for the first time. The results support the decades-old theory that suggests selectin binding preferences with non-sulfated glucosamine residues containing HS oligosaccharides. In addition, the microarray binding profiles of HT ligands with Tau protein are summarized and discussed in detail.