Synthesis and evaluation of multivalent ligands for macrophage galactose C-type lectin. Development of new chemical tools for immunological research and structural investigation of the lectin with GalNAc and derivatives

Abstract

Synthetic chemistry can provide powerful tools to explore the correlation between an antigen’s chemical structure and the type of immune response triggered. This thesis work has focused on the design, synthesis and evaluation of multivalent molecules which target a relevant protein of the immune system, the human macrophage galactose C-type lectin (hMGL). In chapter 2 the synthesis of multivalent glycomimetics is described as well as measurements of their affinity for hMGL. The main objective was to learn more about the structure activity relationship with regard to ligands based on tetraphenyl ethylene (TPE) for hMGL and other lectins. To achieve this divalent, trivalent and tetravalent ligand, based TPE geometry as well as trans-stilbene presenting α- mannopyranoside, β-lactose and α-thio-GalNAc as headgroups, were synthesised. The identified synthetic route to such compounds is reproducible and scalable. The thermodynamics of the interaction of α-thio-GalNAc derivatives were studied using the extracellular domain of hMGL and hMGL CRD, revealing insights into the possible interaction of these compounds with the lectins. In chapter 3 the synthesis of an extensive anti-cancer synthetic vaccine candidate library based on MUC1/MUC4 peptide backbone is described. The main objective of this work was to enhance antigen presentation by hMGL via the major histocompatibility complex II. To achieve this objective mucin epitopes were decorated with a trivalent glycocluster selective for hMGL. Through solid phase synthesis synthetic peptides, including those presenting the glycocluster were obtained. These were found to bind hMGL from 10 to 200- fold better than the natural peptides in an ELISA performed in the S. van Vliet laboratory in Amsterdam. The next step of this project will be to identify peptides which induce the desired signalling cascade and test them on mouse model for the production of long-lasting antibodies IgG. This is the first reported attempt to target a receptor with neoglycosylated peptides baring a fragment specifically designed to selectively target hMGL.In chapter 4 the synthesis of a (neo)peptide containing a glycocluster with potential to give a trimeric coiled-coil sequence is described. The glycocluster is based on the tetraphenylethene residue. The peptide was successfully synthesised and purified. While some evidence for helix formation was obtained by CD spectroscopy, the refolding of the peptide after heating/denaturing was not observed and may have occurred partially. In chapter 5 the first structural elucidation of hMGL carbohydrate binding mode is reported using X-ray crystallography. hMGL is the only lectin on human DCs that selectively recognise carbohydrate antigens present on many cancer cell lines and was proven to have the ability to lead the immune system to produce permanent antibody against cancer. The aim of this work was to understand the structural basis of the hMGL/carbohydrate interaction and to allow a more reliable in silico ligand design. To achieve this the protein was expressed in bacterial cell lines. As the expression resulted in insoluble protein, a refolding screen optimisation was carried out, which facilitated recovery of the protein in high purity and good yield for crystallisation screening. As a result of screening and optimisation, suitable crystals for diffraction were obtained. This led to the elucidation of the first crystal structure of hMGL carbohydrate recognition domain (CRD), binding with several monovalent GalNAc derivatives. The structure offers a possible explanation of hMGL binding mode and selectivity.