Covalent modification of peptide libraries to target the active site of heparanase in mRNA display

Abstract

Heparanase (HPSE) cleaves the chains of heparan sulfate proteoglycans at cleavage sites between glucuronic acid and sulfated glucosamine residues (endo-β-glucuronidase). Heparan sulfate proteoglycans are crucial in maintaining structural integrity of basement membranes, cell surfaces and extracellular matrixes (ECM). Heparan sulfate chains bind to a vast array of signaling molecules, such as growth factors, chemokines and cytokines. By cleaving heparan sulfate chains and proteoglycans, these signaling molecules will be released and ECM and membranes will be remodelled. In adults, the basal level of HPSE activity is rather low. HPSE expression and activity are increased in many diseases, and more importantly in most types of tumors. In tumors, elevated HPSE activity plays a role in angiogenesis, metastasis, cell proliferation and cell invasion. This makes HPSE an interesting target for anti-cancer therapies. Current HPSE inhibitors in clinical trials are all sulfated polysaccharides, which entail anticoagulant side-effects. Peptides are a potential class of HPSE inhibitors without anticoagulant effect, but they remain understudied. In this thesis, a method for chemical modification of peptide libraries to selectively target the enzyme’s active site in high-throughput screenings was developed and optimised. The modification is a heparan-sulfate disaccharide with an azide-bearing aromatic aglycone, being attached to the peptide libraries by a well-established copper(I)-catalysed alkyne-azide cycloaddition (CuAAC). The glycan-modified libraries were used in RaPID mRNA display, and will be used in the future in cDNA-TRAP display, to screen for potential HPSE inhibitors. RaPID mRNA display against HPSE did not lead to hits because of recurring truncation issues with the libraries, but the cDNA-TRAP display system could overcome this. The optimised click conditions can also be exploited in future selections with similar goals to modify libraries with different substrates for more specific targeting of the target.