Genetically Encoded Fragment-Based Discovery of Inhibitors for Glycan-Binding Proteins

• Author / Creator

  Ng, Simon

• Carbohydrates play vital roles in many disease pathologies, including inflammatory disease, cancer metastasis, autoimmune disease and pathogenic infection. Despite their significance and immense opportunity for pharmaceutical application, carbohydrate-derived drugs only constitute a relatively small portion of therapeutics today. One of the major challenges in drug discovery for glycan-binding proteins is their relatively weak binding affinities for the glycan. The issue is further aggravated by the structural complexity of glycans, which often leads to a bottleneck in generation of various different analogues in sufficient quantity for in vitro and in vivo studies. Fragment-based lead discovery was used to design better lead compounds for therapeutic targets and demonstrated its potential for challenging targets such as glycan-binding proteins. However, there remains an unmet need for simpler, less expensive, and more efficient routes to identify superior lead compounds. This thesis approaches these challenges, i.e., the need for simpler and more efficient method to identify lead compounds for glycan-binding proteins, by combining fragment-based lead discovery with phage display. Through the linking of each “displayed peptide” to its encoding DNA, phage libraries are several orders of magnitude (~10^8–10^9) larger than chemical libraries, but still can be screened against a target in a very efficient way. I pioneered a methodology to selectively and covalently attach a small-molecule fragments to a phage-displayed peptide libraries (Chapter 2). The resulting bivalent fragment-peptide library demonstrated its application for the selection of potent inhibitors for glycan-binding proteins. By using ConA as a model target of a glycan-binding protein, I discovered novel monosaccharide-peptide conjugates with Kd and IC50 values 30–50-fold better than that of the monosaccharide itself (Chapter 3). The conjugates bound competitively to the desired glycan-binding site and functioned as potent inhibitors. The most potent hit, Man-WYDLF, has similar affinity and selectivity as a trimannoside. Interestingly, the peptide bound to a secondary site different from that bound by the disaccharide portion of the trimannoside. The selection identified a novel binding pocket and suggested the potential use of this technology for “hot spot” mapping. I further applied this technology for the selection of mannose-based inhibitors for DC-SIGN, albeit with partial success (Chapter 4). I applied computational solvent mapping (FTMap) to analyze the protein surface of DC-SIGN to identify putative fragment-binding “hot spot”. The results suggested that a “hot spot” is available at ~10 Å away from the Ca2+ glycan-binding site and could be accessible for library attached to 2-OH position of mannose or anomeric position of fucose. Future work is required to confirm this hypothesis. Finally, I demonstrated a strategy to create the first macrocyclic glycopeptide library displayed on phage (Chapter 5). The disulfide-containing peptide library could be alkylated chemo- and regio-selectively using a dichloro-oxime derivative to form macrocycles stable toward reduction. These libraries, both linear and cyclic glycopeptides, show potential for use in fragment-based ligand discovery, “hot spot” mapping, and selection of potent ligands for many protein targets. Ligands resulting from the selection will strongly complement and “guide” the rational design of more potent and “drug-like” compounds or vice versa.

• Subjects / Keywords

  • Phage display
  • Glycopeptide
  • Inhibitors
  • Fragment-based discovery

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3BZ61M68

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.