Genetically Encoded Fragment-Based Discovery of Inhibitors for Galectin-3

  • Author / Creator
    Ferrer Vinals, Daniel
  • Genetically-encoded fragment-based discovery (GE-FBD) holds the promise for accelerate ligand discovery for protein targets. By chemical bridging of readily available genetically encoded (GE) libraries of polypeptides with constant fragments, this methodology opens a fast route to discover superior lead compounds. In GE-FBD the constant fragment guides the interactions of peptide and target to the close vicinity of the binding site on the target protein. Then, biochemical screening can sample millions of peptide sequences and filter those that provide synergistic binding contributions and enhance the overall binding affinity towards the target. Glycan binding proteins (GBP) represent an important target, that is challenging to address for ligand development with conventional high-throughput screenings (HTS). Conveniently, glycans are in fact, excellent candidates for GE-FBD, however, to tackle challenging targets such as GBP, the need for optimization remains unmeet on GE-FBD.
    This thesis presents an optimization study for GE-FBD to enable the discovery of glycopeptides that inhibit human galectin-3(Gal3). First, I interrogated the limits of GE-FBD with respect to the initial affinity of the constant fragment (Chapter 2). To this end, GE-libraries of glycopeptides displayed on phage and expressing combinations of redundant Ser and Gly codons, enabled to track libraries individually by DNA sequencing. Next, aldehyde-based oxime ligation with hydroxylamine-containing monosaccharides, decorated each barcoded library with glycans of variable affinity towards Gal3. GE-FBD campaigns of those libraries, against plate-immobilized Gal3 discovered glycopeptide ligands that bind to Gal3 in a sequence dependent manner. The sequence Gal-WHVP exhibited a 40-fold increase in binding potency for Gal3 compared to random Gal-peptide but was inactive as monovalent ligand. GE-FBD of fragments with no detectable initial affinity discovered ligands that bind Gal3 equally well with or without the fragment. Further implementations of GE-FBD employed a glycan fragment of higher affinity towards Gal3(Chapter 3). The glycan fragment was chemically attached to a library of 108 diverse phage displayed heptapeptides. Solution phase panning allowed to screen library-target interactions in 1:1 correspondence and in using selective elution of the bound phages I discovered monovalent ligands for Gal3. Next, the use of “silent barcodes” technology allowed to develop a genetically-encoded glycan array technology from which I performed the screening of 80+ glycan fragments (Chapter 4). This study permitted to identify new potential starting point glycan that could maximize the success of future GE-FBD campaigns on Gal3. This approach could be extended to other protein targets for which the initial fragments selection might not be obvious.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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