Genetically Encoded Discovery of Proteolytically Stable Macrocyclic Peptides

• Author / Creator

  Wong, Ying Kit Jeffrey

• Phage display libraries bearing natural amino acids are commonly used for discovering potential therapeutic macrocycles; however, direct application of those linear peptides often leads to poor pharmacokinetics, including low serum stability, lower cell membrane permeability, and rapid renal clearance. Chemical macrocyclization of readily accessible phage display libraries can potentially improve the pharmacokinetic properties of peptides. In this thesis, I aim to expand the current chemical space of the phage displaying peptides with chemical post-translational modifications to discover bioactive ligands for therapeutically important proteins with modified phage display libraries.In chapter 1, I briefly review the current state of the art in macrocyclization for peptide phage-displayed libraries. In Chapter 2, I develop a two-fold symmetric linchpin (TSL) that converts readily available phage-displayed peptide libraries made of 20 natural amino acids to genetically encoded bicyclic libraries. TSL combines an aldehyde-reactive group and two thiol-reactive groups by bridging two side chains of cysteine C with an N-terminal aldehyde group derived from an N-terminal serine S, yielding a novel bicyclic topology that lacks a free N-terminus. Phage display library of SXCX6C sequences, where X is any amino acid but cysteine, were converted into a library of bicyclic TSL-SXCX6C peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signalling in cancer cells. At 10 μM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma cells. The TSL-SYCKRAHKNC bicycle inhibited NODAL-induced proliferation of NODAL-TYK-nu ovarian carcinoma cells with an apparent IC50 of 1 μM. The same bicycle at 10 μM concentration did not affect the growth of the control TYK-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase™) for 21 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides to expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking cysteine side chains. We anticipate that the TSL will aid in the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets. In Chapter 3, I describe discovery of a low molecular-weight, chemically modified macrocyclic peptide that binds to albumin with low micro-molar affinity for prolong circulation time. We modified SXnCXmC phage-displayed libraries, where X is any amino acid but cysteine, n = 1 and m = 3-5 amino acids, with decafluoro diphenylsulfone (DFS). Using these macrocyclic libraries and human serum albumin (HSA) as bait, we identified 8 macrocyclic peptides through three different discovery campaigns. The peptides were modified with DFS and a more stable linchpin pentafluorophenyl sulfide (PFS). The poly-fluorobenzene groups in the DFS and PFS modified peptides made it possible to a use 19F NMR binding assay to determine and rank their bindings capacities. PFS-SICRFFC binds the strongest to HSA, and PFS-SICQGEC binds the weakest to HSA. I determined the PFS-SICRFFC binds to HSA with an affinity of Kd = 6 µM via fluorescence polarization.

• Subjects / Keywords

  • Chemistry
  • Phage display
  • Peptide
  • Protein-protein interaction
  • Bicycles
  • Genetically encoded

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-y2zf-3t90

• 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.