Young Investigator Highlight

Arunika Ekanayake, member of the Derda lab at the University of Alberta, is one of the winners of the 2022 Schram Young Investigators Oral Presentation Competition. She presented her work at the American Peptide Society Symposium in Whistler, B.C., Canada this past June. The title of her talk was Genetically Encoded Fragment-Based Discovery (Ge-Fbd) from Covalent and Non-Covalent Pharmacophores.

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Peptide-based therapeutics present many advantages with their ability to bind to targets that are not accessible with small molecules, such as proteins without defined binding pockets. Peptides can generate highly specific interactions with their targets, with their ability to mimic the natural ligands of proteins. Phage display is one of the most desirable platforms for peptide-based drug discovery because it utilizes amino acids' natural diversity to generate millions-to billions of peptide ligands. However, linear peptides present several challenges, including short half-life, low bioavailability, and difficulty passing the cell membrane. Macrocyclization is one of the strategies that can aid in mitigating some of these caveats presented by linear peptides. We employed a chemical linchpin-based macrocyclization approach that can also provide a handle for further functionalizing these macrocycles. The goal was to graft unnatural chemical fragments to the macrocycles to expand their diversity beyond the natural diversity of amino acids.

We used a 1,5-dichloro-2,4-pentadione linchpin to form macrocycles on phage display libraries with 1,3-diketone handles. Converting these phage-displayed peptides to 1,3-diketone bearing macrocycles provides a shelf-stable precursor for further functionalization with hydrazine through a well-established Knorr-pyrazole synthesis reaction. These hydrazine-bearing chemical fragments often interact with a known site of the target protein but often with low potency and specificity. Covalent incorporation of unnatural fragments or ‘pharmacophores’ into conventional peptide libraries expands the chemical space and facilitates the discovery of molecules with favorable properties not offered by the elements alone. This strategy can be applied to linear and cyclic peptide libraries, using pharmacophores with covalent and non-covalent reactivities towards target proteins. Ligating diverse hydrazine derivatives onto diketone macrocyclic peptide libraries displayed on a phage that carries silent DNA barcodes enables genetic encoding of these post-translational chemical modifications. Therefore, the methodology can be called genetically encoded fragment-based discovery (GE-FBD). Genetically encoded fragment-based discovery (GE-FBD) is a promising approach for selecting ligands and drug leads from existing GE libraries displayed on phage, DNA, or mRNA.

Traditionally, the generation of GE-FBD libraries employs “early-stage” incorporating unnatural building blocks into the chemically or translationally produced macrocycles. The current approach utilizes the binding affinity of the chemical fragment together with the amino acids in the macrocycle to employ the synergistic effects at the discovery stage. These libraries can be applied against “undruggable” protein targets to discover ligands with improved affinity and specificity.

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