Molecular evolution of peptide inhibitors targeting the chemokine network

Thesis

Aberrant inflammation is implicated in a vast range of illnesses including cardiovascular disease, inflammatory bowel disease, cancer, and autoimmune disorders. Chemokines regulate inflammation by driving chemotaxis of leukocytes to sites of tissue injury and disease. Pharmacological targeting of chemokine-driven inflammation has been challenging due to redundancies in the chemokine network. Promiscuous chemokine-binding proteins such as tick salivary evasins and short peptides derived from them have shown potential in models of immune cell migration but require enhanced binding promiscuity and inhibitory potency for therapeutic use. We hypothesised that in vitro molecular evolution could identify promiscuous therapeutic peptide variants capable of inhibiting a broad spectrum of chemokines.

Starting with a lead peptide candidate, we demonstrated that affinity maturation, traditionally used to enhance affinity to a single target, could improve peptide affinity to multiple chemokines. Using NNK saturation mutagenesis and phage-display selection against multiple chemokines, we identified mutations that enhanced binding to several chemokines and engineered de novo binding to additional chemokines. We further screened peptides with all possible combinations of enhancing mutations, identifying those with significantly improved potency of inhibition in chemotaxis assays. We have expanded the number of chemokines targeted with a single peptide from 6 to 12 and demonstrated peptide efficacy against a complex pool of chemokines mimicking their expression in disease. Additionally, a select HD2 point mutant exhibited a broad profile of chemokine inhibition, emerging as the most potent inhibitor for several of them, an effect not predicted from phage display. Biophysical investigation into the mutant peptide mechanism of action revealed a potentially novel molecular method of targeting chemokines and other circulating signalling proteins.

We show that we can iteratively generate effective promiscuous binders for redundant biological networks, advancing the engineering of precise peptide therapeutics for specific chemokine expressions and bringing us closer to precision therapeutics for inflammatory indications.

Authors: Kryukova, J

• DOI: 10.5287/ora-bp5mavzgy
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: saturation mutagenesis; affinity maturation; autoimmunity; precision medicine; biologic drug development; drug design; evasins; chemokine; peptide drug; peptide therapeutics; chemotaxis inhibition; inflammation
• Subjects: Peptide drugs; Chemokines; Anti-inflammatory agents