Probing Site Specific Protein PEGylation to Improve Therapeutic Potential

Emma Mulry, Xzaviar Solone, Sreyashi Das, Lizi Wu, Matthew Eddy

University of Florida

Biologics, or protein drugs, are potent therapeutics because of their diversity, specific conformations, and biocompatibility, yet they suffer from rapid degradation and low circulation times. To address this, covalent attachment of polyethylene glycol (PEG) to proteins has been used to improve their robustness and stability, aiding in developing new pharmaceutical applications. Despite the success of PEGylating proteins, we lack comprehensive and predictive understanding of protein-polymer interactions at a detailed molecular and atomic level as well as their impact on function. Motivated by this need, we investigated a PEGylated carbohydrate recognition domain of galectin 3 (Gal3C), a lectin protein required for cellular adhesion and potential cancer therapeutic. In our previous study1, we demonstrated that PEGylation at a specific residue position in Gal3C improved the protein’s thermal stability without significantly disrupting the protein structure. We have expanded on this work to systematically explore the role of the position of chemical conjugation on the physical and functional properties of PEGylated Gal3C. We designed a series of Gal3C variants with single extrinsic cysteines introduced at different discrete locations throughout the protein structure and conjugated these variants with PEG. We then compared the thermal unfolding and ligand binding properties of all variants with and without PEGylation. Additionally, we present preliminary data from lung cancer cell assays to test the impact of Gal3C and PEGylated Gal3C on cancer cell viability and proliferation. Together these data allow us to visualize the impact of PEGylation at different locations and the potential of improving the drug potential, supporting our long-term goal of accumulating sufficient experimental data to inform the de novo rational design of protein-polymer conjugates with predicted chemical properties for therapeutic use.

1. Pritzlaff A, Ferré G., Mulry E., Gopal Pour N., Savin D., Harris M., Eddy M., Atomic Scale View of Protein-PEG Interactions that Redirect the Thermal Unfolding Pathway of Human Galectin-3. Angew Chem Int Ed Engl. 2022 Oct 4.