(97d) Building Better Proteins: Integrating Cell-Free Protein Synthesis and Coarse-Grained Molecular Simulation to Rapidly Determine the Optimal Location for PEGylation

Protein therapeutics are life-saving drugs that combat some of the world’s most debilitating and painful diseases including cancer, multiple sclerosis, rheumatoid arthritis, Hepatitis, and diabetes. Many protein drugs are currently under development, and within five years, the number of protein therapeutics prescribed is expected to eclipse the number of small molecule therapeutics. This is particularly exciting as there is great hope for effective protein therapeutics to combat Alzheimer’s, cancer, and genetic disorders. However, fundamental limitations of protein therapeutics include susceptibility to degradation and aggregation, stimulation of an immune response, and rapid clearance from the body. These impediments have led to the development of second generation drugs in which a protein therapeutic is covalently conjugated to the FDA-approved polymer polyethylene glycol (PEG). This has resulted in significant improvement in overcoming the above limitations; however, PEGylation typically leads to reduced protein activity. To overcome this drawback, our lab has developed a method to 1) site-specifically attach PEG to proteins and 2) find the optimal conjugation location that stabilizes the protein while maintaining its activity. The method utilizes a hybrid computational/experimental approach to determine the locational impact of PEGylation on PEG attachment efficiency, protein stability and activity. This is done by integrating a coarse-grain simulation approach with cell-free protein synthesis experimental technology. Through experiments utilizing T4 Lysozyme as a model protein, we demonstrate the utility of this integrated approach and evaluate the predictive impact of currently held design guidelines for PEGylation including site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings revealed that PEGylation efficiency, protein stability, and activity were not well predicted by the current guidelines, illustrating the need for a rapid screening approach such as the one reported herein. The results of this work have been recently reported in ACS Synthetic Biology: 7(2):510-521.