Development of Cell-Penetrating Monobodies

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Qing Lin of SUNY at Buffalo is developing new approaches for designing cell-permeable antibody-like small proteins called monobodies. While monobodies have provided a versatile research tool for dissecting the intracellular signaling pathways, it remains a challenge to design cell-penetrating monobodies for therapeutic applications. The proposed approaches introduce selective chemical crosslinking into monobodies to reinforce their structure, and together with surface supercharging endow cell permeability to the engineered monobodies. This project will provide multidisciplinary research opportunities to underrepresented undergraduate students through collaborations with several minority-focused programs at SUNY at Buffalo. The results of this project will also be integrated into the Experiential Learning Network for undergraduates to enhance their classroom learning. This research project seeks to optimize orthogonally crosslinked monobody scaffolds for the design of bioactive cell-penetrating monobodies targeting the intracellular proteins. By incorporating a genetically encoded noncanonical amino acid called beta-lactam-lysine into a monobody for selective chemical crosslinking, two complementary approaches will be explored to enhance the capability of the monobody scaffold: a computational approach based on deep learning; and an experimental approach based on bacterial surface display. The utility of the optimized monobody scaffolds will be evaluated by designing potent and selective cell-permeable monobodies targeting oncogenic KRAS mutants and examining efficacy. These studies are expected to provide insight into the relationship between protein topology and cytosolic transport, which is crucial for the use of protein-based ligands to study life processes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.