Research Project
10173147
PROJECT SUMMARY/ABSTRACT: New molecular modalities to target sites of protein-protein interaction with high affinity and specificity are desperately needed in cancer treatment. Blocking protein-protein interactions between cell surface receptors on immune cells and cancer cells is the basis for the design of a new generation of cancer immunotherapy therapeutics. However, targeting protein-protein interactions with small molecules has proven difficult due to the large area of interaction between proteins and the dearth of small molecule binding pockets. Additionally, targeting these site with antibodies can prove difficult due to lack of tumor pentration and immune related adverse effects. We propose to overcome these difficulties with a novel class of multivalent inhibitors designed to perfectly interact with a protein surface by combining two exciting technologies. Protein painting, an in-house IMAT-funded structural biology technique designed to discover hotspots of protein interaction, will be used to identify protein sequences that drive affinity between interacting proteins. DNA Origami will be used to prepare a size-scalable, semi-rigid scaffold for the interfering peptides identified with protein painting. This scaffold can be precisely tuned for ideal interaction with the 3D topology of the target protein and will spatially orient the interfering hot spot targeting peptides for interaction with the protein partner. Additionally, multivalency provides increases in affinity and specificity over interfering peptides alone. For proof- of-principle, we will develop a multivalent inhibitor designed to target myeloid-derived suppressor cells (MDSCs). These cells express a receptor called ST2, which when bound to IL-33 and its co-receptor IL-1RAcP, allow the MDSCs to exert an immunosuppressive function in the tumor microenvironment. Disrupting the IL-33/ST2/IL- 1RACP protein complex represents a new avenue for cancer immunotherapy. Under Aim 1, we will optimize interfering peptide inhibitors we have previously discovered targeting the hotspots of protein-protein interaction between IL-1RAcP and IL-33/ST2. Following truncation, cyclization, and amino acid substitution, we will have three potent interfering peptide inhibitors of the IL-33/ST2/IL-1RAcP complex. Under Aim 2, we will construct a DNA origami scaffold designed for precise interaction with the three-dimensional topology IL-33/ST2 surface, and will attach the interfering peptides generated in Aim 1 to this scaffold to synthesize a multivalent polyligand inhibitor of the IL-33/ST2/IL-1RAcP complex. Immunogenicity will be examined for both the scaffold alone, and for the multivalent inhibitor after interfering peptides are attached to the DNA origami scaffold. Under Aim 3, we will first examine affinity of the multivalent inhibitor as compared to the interfering peptides alone via surface plasmon resonance. Second, we will determine the functional potency of the multivalent polyligand inhibitor at reducing ST2 receptor signaling by using a HEKBLue IL-33 ligand cell line assay. Finally, we will verify the activity of the multivalent inhibitor using myeloid derived suppressor cells derived from the murine 4T1 breast cancer model using flow cytometry.
