Abstract 211: Protein painting identifies PD-1: PDL-1 therapeutic targets at protein-protein interfaces

Abstract

Abstract The next generation of molecular cancer therapeutics will target pivotal protein-protein interaction interfaces participating in immune cell receptor signaling, oncogenes, and suppressor genes. We have created a wholly novel, technology “protein painting” for the rapid direct sequencing of hidden native protein-protein interaction hot spots. Our technology, employs previously unexplored small molecule (12 Å) aryl hydrocarbon dyes or “paints” to cut out, and MS sequence, only the hidden unmodified contact interfaces between two or more interacting native proteins. Novel Technology: Paint chemistries have extremely high affinities (rapid on-rates, and very slow off-rates that are ten to 100 times higher than most protein-protein interactions). When mixed with a native pre-formed protein complex for only 5 minutes at physiologic pH and salinity, the paints non-covalently coat all external sites on the protein without altering the 3D conformation of the complex, but cannot gain access to the solvent inaccessible hidden protein-protein interaction domains. Each paint molecule spans less than 3 amino acids, and has high affinity for protease cleavage consensus sites. Following painting, the proteins are dissociated. This leaves the paint molecules coating surfaces not participating in the interface. Following dissociation, the proteins are linearized, digested with trypsin, and sequenced by standard MS. The paint molecules remain non-covalently bound after the proteins are denatured. Trypsin will not cleave the regions of the protein that are “painted”. Following proteolysis peptides emerging from MS will be generated exclusively from the unmodified opposing points where the proteins were in intimate contact. Results: Protein Painting identified hot spot domains between PD-L1:PD-1, including two surface interface regions that are separated in the linear sequence but adjacent in the 3D structure. We created novel cyclized multivalent inhibitors that block both sides of the PD-L1:PD-1 interface and markedly suppress cell-cell coupling and abolished downstream signaling through this complex in cultured tumor cell immune cell interactions. A very high correlation (p&lt0.0003) was found for known contact points predicted by crystal structure, with a 97% specificity for true positive hot spots: 95% agreement with Robetta prediction software for known complexes. Protein painting outperforms (425%) hydrogen deuterium exchange and cross linking for number of positive hits and % true positive hits. Conclusions: Protein painting is a new tool to identify highly specific drug targets located within protein interaction interfaces, yielding inhibitors that abolish protein signaling relevant to cancer immunotherapy. Citation Format: Alessandra Luchini, Luisa Paris, Virginia Espina, Kelsey Mitchell, Angela Dailing, Lance A. Liotta. Protein painting identifies PD-1: PDL-1 therapeutic targets at protein-protein interfaces [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2017-211