Dissertation Defense Announcement
To: The George Mason University Community
Candidate: Angela Dailing
Program: PhD in Biosciences
Date: Wednesday July 26, 2017
Time: 12:00 PM
Place: George Mason University
Science & Tech campus
IABR Building, Room 1004
Title: "Painting a Target on Osteoarthritis: Protein Painting, IL-1â Inhibitor Development, and Optimization"
Committee Chair: Dr. Alessandra Luchini
Committee Members: Dr. Lance Liotta, Dr. Iosif Vaisman, Dr. Mikell Paige
All are invited to attend the defense.
ABSTRACT:
The next frontier of proteomics is the elucidation of how and where proteins interact. Protein-protein interactions (PPIs) are known to be involved in many important biological processes and diseases, which makes their elucidation extremely important for the evolution of targeted therapeutics. Therefore, we introduce a new structural mass spectrometry method that hinges on the use of small molecule chemistry to help reveal exclusively the sequence contained within the PPI interface. This technique, referred to as protein painting, was used to discover and construct a new peptide inhibitor that abolishes the necessary interaction of IL-1 receptor accessory protein (IL1-RAcP) with the interleukin-1â (IL-1â)/IL-1 receptor I (IL-1R1) complex involved in osteoarthritis. An estimated 27 million Americans each year are afflicted by osteoarthritic with no effective, targeted therapeutics currently available. Our inhibitor, Arg286p, which mimics a key interaction region at the Arg286 beta loop of IL1-RAcP, constitutes a highly novel drug target and therapeutic probe. IL-RAcP multivalent Arg286 peptide inhibitor variants were created for increased binding affinity, specificity, and potential druggability by substitution with natural amino acids and the introduction of cyclized motifs into the sequence. A custom monoclonal antibody against IL-1RAcP was also developed from the painting as well to test targeting this region with various forms of targeted inhibitors. Characterization is performed using our published in vitro pull down assays for protein-protein interactions and interleukin signaling in cell lines. The Arg286 peptide variants abolished interleukin complex formation in the pull down assay (target > 95%) and interleukin signaling in the cell culture model (target > 75%) in comparison to controls. RA_Cycl and Met293 showed an increased ability to inhibit the formation of the 3-way complex, in vitro, in a dose-dependent manner when compared to the original Arg286 peptide. The custom monoclonal antibody 7B8D7 also displayed potent inhibition of IL-1â signaling in both the pull-down assay (target > 95%) and cell culture model (target > 75%). An in vivo mouse model of inflammation was established for future functional testing of the candidate peptides and antibody. The results revealed that the IL-1RAcP Arg286 inhibitors developed from protein painting show increased binding efficacy, specificity, and potential druggability, and will consequently decrease IL-1â-signaling further then current IL-1 targeted osteoarthritis therapies.
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