Identification of Loop Mediated Protein-Protein Interactions and Design of Cyclic Peptide Locked Loop Inhibitors
Abstract: The landscape of drug discovery has recently begun to shift from
traditionally druggable enzymes to protein-protein interactions (PPIs). Enzymes possess
well defined active sites, while PPIs often have large and flat surface areas that small
molecules can not interrupt. As this landscape shifts, it is essential that the tools used
to target these proteins change to meet these demands. ... read moreOne class of molecules particularly
adept at binding proteins are peptides. While rational design of substrate transition state
mimics as inhibitors has led to successful targeting of enzymes, an analogous approach for
rational design PPI inhibitors using peptides is a difficult task. The identification of
hot spot residues at protein interfaces has enabled the characterization of essential amino
acid residues at protein interfaces amenable to targeting or mimicry using peptides. Much
previous work on this subject has used computational approaches to identify epitopes on
proteins that facilitate PPIs containing either well-defined secondary structure, such as
α-helices and β-sheets or have disregarded any structural information searching for purely
extended linear regions. This thesis work describes LoopFinder, a computational tool
developed to identify loops and turns at protein interfaces for design and development of
synthetic cyclic peptide inhibitors of PPIs. This program was used to identify a hot loop
residing on the protein stonin2 at its interface with Eps15, a complex essential for
clathrin-mediated endocytosis. The hot loop was used to inspire the development of a cyclic
peptide "locked loop library". Libraries were prepared using a panel of bis-bromomethy-aryl
linkers for bis-alkylation of cysteine residues introduced to flank the hot loop. Using
this approach, we were able to identify sub-micromolar binding peptides to the Eps15-EH2.
This peptide was then used to develop a novel biochemical assay for Eps15 binding molecules
enabling the search for new inhibitors of endocytosis.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Chemistry.
Advisor: Joshua Kritzer.
Committee: David Walt, Krishna Kumar, and Adrian Whitty.
Keyword: Chemistry.read less