Characterization of the structure and function of NF-KappaB essential modulator and its interaction with inhibitor of KappaB Kinase Beta and development of a screening protocol to discover and validate inhibitors of the interaction
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Protein-protein interactions (PPI) mediate numerous biological processes, but inhibiting these interactions with small molecules has been difficult to achieve in drug discovery. A small number of successes have shown that some PPIs are amenable to inhibition. Computational algorithms designed to measure the druggability of PPIs have been developed based on these successes. These algorithms have identified the interaction between the NF-κB essential modulator (NEMO) and I𝜅B kinase β (IKKβ) as a candidate for inhibition. Furthermore, in vivo peptide-based inhibition of the NEMO-IKKβ interface has shown benefits in attenuating the NF-𝜅B response in cellular and animal models. In addition to its intrinsic interest as a drug target, developing inhibitors against the NEMO/IKKβ interaction may help in the development of improved methods for PPI inhibition. In this thesis, the production of full-length, recombinant forms of soluble NEMO is described. This protein was used in a variety of biochemical assays to advance our understanding of NEMO structure and function. Furthermore, a fluorescence anisotropy (FA) assay was developed to screen for compounds inhibiting the NEMO/IKKβ PPI. Hits from the FA assay were tested by several methods to confirm true inhibition. Additionally, the FA assay was used to accurately measure the affinity of NEMO for IKKβ and to assess the degree of cooperativity in IKKβ binding. The oligomeric state of NEMO has been characterized through the development of a panel of NEMO cysteine to alanine mutants, using polyacrylamide gel electrophoresis analysis, analytical ultracentrifugation, and fluorescence anisotropy. These data represent the first comprehensive characterization of full-length human NEMO, and may provide a path toward development of drug-like inhibitors of the NEMO/IKKβ interaction.