Protein-protein interactions (PPI) represent a challenging target class in contemporary small molecule drug discovery. The difficulty arises because PPI sites are structurally and physicochemically different from conventional drug binding sites. Moreover, we currently lack a good understanding of the druggability of PPI targets: that is, how the structure and properties of a PPI interface site relates to the properties of small molecules that can bind to that site with high affinity. Efforts to achieve potent drug-like small molecule inhibitors of PPI interfaces, involving a wide range targets, historically have largely been unsuccessful, leading to the conclusion that new inhibitor chemotypes are needed to inhibit this class of target. In this thesis, I describe the application of two approaches to identify inhibitors of the PPI interface between Kelch-like ECH associated protein 1 (KEAP1) and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2): (i) screening a library of synthetic macrocycles, and (ii) fragment-based lead discovery. I validate and characterize the hit compounds obtained. In the case of the fragment hits, I investigate what features of the compounds are required for binding to the target (Chapter Two). In parallel, I investigate the structure of the hot spot ensemble at the KEAP1/Nrf2 binding interface using three complementary methods: alanine scanning mutagenesis, fragment screening, and in silico probe mapping using the FTMap algorithm (Chapter Three). This analysis brings insight into the druggability of KEAP1, and advances our understanding of the utility and limitations of those three widely used methods for characterizing the hot spot ensembles at PPI interfaces (Chapter Three). Finally, to gain additional insight into the energetics of KEAP1/Nrf2 binding, I probe the additivity of combinations of alanine mutants (Chapter Four). I use the results to propose a quantitative approach to categorizing the various degrees of additivity that can be observed at PPI interfaces, and discuss the possible structural basis for these behaviors. The model potentially provides a more general framework for understanding the binding energetics at PPI interfaces using combinations of mutations.

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