Abstract In the past 20 years, great strides have been made in identifying and developing small molecules that target and modulate disease-relevant protein-protein interactions (PPIs). Initially, research focused on identifying molecules with a mode of action that depends on preventing specific PPIs. These molecules bind to protein interaction interfaces and thus block PPIs from occurring. Consequently, these molecules inhibit or attenuate the subsequent flow of information through a particular signaling network. Targeting PPIs is challenging as interaction interfaces are typically large planar surfaces that donā€™t present obvious pockets, such as the ligand binding pocket of a G protein-coupled receptor or the active site of an enzyme. An alternative mode of action that has emerged is the development of PPI stabilizers. Unlike PPI inhibitors, stabilizers function as a molecular glue to stick protein interaction partners together. Modulation then occurs by prolonging or amplifying interactions that result in disease inhibitory signaling, such as shifting the signaling balance from pro-survival to apoptosis in cancer. Research into developing PPI stabilizers has centered on the scaffolding and regulatory proteins 14-3-3. These proteins are of interest as nature has provided us with a class of compounds, the Fusicoccanes, which target and stabilize specific 14-3-3 PPIs. Fusicoccanes have thus served as tool compounds that have enabled the identification and characterization of novel 14-3-3 interaction partners and aided in developing and studying PPI stabilizers. My Ph.D. research focused on two main goals: identifying novel PPI stabilizers targeting the 14-3-3/Estrogen receptor alpha (ERĪ±) interaction interface and the identification of novel 14-3-3 interactions sensitive to PPI stabilization by Fusicoccanes. In Chapter I, a general introduction is given into the concept of PPI stabilization and the scaffolding proteins 14-3-3, which made up the primary protein target in this thesis. Chapter II presents a brief historical overview and review of Fusicoccanes and their utility as PPI stabilizers targeting 14-3-3 protein interaction complexes. Chapter III discusses our efforts to identify novel PPI stabilizers targeting the 14-3-3/ ERĪ± interaction complex using a fragment library. In chapter III, we identify Fragment 2 as our lead molecule, and the remainder of the chapter discusses our attempts to delineate its mechanism of action. Chapter IV describes a small molecule library screening campaign at the European Lead Factory to identify 14-3-3/ERĪ± targeting PPI stabilizers. Chapter V identifies the oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) as a novel 14-3-3 interaction partner. We subsequently show that the 14-3-3/CIP2A interaction complex is sensitive to Fusicoccin-mediated stabilization. Chapter VI identifies the serotonin receptor 5-HT2C-R, a G protein-coupled receptor, as a mode III 14-3-3 interaction partner that Fusicoccin can target. Moreover, we discuss the presence of a possible phosphorylation switch on the C-terminal tip of the 5-HT2C-R regulating binding of 14-3- 3 and PDZ domain-containing proteins to the receptor. Finally, chapter VII discusses the results of this thesis within the context of the PPI stabilization field and provides perspectives for future studies.