Dissecting Transcriptional Coactivator Binding Networks To Enable Small Molecule Modulation

The activation of transcription is reliant on the coordinated assembly of multiple dynamic protein complexes at promoter regions of DNA. The composition of this transcriptional machinery varies with cell type and cell cycle allowing for differential gene expression associated with different cellular processes and pathways. Central mediators of transcription are coactivator proteins that form key protein-protein interactions (PPI) with DNA-bound transcriptional activators via activator binding domains (ABD). Due to the requirement of these activator-coactivator interactions for gene expression, dysregulation of both activators and coactivators is associated with many diseases including cancers, metabolic disorders, and developmental defects. Thus, there is great interest in the development of small molecules capable of targeting these dynamic protein interfaces in diseases linked to aberrant activator-coactivator activity. Activator-coactivator interactions are challenging small molecule targets because of the broad, dynamic surfaces that mediate their interactions. Successful targeting of these PPIs has been facilitated by detailed structural and mechanistic characterization of individual activator-coactivator complexes. However, only a small subset of activator-coactivator interactions have been mechanistically characterized leaving many open questions surrounding how differences in ABD structure alter activator-coactivator complex formation.;In this dissertation, we characterize the mechanistic and structural properties of activator interactions with the Activator Interaction Domain (AcID) of Med25. AcID is a structurally unique ABD and we trace the mechanistic features of its interactions with activators to other more characterized coactivator ABDs including the KIX domain of CBP. We show that although it has a unique protein fold, many of the mechanistic features driving AcID activator interactions are observed in structurally diverse ABDs.;Med25 AcID interacts with multiple transcriptional activators demonstrated to drive gene expression linked to multiple diseases including metastatic cancers. Disrupting these AcID-activator complexes can lead to downregulated expression of these disease-associated genes underscoring the potential of small molecule inhibitors of these interactions. We demonstrate that two native cysteines within the AcID domain of Med25 can be targeted by small molecules and are functionally positioned such that targeting these cysteines with small molecules can inhibit activator-AcID complexes. We next describe the use of Tethering to identify covalent small molecule modulators of AcID function. Through this screen we identified the compound A6, which induces allosteric modulation of the AcID domain and the use of a cell active analog of A6 to inhibit Med25 AcID-dependent gene expression in live cells.;Activator-coactivator interactions that have yet to be fully characterized evade targeted inhibitor discovery strategies. We show that transcriptional activity can be modulated via two different methods of targeting the NF-kappB transcriptional activation pathway beyond activator-coactivator interactions. One approach selectively inhibits canonical NF-kappaB signaling through the use of a novel stabilized peptide (NBD2) that inhibits the IKK-NEMO interface of the IKK kinase complex. We also describe the mechanistic characterization of ketogibberellic acid methyl ester which acts as a potent inhibitor of NF-kappaB activity.