Mechanistic Investigation of the Transcriptional Coactivator Med25 and its Protein-Protein Interaction Network

Abstract

Regulation of transcription through protein complex assembly is critical for the maintenance of cellular homeostasis. Dysregulation of transcription is unexpectedly a cause or consequence of most human disease. Thus, there is significant therapeutic potential in discovering novel mechanisms to restore normal protein-protein interaction (PPI) networks in transcription. Targeting these transcriptional PPIs with small molecules has historically been challenging as the interactions occur over broad surface areas with relatively weak binding affinities. Within this dissertation, I describe two emergent strategies, namely natural products screening and protein-observed 19F-NMR, that allowed for the identification of small molecule inhibitors of the activator interaction domain (AcID) of Med25, a critical coactivator protein in transcription. Med25 AcID is a transcriptional coactivation domain that interacts with several transcriptional activators that have been implicated in cancer and disease, including the ETV/PEA3 family and the oxidative stress response factor ATF6α. An overarching goal of this dissertation was to identify small molecule inhibition of Med25 AcID that will allow for future study of the role of Med25 in cancer processes such as metastasis and tumorigenesis. This dissertation first describes the mechanistic details that define the interactions between Med25 AcID and its native protein partners to enable the identification of small molecules that selectively inhibit Med25 AcID. As demonstrated, the AcID motif interacts with each of its binding partners using discrete modes of molecular recognition. The significant differences between the interactions of Med25 AcID with the ERM and ATF6α activators (e.g. differences in binding locations and relative dependence on electrostatic interactions) highlight the diversity of molecular mechanisms through which Med25 AcID functions. Small molecule inhibitors of Med25 AcID were then identified, guided by these mechanistic details. Natural products discovery and protein-observed 19F-NMR were leveraged to target Med25 AcID, which interacts with its protein partners over broad surface areas with relatively weak binding affinities. These two strategies have provided recent successes for other transcriptional PPIs. The 34913 lipopeptide, which exhibits effective inhibition of the Med25 ATF6α PPI in a cellular context, and nine preliminary hit fragments were successfully identified using those strategies. Collectively, this dissertation represents a tremendous advance in the identification of small molecules that target a challenging transcriptional coactivator, Med25 AcID, that will allow for subsequent determination of the role of Med25 in disease.