Natural Product-Derived Lipopeptides Are Selective and Effective Modulators of the Coactivator Med25

Abstract

Transcription is an important cellular process that plays a vital role in the regulation of gene expression. A key component of this process is the protein-protein interactions (PPIs) that occur between transcriptional coactivators and activators. Med25 is an example of one of these transcriptional coactivators that interacts with the transcriptional activation domain (TAD) of several different activators through its activator interaction domain (AcID). Several lines of evidence implicate Med25 and its activator binding partners, ATF6α, ETV1, ETV4, and ETV5 in human disease, suggesting this specific PPI would be an attractive target for molecular intervention. Unfortunately, Med25 has been difficult to inhibit due to its larger, shallow binding surfaces and modest binding affinities. Recently, natural products have been a rich source of molecules that have demonstrated success in inhibiting Med25-activator PPIs. This dissertation work focuses on the discovery of one of these natural products, lipopeptide 34913-3 and its analogs as inhibitors of the Med25-ATF6α PPI. It was first revealed that 34913-3 inhibits the Med25•ATF6α PPI in vitro and in cellulo. The structure of this lipopeptide allowed for the synthesis of several analogs that were tested via competition fluorescence polarization assays to identify important components of the lipopeptide for inhibition of Med25. This guided the synthesis analog 34913-8, which demonstrated inhibition of Med25 AcID in vitro and full length Med25 in mammalian cell extracts, while selectively inhibiting Med25 AcID PPIs over other coactivator-activator PPIs. The resemblance of the peptide sequence of 34913-8 to that of TADs suggest these lipopeptides interact with Med25 AcID similarly to amphipathic TADS, suggesting a possible nonspecific model of binding. This led to the synthesis of several substitution analogs that were tested to determine the role of specific residues of the 34913-8 peptide sequence for its inhibitory activity and selectivity for the Med25 PPIs. It was determined that several residues within the peptide sequence do serve an important purpose in selectively inhibiting these interactions, highlighting the importance of sequence specificity for binding of Med25. The overall results in this dissertation demonstrate the potential use of lipopeptide 34913-8 as a template for further development of analogs that can effectively and selectively target coactivator-activator PPIs.