Norstictic Acid Is a Selective Allosteric Transcriptional Regulator.

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å2) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprising a highly dynamic loop flanking one canonical binding surface, and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple-negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators.

Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a ß-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences. The ABD of the coactivator Med25 (activator interaction domain or AcID) is unique in that it contains secondary structural elements that are on both ends of the spectrum: helices and loops that display significant conformational mobility and a seven-stranded ß-barrel core that is structurally rigid. Using biophysical approaches, we build a mechanistic model of how AcID forms binary and ternary complexes with three distinct activators; despite its static core, Med25 forms short-lived, conformationally mobile, and structurally distinct complexes with each of the cognate partners. Further, ternary complex formation is facilitated by allosteric communication between binding surfaces on opposing faces of the ß-barrel. The model emerging suggests that the conformational shifts and cooperative binding is mediated by a flexible substructure comprised of two dynamic helices and flanking loops, indicating a conserved mechanistic model of activator engagement across ABDs. Targeting a region of this substructure with a small-molecule covalent cochaperone modulates ternary complex formation. Our data support a general strategy for the identification of allosteric small-molecule modulators of ABDs, which are key targets for mechanistic studies as well as therapeutic applications.