Benzothiophene-based Fragments Act as Reversible and Irreversible Covalent Probes for Dynamic Coactivator Med25

Abstract

Transcriptional coactivators mediate transcriptional activity within cells by forming transient protein-protein interactions (PPIs) to facilitate the assembly of the RNA polymerase machinery. Dysregulation of these PPIs results in disease. Transcriptional coactivators consist either of multiple protein subunits or multiple subdomains that assist the transcriptional activation process through a PPI network with transcriptional activators. For example, Med25, a subunit of the Mediator complex, has been shown to play a role in bridging the Mediator complex and RNA Polymerase II at Med25-dependent gene targets, ultimately resulting in transcriptional upregulation. Mis-regulation of Med25-transcriptional activator PPIs contributes to viral infection, oncogenesis, and stress response disorders. For this reason, there is great interest in identifying druglike modulators of Med25. The motif that Med25 uses to form activator PPIs is the Activator Interaction Domain (AcID). It contains two binding surfaces, the H1 and H2 faces, that are large (900 A2) and have little topology for small-molecule interactions. However, Med25 AcID contains two solvent-exposed cysteine residues adjacent to the H1 binding surface, suggesting that a site-directed screening approach might be an effective method for ligand discovery. Towards that end, the Mapp lab identified reversible covalent modulators of Med25 AcID using a disulfide Tethering approach in collaboration with the Wells lab. When the hits from the screen were examined, we noted the unusual structure of a benzothiophene-containing ligand that Tethered Med25 AcID at C506 selectively and potently and thus appeared to be an excellent candidate for further development. The lead fragment contains both a benzothiophene moiety and an isonipecotic acid moiety, along with a disulfide used for Tethering. In Chapter 2 a study dissecting the roles of each of these functional groups and their contribution to the affinity of the lead fragment for Med25 was carried out. Each of the molecules was assessed using single-point Tethering experiments in a time-dependent fashion. The results indicate that both the benzothiophene and the nipecotic acid groups have some affinity for Med25 AcID but that neither recapitulates the affinity of the lead fragment. Taken together, the data suggest that both moieties make important contacts with Med25 AcID. In Chapter 3 we transformed the reversible ligands from Chapter 2 into irreversible covalent ligands through the incorporation of α-bromoacetamide into each of the ligands. Each of the ligands was synthesized using standard methods and then tested in single-point alkylation experiments. We also used these experiments to evaluate the effects of stereochemistry on small molecule binding to Med25. Through this we demonstrate that benzothiophene-based ligands discovered through disulfide Tethering can be transformed into irreversible probes that target Med25 AcID. We show that both (R)- and (S)-benzothiophene nipecotic acetyl bromides not only irreversibly bind to Med25 AcID but also shift the melting temperature of Med25 AcID, suggesting that these two compounds stabilize particular Med25 AcID conformations. Thus, these two molecules are useful probes for us and others for the study of Med25 function. Future efforts will examine the effect of the conformational stabilization on Med25 PPI networks in vitro and in cells.