Search

Transcription is the first key step in gene activation, and it is controlled in part by protein-protein interactions (PPIs) between transcriptional activators and coactivators. Activator-coactivator PPIs are thus excellent targets for modulating gene expression and potential therapeutic development. However, transcriptional activators and coactivators are typically considered ‘undruggable’ because of their shallow binding surfaces lacking significant topology and promiscuous binding profiles. For decades much effort has been exerted to develop inhibitors targeting activator-coactivator PPIs, but the selectivity of the inhibitors remains a significant challenge. The overarching goal of the work described in this dissertation is to develop a selective inhibitor targeting the master transcriptional coactivator CBP/p300. CBP/p300 is a master transcriptional coactivator regulating gene activation by interacting with multiple transcriptional activators. KIX, which is one of the activator binding domains of CBP/p300, interacts with over 15 distinct transcriptional activators through two binding sites. Dysregulation of the PPIs between CBP/p300 KIX and its activators is involved in many cancers including breast, leukemia, and colorectal cancer. Although inhibitor discovery campaigns targeting KIX have led to some success, the existing modulators exhibit modest potency and low selectivity for KIX. Here we demonstrate a novel dual-targeting peptide that selectively inhibits the PPIs of the CBP/p300 KIX domain with nanomolar potency. The design of a dual-targeting inhibitor (named CREBLL-tide) relied upon connecting the transcription activation domain of MLL and pKID with a covalent linker. CREBLL-tide binds to the two binding sites of KIX simultaneously and exhibits much higher binding affinity over other reported KIX inhibitors. More significantly, it exhibited thousands-fold selectivity for CBP/p300 KIX over other KIX motifs and coactivator domains using several biophysical techniques. Also, cell lysate pull-down assays indicate the CREBLL-tide interacts with full-length endogenous CBP. After validating the high binding affinity and selectivity of CREBLL-tide, the cellular permeability of CREBLL-tide and its analogs as well as their proteolytic stability were characterized. CREBLL-tide was stable in human serum with a 28-hour half-life, and the cell permeability of CREBLL-tide was much improved by the incorporation of a cell-penetrating peptide. The best of these CREBLL-tide analogs shows excellent and selective anti-proliferation activity in breast cancer cells. Additionally, the transcription of KIX-CREB-dependent genes was regulated by the CREBLL-tide analog. In conclusion, these results indicate CREBLL-tide is an effective and potential tool for assessing the role of KIX-activator interactions in breast cancer.