(176o) Hitting Undruggable Targets: Design of Double-Click Stabilized p53-like Peptides with Serum-Independent Efficacy

Nearly two-thirds of all disease-associated genes are ‘undruggable’ by modern therapeutics, meaning they are inside cells, out of the reach of biologics, but lack small molecule binding pockets. Stabilized peptides have the potential to hit these targets, which would open a vast array of potential new therapies. However, the design criteria for developing agents that can reach and disrupt their target are currently unknown. One such target is the p53/MDM2 interaction—a protein-protein interaction central to many cancers. Several inhibitors have been developed against the MDM2 protein because this target degrades the “the guardian of the genome” protein, p53. However, few of these peptides demonstrate the serum-independent, on-target efficacy required for clinical translation. This work focuses on the physicochemical properties of newly-developed high affinity double-click stabilized MDM2-binding peptides that promote favorable in vitro outcomes including cytosolic access, protease stability, and target-specific cell killing. Using a directed-evolution platform developed in our lab, we show that several of these agents are capable of targeting the MDM2 protein inside cells. Furthermore, we highlight a promising lead peptide variant with an added disulfide-bond backbone modification that demonstrates sub-micromolar inhibition in the presence of serum. We show how stabilization improves the protease stability, binding affinity, and impacts the cytosolic delivery, all affecting target engagement. A simple model of subcellular distribution is used to integrate the results and help design future agents.