Development of Caspase Cleavage Targeting Chimeras (CACTACs) for Targeted Protein Cleavage

PROJECT SUMMARY Caspases are a family of cysteine proteases which can cleave proteins containing cleavage sites on the flexible region to trigger cell death through apoptosis or pyroptosis. Recently, bifunctional small molecules, such as proteolysis targeting chimeras (PROTACs), lysosome targeting chimeras (LYTACs), or autophagy targeting chimeras (AUTACs), have emerged as potential therapeutic modalities by hijacking the ubiquitin-proteasome system (UPS), lysosome or autophagy to degrade proteins of interest (POIs). Caspase cleavage is a one-step process that instantly terminate protein function. Here, we discovered that piperlongumine (PL), a dietary natural product, can recruit caspase(s) to cleave Bcl-xL when conjugated with a Bcl-xL/2 inhibitor ABT263. This finding motivated us to further develop bifunctional small molecules for targeted protein cleavage by recruiting caspases, which could be superior to or complementary to the known degraders. We termed this kind of compounds caspase cleavage targeting chimeras (CACTACs). In this proposal, we will apply a series of state-of-the-art technologies to find the caspase(s) recruited by the current PL-based Bcl-xL CACTAC and confirm the mechanism of action. In addition, we will build a Halo-FKBP system to further genetically explore the potential of CACTAC-mediated targeted protein cleavage by various caspases. The caspase-cleavage site pairs will be systematically identified to inform the future CACTAC design. We will also further develop new caspase-2 recruiters through PL analog library screening and structure/computer modeling-based optimization. More CACTACs targeting other POIs will be synthesized and tested to expand the application of CACTACs. The CACTAC platform proposed here will offer a revolutionary weapon against oncoproteins for cancer therapy.