Project Summary Up to 75% of human-disease causing targets are considered “undruggable” using the current drug classes available: small molecules and biologics. Many of these “undruggable” targets are involved in intracellular protein-protein interactions (PPIs), which are challenging to inhibit with small molecules and antibodies. Small molecules require hydrophobic pockets for binding, which PPIs generally lack. Proteins are capable of disrupting PPIs, but they generally cannot cross the cell's plasma membrane. Miniproteins are small proteins (<10 kDa) with defined tertiary structure. They are a promising class of protein therapeutics that have shown great success at inhibiting extracellular targets and several cell-penetrant miniproteins have been identified. However, we still do not know the design rules needed to develop miniproteins with intracellular delivery. The main bottleneck to developing cell-penetrating miniproteins is that measuring cytosolic delivery is slow and expensive because each protein must be purified and tested individually. To overcome this experimental limitation, we will develop an entirely new multiplexed approach to measure the cytosolic delivery of many candidate cell-penetrant miniproteins at once. We hypothesize that a high-throughput approach will rapidly uncover new cell-penetrant miniproteins to study the determinants of cytosolic delivery. In Aim 1, we will develop a cytosolic capture and pull-down approach to screen cell-penetrant miniproteins. We will create an entirely new approach that quantifies the cytosolic abundance of each miniprotein in a mass spectrometry-based approach. The goal of this aim is to extensively validate this method and see enrichment of our known cell-penetrant protein from a mixture of inactive proteins added to cells. In Aim 2, we will define the protein features that contribute to the cytosolic delivery of miniproteins. We will perform a second round of protein design to validate the features found in our initial screen. The goal of this aim is to apply a multiplexed approach for measuring cytosolic delivery and identify the protein features of successful cell-penetrant miniproteins. The long-term goal of the proposed work is to determine the protein features needed for cytosolic delivery to develop new cell-penetrant miniproteins capable of inhibiting PPIs implicated in disease.