Translation of Hot Loops into Macrocyclic Inhibitors of Protein-Protein Interactions Macrocyclic natural products include many of our most potent drugs and most useful biological probes, but there are no reliable design tools for developing macrocycle drugs. The Kritzer lab uses synthesis, biophysics, and cell biology to discover and apply macrocyclic compounds. Recently, we reported LoopFinder, an algorithm that analyzes protein-protein interactions (PPIs) and identifies critical “hot loops”. We hypothesize that hot loops can be translated into inhibitors of their associated PPIs by stabilizing their bioactive conformation. This hypothesis is supported by preliminary results, but there remains a critical gap in knowledge: how can we stabilize a desired loop conformation within a macrocycle? In this project, we use complementary synthetic, computational and biochemical approaches to address this question. In one approach, we apply a new “diversity-oriented stapling” strategy that prepares and tests large libraries of macrocycles with varied 3D shapes. In a complementary approach, we are collaborating with world-leading computational scientists to apply new Rosetta algorithms that search for highly structured macrocycles, and to refine predictions using explicit-solvent MD. For each PPI inhibitor we discover, we will analyze its solution structure and quantify its target affinity, selectivity, metabolic stability and cell penetration. This represents an unprecedented data set for correlating macrocycle sequence, structure, and function. These data will also provide continual feedback for the synthetic and computational design approaches. As targets for macrocycle design, we have identified loop-mediated PPIs that control vesicle trafficking pathways that are involved in cancer and infectious disease. These include: Eps15, which is required for endocytosis; EHD1, which is involved in receptor recycling; the ESCRT-II complex, which mediates endosomal sorting; and the COP-I complex, which controls endosome budding from the Golgi. There are no selective inhibitors for any of these pathways, making our macrocycles immediately useful to biology and medicine. This project develops innovative computational and synthetic tools that are specific to the development of macrocycle drugs. We cannot currently design the next cyclosporine, but we anticipate being able to do so using this a new generation of methods, tools and molecules. This project represents a timely opportunity to bring macrocycle drug design closer to reality.