Intramembrane proteases control the fate of many integral membrane proteins, regulate a number of signaling processes, are key for maintaining homeostasis, and their dysregulation is associated with pathological states. Despite their biological importance, the mechanism of membrane-immersed catalysis, the basis of their substrate specificity and tools for their selective inhibition are mostly lacking. The main structural and mechanistic models for investigating intramembrane proteolysis are the serine intramembrane proteases of the rhomboid family. Rhomboids are evolutionarily widespread and regulate growth factor secretion, biogenesis of mitochondrial proteins and mitochondrial dynamics, invasion of the malaria parasite and membrane protein quality control. Beyond being model intramembrane proteases, rhomboids are increasingly being explored as potential drug targets, but selective and potent rhomboid inhibitors are not available.
We investigate rhomboid protease mechanism, specificity and substrate-enzyme interactions using enzymology and X-ray crystallography (Zoll et al EMBO J 2014) with the aim to understand the catalytic events along the whole reaction coordinate structurally. Based on this, we have developed novel mechanism-based covalent reversible inhibitors of rhomboid proteases that are specific and have high affinity. We demonstrate the mode of binding of these compounds by co-crystal structures with a rhomboid protease, and we provide a general modular platform for the design of isoform-specific inhibitors.