Identification of inhibitors of the transmembrane protease FlaK of Methanococcus maripaludis

GxGD‐type intramembrane cleaving proteases (I‐CLiPs) form a family of proteolytic enzymes that feature an aspartate‐based catalytic mechanism. Yet, they structurally and functionally largely differ from the classical pepsin‐like aspartic proteases. Among them are the archaeal enzyme FlaK, processing its substrate FlaB2 during the formation of flagella and γ‐secretase, which is centrally involved in the etiology of the neurodegenerative Alzheimer's disease. We developed an optimized activity assay for FlaK and based on screening of a small in‐house library and chemical synthesis, we identified compound 9 as the first inhibitor of this enzyme. Our results show that this intramembrane protease differs from classical pepsin‐like aspartic proteases and give insights into the substrate recognition of this enzyme. By providing the needed tools to further study the enzymatic cycle of FlaK, our results also enable further studies towards a functional understanding of other GxGD‐type I‐CLiPs.