Molecular Editing Reveals a Bilobalide Chemotype that Attenuates Prolyl Endopeptidase (PREP)‐Linked Neuroinflammation

Natural products offer privileged three‐dimensional chemotypes for drug discovery, yet scaffold lability and limited analogue space often impede mechanism‐guided optimization. Herein, we show that molecular editing can reveal a natural product‐derived chemotype that attenuates neuroinflammation via prolyl endopeptidase (PREP) inhibition. BB10 , a chemically stabilized bilobalide analogue generated via lactone‐to‐lactam molecular editing, significantly suppressed LPS‐induced microglial activation and reduced levels of key inflammatory mediators. Unbiased cellular target identification (CETSA‐MS) identified PREP as the top thermally stabilized target, supporting cellular target engagement. BB10 inhibited PREP activity in both cellular and recombinant enzyme assays, implicating PREP inhibition as a mechanism contributing to its anti‐neuroinflammatory activity. Guided by this target‐based insight, structural optimization afforded a fluorinated analogue, BB56 , with improved PREP inhibition and superior anti‐inflammatory efficacy. Mechanistically, BB56 attenuated p38/NF‐κB signaling and reduced NLRP3 upregulation, and PREP knockdown abrogated the anti‐inflammatory effects. Finally, BB56 provided functional rescue in a zebrafish model of neuroinflammation. Collectively, this work establishes a molecular editing‐enabled approach to a natural product chemotype that modulates neuroinflammation through PREP inhibition.