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.