Ultra large docking reveals novel chemotypes and a distinct binding mode for Porcupine inhibition

Wnt signaling is essential for embryonic development and adult tissue homeostasis, and its dysregulation contributes to cancer and other human diseases. Porcupine (PORCN), a membrane-bound O-acyltransferase (MBOAT) required for Wnt palmitoylation and secretion, has emerged as an important therapeutic target. Despite promising preclinical results, PORCN inhibitors have shown limited clinical activity to date, motivating the search for new chemotypes and binding modes. Here, we docked a library of 1.3 billion molecules against PORCN and identified 16 novel compounds that inhibit Wnt signaling. These hits are structurally distinct from known PORCN inhibitors, and structure-based optimization of one series yielded sub-micromolar leads. Cryo-electron microscopy revealed that the lead inhibitor binds PORCN in an unreported mode within the palmitoleoyl-CoA chamber, positioned closer to the cytosol than existing inhibitors. Together, our findings expand the chemical and structural space available for targeting PORCN and provide new starting points for development of Wnt signaling antagonists.