A relatively small number of proteins have been suggested to act as morphogens, signalling molecules that spread within tissues to organize cell fate specification during development and tissue repair. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity 1-3 . How can a hydrophobic lipoprotein spread in the aqueous extracellular space? A variety of mechanisms, some invoking lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem 4-6 . Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnts from the aqueous environment and serve as a reservoir from where Wnts can be handed over to signalling receptors.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease supporting the need to discover new small molecule inhibitors. A crystallographic x-ray fragment screen was performed, which identified fragment hit 1,2,3triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity) making it suitable as a new chemical tool for exploring the role of Notum mediated regulation of Wnt signaling.

Optimization of fragment hit 3 identified isoquinoline 45 as a potent inhibitor of NOTUM with an unexpected flipped binding mode.