Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC 50 < 500 nM. A critical selection process was then applied with two clusters and two singletons ( 1 - 4d ) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/β-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3- b ]pyradizin-3(2 H )-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.