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 IC50 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(2H)-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.

Notum是一种通过水解棕榈油酸酯发挥作用的Wnt信号通路（Wnt signaling）负调控因子，而该棕榈油酸酯修饰是Wnt活性所必需的。针对以Notum功能异常为潜在病因的疾病，Notum抑制剂具有潜在应用价值。研究人员针对大型商用化合物库开展了基于对接的虚拟筛选（virtual screen, VS），最终筛选出952种化合物用于Notum抑制剂的实验验证。本次虚拟筛选取得了理想效果，共获得31种半数抑制浓度（IC50）处于1~4 μM范围的化合物，其中4d被选为命中化合物进行验证。通过结构生物学指导对4d进行优化，最终获得了强效Notum活性抑制剂，可在细胞模型中恢复Wnt/β-连环蛋白（β-catenin）信号通路的活性。[1,2,4]三唑并[4,3-b]吡嗪-3(2H)-酮（[1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one）系列4代表了一类全新的Notum抑制剂化学骨架，也是首个通过虚拟筛选项目发现的该类抑制剂。上述研究结果证实了基于X射线晶体结构（X-ray structures）构建的优化对接模型开展虚拟筛选的应用价值。