Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling

Wnt
proteins are secreted morphogens that play critical roles in
embryonic development and tissue remodeling in adult organisms. Aberrant
Wnt signaling contributes to diseases such as cancer. Wnts are modified
by an unusual O-fatty acylation event (O-linked palmitoleoylation of a conserved serine) that is required
for binding to Frizzled receptors. O-Palmitoleoylation
of Wnts is introduced by the porcupine (PORCN) acyltransferase and
removed by the serine hydrolase NOTUM. PORCN inhibitors are under
development for oncology, while NOTUM inhibitors have potential for
treating degenerative diseases. Here, we describe the use of activity-based
protein profiling (ABPP) to discover and advance a class of N-hydroxyhydantoin (NHH) carbamates that potently and selectively
inhibit NOTUM. An optimized NHH carbamate inhibitor, ABC99, preserves
Wnt-mediated cell signaling in the presence of NOTUM and was also
converted into an ABPP probe for visualizing NOTUM in native biological
systems.

Wnt蛋白是一类分泌型形态发生原，在胚胎发育以及成体生物体的组织重塑过程中发挥关键调控作用。异常激活的Wnt信号通路与癌症等多种疾病的发生发展密切相关。Wnt蛋白会经历一种罕见的O-脂肪酰化修饰，即保守丝氨酸残基的O-棕榈油酰化修饰，该修饰是Wnt与Frizzled受体结合所必需的。Wnt蛋白的O-棕榈油酰化修饰由porcupine（PORCN）酰基转移酶催化添加，而丝氨酸水解酶NOTUM可将该修饰去除。目前，PORCN抑制剂正处于肿瘤治疗的研发阶段，而NOTUM抑制剂则有望用于治疗退行性疾病。本研究介绍了利用活性导向蛋白质谱分析（activity-based protein profiling, ABPP）技术，发现并优化一类强效且选择性抑制NOTUM的N-羟基海因氨基甲酸酯（N-hydroxyhydantoin, NHH）类化合物。经过优化的NHH氨基甲酸酯类抑制剂ABC99，可在NOTUM存在的条件下维持Wnt介导的细胞信号通路活性，同时该化合物还可被衍生为ABPP探针，用于在天然生物系统中可视化检测NOTUM。