5-Phenyl-1,3,4-oxadiazol-2(3H)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit

Abstract

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.

Notum deacylates octanoylated ghrelin

“…A competitive inhibition assay using the chromogenic pNP8 ester as a substrate and palmitoleic acid as an inhibitor suggested that the Wnt-associated C16 lipid could compete with the ghrelin-associated C8 lipid; however, there was no direct evidence for either being the strongly preferred Notum substrate [ 16 ]. Irrespective of substrate, the Notum is eminently druggable [ 18 , 21 , [44] , [45] , [46] ] and thus provides a potential route for pharmacological treatment of metabolic diseases.…”

Small-Molecule Inhibitors of Carboxylesterase Notum

“…The shorter ghrelin octanoyl (C8) lipid adopts a linear conformation in the center of the pocket (PDB: 6ZYF) [18]. There are a number of x-ray structures with small-molecule inhibitors bound to Notum, with most bound in this pocket (e.g., 15 [PDB: 6ZUV]) [35], but with significant variation in position and orientation of the ligand [36].…”

“…The screening of fragment libraries against Notum's druggable hydrophobic pocket is an attractive strategy and has led to the discovery of a number of hits with orthogonal chemical structures (Figure 4 & Table 1). Fragment screening is performed using both x-ray crystallographic and biochemical platforms in close succession, supported by biophysical screening methods, target occupancy and cell-based TCF/LEF reporter assays [33,35,42,44]. • Saturated C8-12 linear carboxylic acids inhibit activity, whereas longer fatty acids have no effect.…”

“…A standout hit from this screen is 1,2,3-triazole 15 (PDB: 6ZUV) (Figure 4A). Optimization of 15 by structure-based drug design led to the discovery of ARUK3001185 (17) as a potent, selective brain-penetrant inhibitor of Notum activity suitable for use in both cellular and in vivo models of CNS disease (Table 1) [35,43].…”

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity

“…Oxadiazolone 30 was of particular interest as a lead to explore the potential of weak acids to penetrate the brain as molecules with these properties are under-represented in CNS drug discovery. 30,31,32 A set of preferred inhibitors (20z, 26, 28e, 30) were then assessed in in vitro ADME assays to compare their aqueous solubility, microsomal stability (MLM) and cell permeability (MDCK-MDR1)(Table 6). These inhibitors were selected based on their Notum activity but also their chemical structural diversity and complementary physicochemical properties (clogP, pKa).…”