Small-molecule inhibitors of carboxylesterase Notum

Abstract

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.

Keywords: Notum inhibitors; Wnt signaling; activity-based protein profiling; fragment screening; high-throughput screening; hit-finding strategies; structural biology; virtual screening.

Figure 1.. Notum depalmiteoylates Wnt proteins.

(A) Representation of the biochemical reaction that Notum exerts on palmitoleoylated Wnt proteins. (B) A cell-based TCF-LEF reporter assay can be used to assess activity in the Wnt/β-catenin pathway and shows that an increasing concentration of Notum reduces the Wnt3A signaling response. (C) Example of concentration–response curves with inhibitor 5 ± Notum. The Wnt3A signaling response is restored in the presence of Notum by the addition of a Notum inhibitor.

Figure 2.. Notum is expressed in the liver and brain in mice.

(A) RNAscope of Notum (A′ red and A″) showing expression in the liver close to a central vein. A‴ is a zoom image. (B) RNAscope of Notum (B′ red and B″) showing expression in the subventricular zone in the brain close to GFAP-positive cells (green). B‴ is a zoom image. Confocal images. Hoechst indicates nuclear staining (blue) in all cases. Scale bar = 100 μm. Courtesy of S Jolly.

Figure 3.. Cartoon representation of Notum structure.

(A) The enzyme core is shown as a gray cartoon with the lid domain in pale cyan. The lipophilic pocket is outlined as a purple surface. (B) Notum pocket-forming residues (white ball and sticks) and the substrate of palmitoleic acid (gray sticks) within the pocket (purple mesh). (C) Close-up view of pocket (purple) showing the alignment of substrates of Wnt palmitoleate (gray) and ghrelin octanoyl lipid (green/orange), along with representative inhibitor 15 (teal).

Figure 4.. Examples of small-molecule inhibitors of Notum discovered by fragment-based drug design.

(A) Fragment hit 15 (green, Protein Data Bank [PDB]: 6ZUV) and optimized lead 16 (gold, PDB: 6ZVL). Oxadiazole 16 makes an effective interaction with the oxyanion hole while still filling the pocket (purple). (B) Fragment hit 11 (cyan, PDB: 6R8P) and derived inhibitor 12 (salmon, PDB: 6R8R). Isoquinoline 12 shows a flipped binding mode. (C) Natural alkaloids caffeine 22 (wheat, PDB: 6TV4) and theophylline 23 (teal, PDB: 6TUZ). Both compounds bind at the center of the pocket but with quite different orientations. (D) Overlay of all Notum–inhibitor structures (wire) aligned with O-palmitoleate serine (gray, PDB: 4UZQ).

Figure 5.. New covalent inhibitor of Notum.

High-resolution crystal structure of the Notum–inhibitor complex reveals a covalent adduct formed between Notum and 24, which resembles the substrate acyl-enzyme intermediate (Protein Data Bank: 7ARG). The indolinyl rings of 24 (green) bind in the pocket (purple surface), whereas the oxobutanoyl chain acylates Ser232 of the catalytic triad (gray).