Structural Analysis and Development of Notum Fragment Screening Hits

Abstract

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC 50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit

“…Compounds were individually soaked into crystals of Notum and high-resolution structures were determined for each of these putative Notum–fragment complexes. The electron density maps provided evidence of compound binding associated with the active site of Notum for 59 of the crystal structures (hit rate: 7.7%). ,, The complete output of the hits from this crystallographic screen with the DPSL will be published separately. , Hit validation was then performed by demonstration that these binders were able to inhibit Notum carboxylesterase activity in a biochemical assay (up to 1 mM).…”

“…23,26,41 The complete output of the hits from this crystallographic screen with the DPSL will be published separately. 42,43 Hit validation was then performed by demonstration that these binders were able to inhibit Notum carboxylesterase activity in a biochemical assay (up to 1 mM).…”

Notum enhances gastric cancer stem-like cell properties through upregulation of Sox2 by PI3K/AKT signaling pathway

“…Notum (Wingful), a palmitoleoyl-protein carboxylesterase, is a secreted antagonist of Wingless (Wg, Drosophila Wnt1), encoding an α/β-hydrolase enzyme with similarity to pectin acetylesterases from plants [ 6 – 8 ]. It is reported that Notum influences Wingless protein distribution by modifying cell surface proteoglycans and is induced by high levels of Wingless signaling at the same time [ 6 ].…”

Design, synthesis, and biological activity of heterocyclic methyl esters

“…The heterocyclic compounds, characterized by five or sixmembered rings containing at least one, but not limited to, an atom of nitrogen, oxygen, and sulfur, generally display bioactivity as medicine and pesticides. The heterocycle moieties were demonstrated for interaction with the target site in medicinal chemistry and biochemistry [1][2][3]. Chemical active ingredients usually give excellent antibacterial, anti-inflammatory, and anti-tumor activities if structurally introduced or replaced by heterocyclic fragments [4][5][6].…”