Structural characterization of melatonin as an inhibitor of the Wnt deacylase Notum

Abstract

The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β‐catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N‐[2‐(5‐fluoro‐1H‐indol‐3‐yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N‐acetylserotonin into Notum crystals and obtained high‐resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial.

Caffeine inhibits Notum activity by binding at the catalytic pocket

“…3f). This conformation has not been observed in previously reported apo, inhibitor-bound or substrate-bound structures 16,22,23,25 and, as residue H389 is one of the catalytic triad (the other two residues being S232 and D340), β8-αF loop conformations are of interest for inhibitor design. However, the theophylline makes no interactions with the β8-αF loop.…”

“…Caffeine only differs by one methyl group to each of its metabolites, however, all metabolites lost their Notum inhibitory effects. The overall Notum inhibitory potency of caffeine is relatively low (IC 50 19 µM), although slightly greater than the reported potency of melatonin (IC 50 75 µM) 25 . The natural Wnt substrate-based luciferase assay suggests an effective concentration of EC 50 46 µM for restoring Notum inhibited Wnt signalling, while the biophysically determined affinity Kd is 85 µM.…”

“…OPTS is a common lipase substrate with an octanoyl lipid linked to a fluorescent moiety. It is not a natural Notum substrate; however, it can be used to quantify the enzymatic activity of purified Notum protein 23,25,28 . As shown in Fig.…”

“…The Notum lid domain has been reported to adopt either an open or a closed conformation 25 . In comparison, the caffeinebound structure reveals an intermediate conformation ( Fig.…”

“…To guide the development of brain-accessible Notum inhibitors, we turned our attention to the identification of natural products that can freely cross the blood-brain barrier. Our recent X-ray crystallography-based fragment screen identified a hit with similar chemical structure to melatonin, which led to the finding that this pineal gland secreted hormone can act as an inhibitor for Notum 25 . Caffeine and melatonin both show neuroprotective effects and caffeine can potentiate the effects of melatonin in the inhibition of Aβ oligomerization and modulation of the Taumediated pathway 26 .…”