Caffeine inhibits Notum activity by binding at the catalytic pocket

Abstract

Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer’s disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors.

Structural Insights into Notum Covalent Inhibition

“…Notum is a druggable target for Wnt signaling modulation . We have previously determined a number of structures of Notum inhibitor complexes. ,,,,, All of these inhibitors bind noncovalently at the Notum enzyme catalytic pocket, where the natural substrate PAM binds. For searching covalent inhibitors, we used this noncovalent inhibitor binding information to limit compound docking into the enzyme active site.…”

“…Notum also plays a key role in adult brain ventricular–subventricular zone neurogenesis . Notum inhibitors can rejuvenate aged colon stem cells, increase cortical bone thickness and strength, and increase adult neuronal progenitors and are being investigated for potential treatment of neurodegenerative pathologies such as Alzheimer’s disease, − in which Wnt signaling is commonly downregulated. , More recently, Notum has been identified as a key mediator for adenomatous polyposis coli (Apc)-mutated tumor cell fixation and tumor formation, while Notum inhibitors abrogate the ability of Apc-mutant cells to expand . These pieces of evidence highlight the importance of Notum as a novel target for drug discovery.…”

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.…”

Methylxanthines and Neurodegenerative Diseases: An Update

“…Recently, another additional mechanism by which caffeine might exert its potential therapeutic benefits in AD has been suggested by Zhao and colleagues [ 45 ]. In the presence of caffeine, Wnt signaling was found to be upregulated.…”

“…Interestingly, only caffeine, but not its demethylated metabolites paraxanthine, theobromine or theophylline exerts this inhibitory property. Using structure analysis, the authors were able to show that caffeine binds at the enzymatic pocket and overlapped the position of notum´s natural substrate palmitoleic lipid [ 45 ]. The relatively low inhibitory potency of caffeine (IC 50 19 µM) indicates that in adults consuming moderate amounts of three or four cups of coffee per day, resulting in plasma concentrations of about 15 µM [ 46 ], physiological notum inhibition seems unlikely.…”