Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

Atkinson, Benjamin N.; Steadman, David; Mahy, William; Zhao, Yuguang; Sipthorp, James; Bayle, Elliott D.; Svensson, Fredrik; Papageorgiou, George; Jeganathan, Fiona; Frew, Sarah; Monaghan, Amy E.; Bictash, Magda; Jones, E.Y.; Fish, Paul V.

doi:10.1016/j.bmcl.2019.126751

Cited by 15 publications

(41 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Resultsmentioning

confidence: 60%

“…Thus, small molecules that inhibit Notum could be used to restore impaired Wnt signalling and increase adult neurogenesis 20 , which may be beneficial in pathologies such as Alzheimer's disease. A number of Notum inhibitors have been identified recently, however these molecules are unable to cross the blood-brain barrier [22][23][24] . 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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Caffeine inhibits Notum activity by binding at the catalytic pocket

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 60%

mentioning

confidence: 99%

Caffeine inhibits Notum activity by binding at the catalytic pocket

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…A helpful design metric was lipophilic ligand efficiency (LLE), 24 used to track improvements in Notum inhibition activity against lipophilicity of the compounds as determined by calculated clogP and measured CHI logD7.4 values. 25 The synthetic routes to new inhibitors of Notum in the pyrrole-3-carboxylic acid (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) and pyrrolidine-3carboxylic acid series (25)(26)(27)(28)(29)(30) are presented in Schemes 1 and 2 respectively. The design of new inhibitors with improved Notum activity initially focused on the SARs of the pyrrole-3carboxylic acids by exploring the substitution on the pyrrole ring 11-24 (Table 2).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions.Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, optimization of 1-phenylpyrrolidine 8 gave acid 26.This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.

show abstract

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…The structure of Notum in a complex with a 14-carbon myristoleic acid lipid has been reported, and Notum efficiently cleaves an 8-carbon-linked chromogenic p-nitrophenyl (pNP) ester substrate [ 16 ]. Indeed, OPTS (8-octanoyloxypyrene-1,3,6-trisulfonate) is a convenient fluorogenic substrate for in vitro Notum activity assays [ [18] , [19] , [20] , [21] ]. These observations inspired us to investigate if Notum could deacylate octanoyl lipid-linked ghrelin, which is the only extracellular protein besides Wnts known to be modified by O-linked serine lipidation.…”

Section: Introductionmentioning

confidence: 99%

Notum deacylates octanoylated ghrelin

Zhao

Schuhmacher

Roberts

et al. 2021

Molecular Metabolism

Self Cite

View full text Add to dashboard Cite

Objectives The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. Methods We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme–substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. Results Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. Conclusions Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.

show abstract

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

Cited by 15 publications

References 24 publications

Caffeine inhibits Notum activity by binding at the catalytic pocket

Caffeine inhibits Notum activity by binding at the catalytic pocket

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

Notum deacylates octanoylated ghrelin

Contact Info

Product

Resources

About