Structural Insights into Notum Covalent Inhibition

The carboxylesterase
Notum hydrolyzes a palmitoleate moiety from
Wingless/Integrated­(Wnt) ligands and deactivates Wnt signaling. Notum
inhibitors can restore Wnt signaling which may be of therapeutic benefit
for pathologies such as osteoporosis and Alzheimer’s disease.
We report the identification of a novel class of covalent Notum inhibitors,
4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures
of the Notum inhibitor complexes reveal a common covalent adduct formed
between the nucleophile serine-232 and hydrolyzed butyric esters.
The covalent interaction in solution was confirmed by mass spectrometry
analysis. Inhibitory potencies vary depending on the warheads used.
Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom
is positioned at an unfavorable angle for the approach of the active
site water, which, combined with strong hydrophobic interactions with
the enzyme pocket residues, hinders the intermediate from being further
processed and results in covalent inhibition. These insights into
Notum catalytic inhibition may guide development of more potent Notum
inhibitors.