Structural Insights into Notum Covalent Inhibition.

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• The enzyme Notum negatively regulates Wingless/Integrated (Wnt) signaling by hydrolyzing a palmitoleate group from Wnt ligands.
• Dysregulation of Wnt signaling is implicated in various pathologies, including osteoporosis and Alzheimer's disease.
• Inhibition of Notum presents a potential therapeutic strategy to restore Wnt signaling.

Purpose of the Study:

• To identify and characterize a novel class of covalent inhibitors targeting the carboxylesterase Notum.
• To elucidate the mechanism of action and structural basis for covalent inhibition of Notum.

Main Methods:

• Synthesis and screening of 4-(indolin-1-yl)-4-oxobutanoate esters as potential Notum inhibitors.
• High-resolution crystal structure determination of Notum-inhibitor complexes.
• Mass spectrometry analysis to confirm covalent adduct formation in solution.

Main Results:

• A novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters, was identified.
• Crystal structures revealed a common covalent adduct formed via nucleophilic attack by serine-232 on the ester carbonyl.
• Mass spectrometry confirmed covalent modification of Notum by the inhibitors in solution.
• Inhibitory potency was dependent on the specific warhead employed.
• The mechanism involves formation of a stable acyl-enzyme intermediate, hindered from further hydrolysis by unfavorable geometry and hydrophobic interactions.

Conclusions:

• The identified 4-(indolin-1-yl)-4-oxobutanoate esters are effective covalent inhibitors of Notum.
• Structural and mechanistic insights explain the basis of covalent inhibition, highlighting the role of serine-232 and the acyl-enzyme intermediate.
• These findings provide a foundation for the rational design of more potent Notum inhibitors for therapeutic applications.