Abstract
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.
Highlights
Secreted Wingless/Integrated(Wnt) morphogens are key components of Wnt signaling.[1]
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.[31]
We have previously determined a number of structures of Notum inhibitor complexes.[21,22,24,25,27,28]
Summary
Secreted Wingless/Integrated(Wnt) morphogens are key components of Wnt signaling.[1]. The electron density for the butyric carbons was restored when 2 was soaked into Notum S232A mutant crystals, consistent with the disorder arising from S232 sterically hindering optimal positioning of the acid form (Figure 4B) Superposition of these two complexes with the 1 complex shows the interplay between nucleophile position and covalent or noncovalent inhibitor binding, with a shift in the S232 Cα of 0.6 Å between complexes involving 1 or 2 (Figure 4C). These data further demonstrate that the methyl ester warhead is important for forming a covalent bond with the nucleophile S232. This carbonyl atom positional difference may be important for 1 as a covalent inhibitor
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