Structural Insights into Notum Covalent Inhibition

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.

Computational Analysis of the Inhibition Mechanism of NOTUM by the ONIOM Method

“…Calculations involving the 6-311+g(2d,2p) basis set predicted a more stable IC-2 structure, and the overall acylation process turned out to be an exergonic process. This result is in agreement with the experimental results 14 in that the crystal structure of Notum together with inhibitor 1 resulted in the irreversible covalent adduct, which correspond to an IC-2 structure. Of all the calculated complexes, IC-2 is predicted to be the most stable species.…”

“…This observation is in agreement with the fact that methyl ester 1 acts as an irreversible inhibitor by forming a covalent adduct as a result of transesterification between 1 and Ser232. 14 On the basis of the crystal structure of this adduct ( Figure 2 ), it was suggested that the deacylation process is unfavorable because of the strong hydrophobic interactions and the unfavorable position of the active site water molecule for the nucleophilic addition step. It was also stated that the nucleophilic attack is hindered, since the angle between the O atom of the water molecule with carbonyl C and O atoms is 87° in the crystal structure ( Figure 2 ); however, the same angle is 114° for the native substrate O -palmitoleate ester.…”

“…For the calculations involving the acylation process (Steps 1 and 2 in Figure 2 ), a model enzyme–substrate complex was generated from the crystal structure of Notum with 5 (PDB accession code: 7B37) 14 using the VMD program. 29 This complex was found inactive and provided a starting point to obtain a reactive enzyme–substrate complex.…”

“…Part of the QM region residues were included into the MM region, and Figure 3 shows the QM region of these residues. For the calculations involving the deacylation process (Step 3 and Step 4 in Figure 2 ), the model enzyme–substrate covalent adduct was generated from the crystal structure of Notum covalently modified with inhibitor 1 (PDB accession code: 7ARG) 14 following the same protocol as outlined for the acylation process.…”

“… 13 Recently, Zhao et al has reported an irreversible Notum inhibitor, methyl 4-(indolin-1-yl)-4-oxobutanoate. 14 The crystal structure of the inhibitor-enzyme complex showed a covalent adduct formation between catalytic Ser232 residue and inhibitor through an ester bond ( Figure 1 ). The indole part of the inhibitor is surrounded with Trp128 and Phe268 through π-stacking interactions.…”