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

Notum is a member
of serine hydrolyses that cleaves the palmitoleate
moiety from Wingless-related integration site (Wnt) ligands. This
enzyme plays crucial functions through modulating the Wnt signaling
pathway. Inhibition of Notum carries therapeutic effects against a
number of maladies including osteoporosis, cancer, and Alzheimer’s
disease. Recently, a class of irreversible inhibitors based on esters
of 4-(indolin-1-yl)-4-oxobutanoic acid have been reported. Using the
crystal structures of enzyme-4-(indolin-1-yl)-4-oxobutanoate adduct
and 4-(indolin-1-yl)-4-oxobutanoic acid-enzyme complex, we studied
computationally the proposed inhibition mechanism using model systems
based on the own n-layered integrated molecular orbital and molecular
mechanics (ONIOM) method. In the first place, model systems were formulated
to investigate the transesterification between the catalytic serine
residue, Ser-232, and the methyl ester of 4-(indolin-1-yl)-4-oxobutanoate.
In the second place, the hydrolysis mechanism of the resultant enzyme–inhibitor
adduct was studied. The energetics of these steps were analyzed using
a density functional theory functional in the ONIOM method. In addition,
the roles of active-site residues during these steps were highlighted.
It was found that the hydrolysis of the covalent adduct is highly
endergonic corroborating the irreversible inhibition mechanism. These
results will shed light not only on the inhibition mechanism but also
on the catalytic mechanism.