Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation

Targeting thymidylate
kinase (TMPK) that catalyzes the phosphotransfer
reaction for formation of dTDP from dTMP is a new strategy for anticancer
treatment. This study is to understand the inhibitory mechanism of
a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity
labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK
inhibitors that bear pyridino­[d]­isothiazolone or
benzo­[d]­isothiazolone core structure in lieu of the
dimethylpyridine-fused isothiazolone moiety in 1a can
have access to both the ATP-binding and catalytic sites. The binding
sites of hTMPK inhibitors were validated by photoaffinity labeling
and mass spectrometric studies. Taking together, 1a and
its analogues stabilize the conformation of ligand-induced degradation
(LID) region of hTMPK and block the catalytic site or ATP-binding
site, thus attenuating the ATP binding-induced closed conformation
that is required for phosphorylation of dTMP.