Thromboxane A2 synthase is an off target of the CES1 small-molecule inhibitor WWL113

Thromboxane A2 (TxA2) is an inflammatory lipid mediator released by blood platelets and monocytes/macrophages. TxA2 is unstable (half-life ∼1 min), but it induces platelet aggregation and vasoconstriction of arteries contributing to Cardiovascular Disease. Therefore, inhibiting thromboxane biosynthesis with pharmacological inhibitors may help to limit ischemic events. Carboxylesterase 1 (CES1) is a serine hydrolase with roles in xenobiotic and lipid metabolism. CES1 activity can be perturbed in biological systems with small-molecule inhibitors that covalently modify its active site serine residue. We surprisingly discovered that a CES1 Inhibitor, WWL113, could also inhibit the activity of thromboxane A2 synthase (TBXAS1), which is responsible for converting prostaglandin H2 (PGH2) to TxA2. TxA2 is non-enzymatically converted to a stable inactive metabolite, TxB2, which can be measured by LC-MS/MS. Human monocytic cells (THP-1 cell line), which naturally express TBXAS1 and CES1, were pretreated for 30 min with increasing concentrations of either WWL113 or WWL229 (another CES1 Inhibitor), followed by addition of exogenous PGH2 and the levels of TxB2 and PGE2 determined. WWL113 significantly decreased TxB2 levels, whereas prostaglandin E2 (PGE2) levels were increased. The concentration of WWL113 that inhibited TxB2 production by 50% (IC₅₀) in THP-1 monocyte lysates and intact living macrophages was ∼0.1-0.2 µM. In contrast, WWL229 had no effect on the amounts of either lipid mediator in living cells and lysates. Recombinant human TBXAS1 protein was overexpressed in COS-7 cells and WWL113 was verified to be a bona fide TBXAS1 inhibitor (IC₅₀=226 nM). These findings indicate that WWL113, which inhibits CES1 activity and exerts anti-inflammatory effects in vitro and in vivo, can also target TBXAS1. Thus, the beneficial effects of WWL113 observed in diet-induced obese mice may in part be related to its ability to block proinflammatory TxA2 production. It is, therefore, recommended that WWL229 be used instead of WWL113 to perturb CES1 activity in living cells and animal models.

Carboxylesterase 1; Macrophages; Monocytes; Serine hydrolase; TBXAS1; WWL113; WWL229.