Charles_2025_Redox.Biol_89_103974

The lipid electrophile nitro-oleic acid (NO(2)-OA) and inhibitors of soluble epoxide hydrolase (sEH) limit injury during myocardial ischemia and reperfusion (IR). We investigated if cardioprotection by NO(2)-OA was mediated by inhibitory adduction of this electrophile to C521 of the hydrolase. Indeed, administering NO(2)-OA to wild type (WT) isolated perfused hearts prior to IR limited infarction, but this protection was absent in C521S sEH knock-in (KI) mice - demonstrating the critical importance of this cysteine. To identify more potent and selective inhibitors, we screened a library of electrophiles for their ability to inhibit sEH. A compound, we termed RLC14, had an IC(50) of 6.8x10(-9) M and protected WT, but not KI, isolated hearts from infarction during IR. Systemic administration of RLC14 decreased myocardial sEH activity and increased plasma EET/DHET ratio selectively in WT mice, consistent with inhibition of the hydrolase. In line with these findings, RLC14 protected WT, but not KI, mice from in vivo coronary artery ligation IR-induced infarction. Mass spectrometry analyses showed the novel electrophilic inhibitor, RLC14, which contains a disulfide, adducts to C521 in sEH to mediate its effects. This study identifies RLC14 as a potent cardioprotective agent that limits IR injury through C521-dependent hydrolase inhibition.