Abstract
During the past decade, the thrombolytic enzyme tissue plasminogen activator (t-PA)-based treatment has been the standard therapy for acute ischemic stroke. However, due to its hemorrhagic risk and narrow therapeutic time window (TTW), only limited patients benefit from t-PA-based therapy, and the development of an alternative therapeutic agent is urgently needed. Reducing inflammation within the infarction area to rescue penumbra is particularly important. SMTP-7 is a small molecule that enhances plasminogen activation by modulating plasminogen conformation. SMTP-7 promotes plasmin formation and clot clearance in vivo and it is effective in treating thrombotic and embolic strokes in experimental models in rodents and a nonhuman primate. Unexpectedly, SMTP-7 reduces hemorrhagic transformation and has extended TTW as compared with t-PA. The distinct effects of SMTP-7 are partly explained by suppression of inflammatory responses following thrombolytic reperfusion, unlike t-PA. Experiments with animal inflammatory disease models (ulcerative colitis, Crohn's disease, and Guillain-Barré syndrome models) suggest that the anti-inflammatory action of SMTP-7 is independent of thrombolytic activity, as a thrombolytically inactive congener, SMTP-44D, exhibits anti-inflammatory action in those models. In this study, we searched for anti-inflammatory target of SMTP and found soluble epoxide hydrolase (sEH) as a possible candidate.
We searched for a target protein using an SMTP-conjugated affinity matrix, which was synthesized by coupling SMTP-50, a congener with a primary amino group on the side chain, with gel beads. Mouse liver homogenates were subjected to affinity chromatography on this matrix, and specifically bound proteins were analyzed by peptide mass fingerprint. As a result, 4 major bound proteins were assigned to full length or fragments of soluble epoxide hydrolase (sEH), a hybrid enzyme with epoxide hydrolase activity in the C-terminal domain and lipid phosphatase activity in the N-terminal domain. The sEH hydrolase converts epoxy fatty acids, such as epoxyeicosatrienoic acids (EETs) which are endogenous anti-inflammatory lipid mediators, to less-active diol forms, such as dihydroeicosatrienoic acids (DHETs). The sEH phosphatase is implicated in lipid metabolism and hydrolysis of lysophospatidic acid, whereas its precise biological role is still unclear. SMTP-7 and SMTP-44D inhibited both hydrolase (IC50 20 and 27 µM, respectively) and phosphatase (IC50 6 and 25 µM, respectively) activities of sEH. The simplest congener SMTP-0 (IC50 28 µM for hydrolase and 29 µM for phosphatase), which consists of only the core structure common with all the SMTP congeners, was used to analyze the kinetic mechanism of sEH inhibition. The inhibition of hydrolase by SMTP-0 was competitive with respect to 14,15-EET, and the inhibition of phosphatase is uncompetitive with respect to the synthetic substrate Attophos. The inhibition of phosphatase was unchanged in the presence of a potent competitive inhibitor of hydrolase, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid. Thus, SMTP-0 may bind to two distinct sites in sEH: one is the active site in the hydrolase domain, and the other is an allosteric site that affects the phosphatase domain. Inhibition of sEH hydrolase was also observed in cells in culture. The conversion of 14,15-EET to 14,15-DHET in HepG2 cells was inhibited by SMTP-7, SMTP-44D, and SMTP-0 with IC50 at 4.5, 8.8, and 1.3 mM, respectively. To confirm sEH inhibition in vivo, we traced the fate of intravenously injected EET in the liver. Treatment of wild-type mice with SMTP-7 significantly reduced the 14, 15-DHET level (∼41% reduction, P <0.05), while no significant reduction was observed in sEH KO mice. Thrombolytically inactive SMTP-44D reduced the degree of edema and pro-inflammatory cytokine expression in a mouse embolic stroke model, whereas infarct size and neurological deficits were not ameliorated. Thus, it is possible that both thrombolytic and anti-inflammatory potentials of SMTP are important in its excellent therapeutic activity.
Our present study provides evidence that SMTP-7 targets sEH for anti-inflammatory action. The inhibition of sEH and the profibrinolytic action due to plasminogen modulator activity may synergistically contribute to treatment of ischemic stroke. SMTP-7 is thus a promising alternative therapy for ischemic stroke.
Disclosures:
No relevant conflicts of interest to declare.
Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain
