Characterization of a Novel Inhibitor of Soluble Epoxide Hydrolase and Role in Ocular Neovascularization

Abstract

ObjectiveOcular neovascularization causes blindness in diseases such as proliferative diabetic retinopathy (PDR) and wet age‐related macular degeneration (AMD). Today, the effort to treat wet AMD is hampered by patients who are resistant and refractory to the current standard of care, anti‐vascular endothelial growth factor (VEGF) therapies. Therefore, identification of novel therapeutic targets and inhibitors is desirable to address the unmet needs in antiangiogenic treatment. Previously, we have identified soluble epoxide hydrolase (sEH) as a binding target of an antiangiogenic small molecule, SH‐11037. Here, we aimed to further investigate sEH expression in the eye and the inhibition of its activity by SH‐11037.MethodsThe expression and localization of sEH in retinal sections of laser‐induced choroidal neovascularization (L‐CNV) mice were evaluated by immunohistochemistry. The sEH enzymatic activity in ocular tissue homogenates of L‐CNV and control mice was assayed using trans‐stilbene oxide (t‐SO) as substrate. The sEH inhibition by SH‐11037 and mode of inhibition was assessed by recombinant sEH activity assay using a fluorogenic substrate, PHOME. Docking and molecular dynamics simulations were performed to predict the binding mode of SH‐11037 within the active site of sEH. In addition, intravitreal injections of sEH substrate 19,20‐epoxy docosapentaenoic acid (EDP) and product 19,20‐dihydroxy docosapentaenoic acids (DHDP) were given to L‐CNV mice, and effects were assessed by imaging and choroidal flatmount immunofluorescence.ResultssEH was upregulated in the rod photoreceptors in L‐CNV mice compared to control mice and it did not co‐localize with other retinal cell type markers. Correspondingly, sEH activity was increased in L‐CNV eyes and was normalized by SH‐11037 or a known sEH inhibitor. In a recombinant enzyme assay, SH‐11037 inhibited sEH activity in a dose‐dependent manner, with IC50 = 0.15 μM. Enzyme kinetics analysis demonstrated that increasing concentrations of SH‐11037 decreased Vmax and increased KM, revealing SH‐11037 as a mixed‐type inhibitor of sEH, with Ki = 1.73 ± 0.45 μM. Docking of SH‐11037 to sEH showed an energetically favorable binding mode with the ligand spanning the active site of the enzyme and interacting with key catalytic residues. Intravitreal 19,20‐EDP reduced CNV lesion volume compared to vehicle and 19,20‐DHDP, confirming antiangiogenic properties of this sEH substrate.ConclusionOur studies confirm the relevance of sEH for ocular neovascularization and reveal the mechanism for SH‐11037's inhibition of sEH. Further optimization of SH‐11037's structure could yield more potent, novel sEH inhibitors.Support or Funding InformationNIH/NEI R01EY025641, Retina Research Foundation, Research to Prevent Blindness, Inc.