Soluble Epoxide Hydrolase Inhibition for Ocular Diseases: Vision for the Future

Ocular diseases cause visual impairment and blindness, imposing a devastating impact on quality of life and a substantial societal economic burden. Many such diseases lack universally effective pharmacotherapies. Therefore, understanding the mediators involved in their pathophysiology is necessary for the development of therapeutic strategies. To this end, the hydrolase activity of soluble epoxide hydrolase (sEH) has been explored in the context of several eye diseases, due to its implications in vascular diseases through metabolism of bioactive epoxygenated fatty acids. In this mini-review, we discuss the mounting evidence associating sEH with ocular diseases and its therapeutic value as a target. Substantial data link sEH with the retinal and choroidal neovascularization underlying diseases such as wet age-related macular degeneration, retinopathy of prematurity, and proliferative diabetic retinopathy, although some conflicting results pose challenges for the synthesis of a common mechanism. sEH also shows therapeutic relevance in non-proliferative diabetic retinopathy and diabetic keratopathy, and sEH inhibition has been tested in a uveitis model. Various approaches have been implemented to assess sEH function in the eye, including expression analyses, genetic manipulation, pharmacological targeting of sEH, and modulation of certain lipid metabolites that are upstream and downstream of sEH. On balance, sEH inhibition shows considerable promise for treating multiple eye diseases. The possibility of local delivery of inhibitors makes the eye an appealing target for future sEH drug development initiatives

Pharmacological Potential of Small Molecules for Treating Corneal Neovascularization

Under healthy conditions, the cornea is an avascular structure which allows for transparency and optimal visual acuity. Its avascular nature is maintained by a balance of proangiogenic and antiangiogenic factors. An imbalance of these factors can result in abnormal blood vessel proliferation into the cornea. This corneal neovascularization (CoNV) can stem from a variety of insults including hypoxia and ocular surface inflammation caused by trauma, infection, chemical burns, and immunological diseases. CoNV threatens corneal transparency, resulting in permanent vision loss. Mainstay treatments of CoNV have partial efficacy and associated side effects, revealing the need for novel treatments. Numerous natural products and synthetic small molecules have shown potential in preclinical studies in vivo as antiangiogenic therapies for CoNV. Such small molecules include synthetic inhibitors of the vascular endothelial growth factor (VEGF) receptor and other tyrosine kinases, plus repurposed antimicrobials, as well as natural source-derived flavonoid and non-flavonoid phytochemicals, immunosuppressants, vitamins, and histone deacetylase inhibitors. They induce antiangiogenic and anti-inflammatory effects through inhibition of VEGF, NF-κB, and other growth factor receptor pathways. Here, we review the potential of small molecules, both synthetics and natural products, targeting these and other molecular mechanisms, as antiangiogenic agents in the treatment of CoNV

Measurement of mitochondrial respiration in the murine retina using a Seahorse extracellular flux analyzer

Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models

Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction

The relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis

Ref-1/APE1 Inhibition with Novel Small Molecules Blocks Ocular Neovascularization

Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1–apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-κB and other proangiogenic transcription factors. An existing inhibitor of Ref-1’s function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 μM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI50 APX2009: 26 μM, APX2014: 5.0 μM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-κB activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle (P < 0.0001, ANOVA with Dunnett’s post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization

Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound’s mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD

Small-molecule inhibitors of ferrochelatase are antiangiogenic agents

Activity of the heme synthesis enzyme ferrochelatase (FECH) is implicated in multiple diseases. In particular, it is a mediator of neovascularization in the eye and thus an appealing therapeutic target for preventing blindness. However, no drug-like direct FECH inhibitors are known. Here, we set out to identify small-molecule inhibitors of FECH as potential therapeutic leads using a high-throughput screening approach to identify potent inhibitors of FECH activity. A structure-activity relationship study of a class of triazolopyrimidinone hits yielded drug-like FECH inhibitors. These compounds inhibit FECH in cells, bind the active site in cocrystal structures, and are antiangiogenic in multiple in vitro assays. One of these promising compounds was antiangiogenic in vivo in a mouse model of choroidal neovascularization. This foundational work may be the basis for new therapeutic agents to combat not only ocular neovascularization but also other diseases characterized by FECH activity

Targeting Soluble Epoxide Hydrolase to Treat Choroidal Neovascularization

Indiana University-Purdue University Indianapolis (IUPUI)Neovascular or “wet” age-related macular degeneration (nvAMD) is a leading cause of blindness among older adults, affecting millions of people worldwide. Choroidal neovascularization (CNV) is a major pathological feature of nvAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible loss of vision. Currently, the effort to treat nvAMD is hampered by resistance and refractory responses to the current standard of anti-angiogenic care, anti-vascular endothelial growth factor biologics. Thus, there is a critical need to develop novel therapeutic strategies. Previously, we discovered an anti-angiogenic small molecule SH-11037, and identified soluble epoxide hydrolase (sEH) as a target of SH-11037 through a forward chemical genetics approach. sEH, encoded by the EPHX2 gene, is a lipid-metabolizing enzyme that hydrolyzes epoxy fatty acids into corresponding diols. I hypothesized that sEH is a key mediator of CNV. Given that the kinetic mechanism of sEH inhibition by SH-11037 and the cellular role of sEH in CNV are poorly understood, the objectives of my thesis project were to elucidate drug-target interactions through enzyme kinetics, investigate sEH mediated mechanisms that regulate CNV, and preclinically validate sEH as a therapeutic target. I discovered that SH-11037 is a mixed inhibitor of sEH with a binding affinity for both the enzyme and enzyme-substrate complex. I examined retinal spatial expression of sEH at both the protein and mRNA levels through immunohistochemistry and RNAscope in situ hybridization and investigated the efficacy of adeno-associated virus (AAV) serotype 8 vector expressing shRNA against Ephx2, in the mouse laser-induced (L-) CNV model with features of nvAMD. My study revealed sEH protein and mRNA overexpression in the retinal pigment epithelium (RPE), vasculature and photoreceptors under the disease state. The delivery of AAV8-Ephx2 shRNA, which has tropism towards RPE and photoreceptor cells, significantly reduced CNV. In addition, gene expression analysis showed normalized Vegfc and CNV-related inflammatory markers upon sEH knockdown. Thus, my study demonstrated sEH overexpression in disease-relevant cell types, highlighted a functional role of sEH in AMD pathophysiology, and provided a novel context to target these cell types for developing pharmacotherapies

Towards understanding the role of organic phosphate in diabetes mellitus

The goal of this research is to understand the role of organic phosphates in the initial step of the nonenzymatic glycation process which is related to diabetes mellitus. In protein glycation, the initially bound ring closed glucose must ring open while bound to generate an electrophile that can react with a nucleophilic lysine or an N-terminal residue to form a covalently-bound Schiff base. The Schiff base can proceed to an Amadori intermediate, which then is able to generate advanced glycation end products. This results in a structurally-modified protein that may induce pathology. Glucose-6-phosphate (G6P) and 2,3-bisphosphoglycerate (BPG) are organic phosphates that are glucose metabolites produced in erythrocytes by glycolysis. They are known to accelerate the glycation by facilitating the ring opening of glucose and bind to known glycation sites. Computational modeling and nuclear magnetic resonance (NMR) spectroscopy were used to assess pre-Amadori mechanistic possibilities of organic Pi with implications for model proteins. In addition, purified bovine hemoglobin and albumin were incubated with G6P and BPG to advance understanding under physiological conditions

Calcitriol and non-calcemic vitamin D analogue, 22-oxacalcitriol, attenuate developmental and pathological choroidal vasculature angiogenesis ex vivo and in vivo

Aberrant ocular angiogenesis can underpin vision loss in leading causes of blindness, including neovascular age-related macular degeneration and proliferative diabetic retinopathy. Current pharmacological interventions require repeated invasive administrations, may lack efficacy and are associated with poor patient compliance and tachyphylaxis. Vitamin D has de novo anti-angiogenic properties. Here, our aim was to validate the ocular anti-angiogenic activity of biologically active vitamin D, calcitriol, and selected vitamin D analogue, 22-oxacalcitriol. Calcitriol induced a significant reduction in ex vivo mouse choroidal fragment sprouting. Viability studies in a human RPE cell line suggested non-calcemic vitamin D analogues including 22-oxacalcitriol have less off-target anti-proliferative activity compared to calcitriol and other analogues. Thereafter, the anti-angiogenic activity of 22-oxacalcitriol was demonstrated in an ex vivo mouse choroidal fragment sprouting assay. In zebrafish larvae, 22-oxacalcitriol was found to be anti-angiogenic, inducing a dose-dependent reduction in choriocapillaris development. Subcutaneously administered calcitriol failed to attenuate mouse retinal vasculature development. However, calcitriol and 22-oxacalcitriol administered intraperitoneally, significantly attenuated lesion volume in the laser-induced choroidal neovascularisation mouse model. In summary, calcitriol and 22-oxacalcitriol attenuate ex vivo and in vivo choroidal vasculature angiogenesis. Therefore, vitamin D may have potential as an interventional treatment for ophthalmic neovascular indications.Funding agencies: Health Research Board, HRB-POR-2013-390, Irish Research Council, GOIPG/2015/2061, NIH/NEI R01EY025641, Retina Research Foundation, The foundation Jeanssons Stiftelser, The Magnus Bergvall foundation, and The Swedish Eye foundation.</p