Indiana University-Purdue University Indianapolis (IUPUI) / Choroidal neovascularization (CNV) is the vision-threatening characteristic of wet age-related macular degeneration (AMD), a major cause of blindness affecting almost 2 million elderly Americans. The current approved treatments target the dominant angiogenic mediator, vascular endothelial growth factor (VEGF). However, repeated injections of anti-VEGF drugs can cause ocular and systemic side effects, and about 30% of wet AMD patients are non-responsive. There is thus an unmet need to develop VEGF-independent antiangiogenic molecules to complement or combine with existing medications. I studied SH-11037, a novel homoisoflavonoid with potent and selective antiangiogenic activity against human retinal endothelial cells. Intravitreal SH- 11037 dose-dependently suppressed angiogenesis in the laser-induced CNV (LCNV) mouse model. These effects were prominent as early as 7 days post-laser treatment as measured by a novel ellipsoid quantification method of optical coherence tomography images in vivo. A supratherapeutic dose of 100 μM SH- 11037 was not associated with signs of murine ocular toxicity, and did not interfere with pre-existing retinal vasculature or retinal function. SH-11037 synergized with anti-VEGF therapy in vitro and in vivo, suggesting a VEGFindependent mechanism. By photoaffinity pulldown, I identified soluble epoxide hydrolase (sEH) as an SH-11037-binding target. sEH is a key enzyme in ω-3 and ω-6 fatty acid metabolism. sEH levels were dramatically upregulated in retinal sections from L-CNV mice and a specific sEH inhibitor, t-AUCB, significantly suppressed L-CNV lesion volume. Additionally, SH-11037 inhibited sEH enzymatic activity in vitro and in vivo in L-CNV mice. Given the role of sEH in the metabolism of docosahexaenoic acids (DHA), inhibition of sEH using small molecules like SH-11037 would enhance ocular DHA levels, with beneficial antiangiogenic and anti-inflammatory effects. SH-11037 is thus a novel sEH inhibitor, which could make it an alternative or additive therapy to existing anti- VEGF drugs for treatment of neovascular diseases in the eye and other tissues.

Indiana University-Purdue University Indianapolis (IUPUI) / Prevention of pathological angiogenesis is a key strategy for treatment of common blinding ocular diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The current treatment strategies are associated with partial vision loss and are ineffective in a significant patient population. Hence novel drugs as well as new ways to target ocular angiogenesis are needed for treating these diseases. I pursued a natural antiangiogenic compound, cremastranone, to develop novel drug leads and to find new targets. The objective of my doctoral thesis project was to elucidate cremastranone’s molecular mechanism of action and optimize its structureactivity relationship (SAR). In order to achieve this goal, with the help of chemistry collaborators cremastranone was synthesized for the first time. I showed that cremastranone has 50-fold more potency against endothelial cells as compared to nonendothelial cells, and also tested a novel active isomer, SH-11052. By SAR studies I identified a potent molecule, SH-11037, that has 10-fold more selectivity against retinal endothelial cells as compared to macrovascular endothelial cells. I then elucidated cremastranone’s molecular mechanism using a chemical proteomic approach. I identified ferrochelatase (FECH) as a specific interacting protein partner of cremastranone using photoaffinity chromatography. Hence, I hypothesized that cremastranone exerts its antiangiogenic activities through modulation of the functions of FECH. Cremastranone inhibited the enzymatic activity FECH in endothelial cells. Therefore, I investigated the role of FECH in ocular angiogenesis. Partial loss of FECH, using a siRNA-based knock down approach, decreased retinal angiogenesis both in vitro and in vivo in mouse models. Knock down of FECH decreased the expression levels of key proangiogenic proteins HIF-1α, eNOS, and VEGFR2. This work suggests that ferrochelatase plays an important, previously undocumented role in angiogenesis and that targeting of this enzyme by cremastranone might be exploited to inhibit pathological angiogenesis in ocular diseases.