Ferrochelatase as a mediator of ocular angiogenesis

University Of Toronto, Toronto ON

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Abstract

The neovascular eye diseases retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are major causes of blindness through the lifespan. Not all patients respond to existing therapies, so there is thus a critical need to find novel cellular components that could be targeted to block the pathological angiogenesis that is characteristic of these diseases. Ferrochelatase (FECH) is one such component, necessary for proliferation of endothelial cells in vitro and in vivo. FECH is responsible for inserting ferrous ion into protoporphyrin IX, the final step in heme biosynthesis. The previous grant period yielded findings that FECH is upregulated in murine and human choroidal and retinal neovascularization, that FECH inhibition leads to depletion of hemoproteins in endothelial cells, and that FECH loss blocks heme- dependent oxidative phosphorylation in endothelial cells. Reduced FECH activity also surprisingly blocks glycolysis. In addition, the first drug-like FECH inhibitor, SH-17023 was developed and shown to be antiangiogenic in vitro and in vivo. Building on this work, the current goal is to define the mechanism of how heme synthesis through FECH and glycolysis impacts endothelial cell biology and neovascularization. The rationale for this research is that FECH is a significant mediator of angiogenesis, a potential therapeutic target, and a previously unappreciated regulator of the expression and function of glycolytic enzymes. The overall hypothesis is that FECH, via controlling heme availability, is an integrated master regulator of multiple proangiogenic pathways, including glycolysis. Guided by strong preliminary data, the hypothesis will be tested via two specific aims: 1. Delineate the mechanism of FECH’s influence on glycolysis and angiogenesis. The glycolysis enzymes dysregulated by heme synthesis inhibition in endothelial cells and their heme-dependent transcriptional regulation will be assessed in endothelial cells. Glycolytic function and related metabolic pathways will be assessed by Seahorse, targeted metabolomics, and stable isotope tracing. The influence of this heme-dependent glycolysis regulation on endothelial cell function will be determined, along with the cell- type specificity of this effect. 2. Evaluate the first drug-like small molecule FECH inhibitor in ocular neovascularization. The pharmacokinetics and toxicity of SH-17023 will be quantified. Then, this novel molecule will be tested for efficacy in the oxygen-induced retinopathy, Vldlr-/-, and JR5558 retinal, subretinal, and choroidal neovascularization models, plus synergy with anti-VEGF therapy. Overall, this work is innovative, as it is the first mechanistic study of the links between heme synthesis, glycolysis, and posterior ocular angiogenesis, and the first characterization of direct FECH inhibition with a drug-like small molecule for retinal and choroidal neovascularization. The work is highly significant because it will comprehensively elucidate linkages between heme metabolism and the process of glycolysis, not previously connected, and establish FECH blockade as a viable therapy, leading to development of new ways to prevent blindness.