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Kamakshi Sishtla, San-ha Lee, Jung-Eun Lee, Seung-Yong Seo, Timothy W Corson; Discovery of ferrochelatase inhibitors as antiangiogenic agents. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5405.
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© ARVO (1962-2015); The Authors (2016-present)
Pathological ocular angiogenesis underlies multiple blinding eye diseases. We previously identified and characterized the enzyme ferrochelatase as a mediator of ocular angiogenesis. Ferrochelatase (FECH) is responsible for ferrous ion insertion into protoporphyrin IX (PPIX) in the final, rate-limiting step in heme biosynthesis. FECH inhibition via chemical or genetic means resulted in loss of angiogenic potential in vitro and in vivo, making this enzyme an appealing therapeutic target. However, the classic FECH inhibitor N-methylprotoporphyrin (NMPP) is not particularly drug-like. Thus, we set out to identify novel small molecule inhibitors of FECH as potential therapeutic leads. Potent and selective FECH inhibitors may yield new therapeutic agents to combat ocular neovascularization.
FECH inhibition was tested via an enzymatic activity assay monitoring the increase of recombinant FECH-mediated Ni2+-mesoporphyrin IX production. Nearly 20,000 compounds from the ChemDiv and ChemBridge libraries were assessed in a high-throughput screen to judge their effect at 10 μM on FECH enzymatic activity, with subsequent dose-response testing. Compounds were tested for effects on angiogenesis in vitro in ocular endothelial cells, monitoring proliferation using an alamarBlue assay, tube formation in a Matrigel assay, and migration in a scratch-wound assay.
Of 20,000 compounds screened, 93 (0.46%) decreased FECH activity by more than 50%. Intriguingly, 62 of these had a triazolopyrimidinone skeleton. Of these, 22 underwent IC50 determination and 12 more triazolopyrimidinones were synthesized to study structure-activity relationships. Four potent compounds were chosen for further study based on FECH IC50 < 1 µM. All were antiangiogenic with GI50 in human retinal endothelial cells (HRECs) in the mid-micromolar range and inhibition of tubule formation in HRECs in the same range.
The four compounds studied for anti-angiogenic effects performed similarly to known FECH inhibitor NMPP in proliferation and inhibition of tubule formation. Further SAR may yield even more potent compounds. Small molecules may offer the possibility of therapeutic delivery in eyedrop form, superior to existing antiangiogenic biologics that necessitate intravitreal delivery. FECH small molecule inhibitors may prove a powerful tool in the treatment of ocular neovascularization.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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