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Ref-1/APE1 inhibition with novel small molecules blocks ocular neovascularization

Sardar Pasha Sheik Pran Babu, View ORCID ProfileKamakshi Sishtla, Rania S. Sulaiman, View ORCID ProfileBomina Park, Trupti Shetty, Fenil Shah, View ORCID ProfileMelissa L. Fishel, James H. Wikel, View ORCID ProfileMark R. Kelley, Timothy W. Corson
doi: https://doi.org/10.1101/296590
Sardar Pasha Sheik Pran Babu
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
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Kamakshi Sishtla
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
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Rania S. Sulaiman
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
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Bomina Park
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
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Trupti Shetty
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
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Fenil Shah
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
4Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana 46202
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Melissa L. Fishel
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
4Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana 46202
5Melvin and Bren Simon Cancer Center, Indiana 46202
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James H. Wikel
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
6Indiana University School of Medicine, Indianapolis, Indiana 46202
7Apexian Pharmaceuticals Indianapolis, Indiana 46202
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Mark R. Kelley
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
3Department of Biochemistry and Molecular Biology, Indianapolis, Indiana 46202
4Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana 46202
5Melvin and Bren Simon Cancer Center, Indiana 46202
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Timothy W. Corson
1Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indianapolis, Indiana 46202
2Department of Pharmacology and Toxicology Indianapolis, Indiana 46202
3Department of Biochemistry and Molecular Biology, Indianapolis, Indiana 46202
5Melvin and Bren Simon Cancer Center, Indiana 46202
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Abstract

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 NF-κB and other pro-angiogenic 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 to 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 to 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, both 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 to 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 CNV in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.

Abbreviations
AMD
age-related macular degeneration
ANOVA
analysis of variance
AP-1
activator protein 1
APE1
apurinic/apyrimidinic endonuclease 1
APX2009
(E)-N,N-diethyl-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide
APX2014
(E)-N-methoxy-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide
APX3330
(2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene]-undecanoic acid
ATCC
American Type Culture Collection
BSA
bovine serum albumin
CNV
choroidal neovascularization
DAPI
4′,6-diamidine-2′-phenylindole
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
dimethyl sulfoxide
EBM-2
endothelial basal medium 2
EdU
5-ethynyl-2′-deoxyuridine
EGM-2
endothelial growth medium 2
EMSA
electrophoretic mobility shift assay
FDA
Food and Drug Administration
FA
fluorescein angiography
GCL
ganglion cell layer
GI50
median growth inhibitory concentration
GS-IB4
isolectin B4 from Griffonia simplicifolia
HIF-1α
hypoxia inducible factor 1α
HRECs
human retinal microvascular endothelial cells
INL
inner nuclear layer
L-CNV
laser-induced choroidal neovascularization
NF-κB
nuclear factor κ light-chain-enhancer of activated B cells
OCT
optical coherence tomography
ONL
outer nuclear layer
PBS
phosphate buffered saline
PDR
proliferative diabetic retinopathy
PKT
propylene glycol
Kolliphor HS15
Tween 80
Ref-1
reduction-oxidation factor 1
qRT-PCR
quantitative reverse transcription-polymerase chain reaction
ROP
retinopathy of prematurity
RP2D
recommended Phase II dose
RPE
retinal pigment epithelium
STAT3
signal transducer and activator of transcription 3
TUNEL
terminal deoxynucleotidyl transferase dUTP nick end labeling
VEGF
vascular endothelial growth factor
VLDLR
very low density lipoprotein receptor
Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 23, 2018.
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Ref-1/APE1 inhibition with novel small molecules blocks ocular neovascularization
Sardar Pasha Sheik Pran Babu, Kamakshi Sishtla, Rania S. Sulaiman, Bomina Park, Trupti Shetty, Fenil Shah, Melissa L. Fishel, James H. Wikel, Mark R. Kelley, Timothy W. Corson
bioRxiv 296590; doi: https://doi.org/10.1101/296590
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Ref-1/APE1 inhibition with novel small molecules blocks ocular neovascularization
Sardar Pasha Sheik Pran Babu, Kamakshi Sishtla, Rania S. Sulaiman, Bomina Park, Trupti Shetty, Fenil Shah, Melissa L. Fishel, James H. Wikel, Mark R. Kelley, Timothy W. Corson
bioRxiv 296590; doi: https://doi.org/10.1101/296590

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