Apurinic/apyrimidinic endonuclease (Ape1), an essential Base Excision Repair (BER) enzyme, has been shown to have elevated or altered levels of expression in a number of cancers.(1-8) In addition to its DMA repair activity, Ape1 has a redox function that serves to reduce a number of transcription factors including AP-1 (Fos/Jun), HIF-1a, NF-kB, PAX, HLF, p53, and others (reviewed in 1). Despite the discovery of Apel's redox activity more than 10 years ago, there is currently no detailed mechanism to describe how Ape1 reduces these transcription factors and conflicting data in the literature regarding the role of cysteine 65, thought to be critical for the redox function. We propose to revisit the mechanism of Apel's redox activity and its role in cancer etiology with the specific goal of exploring this unique and as yet unexplored activity as a target for the development of cancer therapeutics in the future. Our ability to detail the mechanism of Apel's redox activity and its role in cancer cells will be aided greatly by the use of 3-[5-(2,3-dimethoxy-6-methyl-1,4- benzoquinoyl)]-2-nonyl-2-propionic acid, (E3330), a quinone derivative, that has been shown (1) to bind specifically to Ape1 in a direct binding assay with high affinity and (2) to inhibit the redox activity of Ape1 both in vitro and in cancer cell lines. As detailed in Specific Aims 1 and 2, we have proposed to use X-ray crystallography, site-directed mutagenesis, chemical cross-linking, and hydrogen/deuterium exchange experiments in order to identify the residues that are required for Apel's redox activity and sites of interactions between Ape1 and the transcription factors that it reduces.
In Specific Aim 3, we propose to inhibit the redox activity of Ape1 with E3330 and thereby determine the role of Apel's redox activity on downstream targets in normal and cancer cell lines. Relevance: Apurinic/apyrimidinic endonuclease (Ape1) has been shown to have elevated or altered levels of expression in a number of cancers. This multifunctional enzyme is an attractive target for the development of chemotherapeutics. We propose here to elucidate the mechanism of the redox function of Ape1 and explore its role in tumor cell response following treatment with DNA damaging agents. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA114571-03
Application #
7424924
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Knowlton, John R
Project Start
2006-07-01
Project End
2011-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
3
Fiscal Year
2008
Total Cost
$233,196
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Zhang, Jun; Luo, Meihua; Marasco, Daniela et al. (2013) Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited. Biochemistry 52:2955-66
Kelley, Mark R; Georgiadis, Millie M; Fishel, Melissa L (2012) APE1/Ref-1 role in redox signaling: translational applications of targeting the redox function of the DNA repair/redox protein APE1/Ref-1. Curr Mol Pharmacol 5:36-53
Luo, Meihua; Zhang, Jun; He, Hongzhen et al. (2012) Characterization of the redox activity and disulfide bond formation in apurinic/apyrimidinic endonuclease. Biochemistry 51:695-705
Cardoso, Angelo A; Jiang, Yanlin; Luo, Meihua et al. (2012) APE1/Ref-1 regulates STAT3 transcriptional activity and APE1/Ref-1-STAT3 dual-targeting effectively inhibits pancreatic cancer cell survival. PLoS One 7:e47462
Kelley, Mark R; Luo, Meihua; Reed, April et al. (2011) Functional analysis of novel analogues of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1. Antioxid Redox Signal 14:1387-401
Fishel, Melissa L; Jiang, Yanlin; Rajeshkumar, N V et al. (2011) Impact of APE1/Ref-1 redox inhibition on pancreatic tumor growth. Mol Cancer Ther 10:1698-708
Su, Dian; Delaplane, Sarah; Luo, Meihua et al. (2011) Interactions of apurinic/apyrimidinic endonuclease with a redox inhibitor: evidence for an alternate conformation of the enzyme. Biochemistry 50:82-92
Jiang, Aihua; Gao, Hua; Kelley, Mark R et al. (2011) Inhibition of APE1/Ref-1 redox activity with APX3330 blocks retinal angiogenesis in vitro and in vivo. Vision Res 51:93-100
Vasko, Michael R; Guo, Chunlu; Thompson, Eric L et al. (2011) The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation. DNA Repair (Amst) 10:942-52
Vascotto, Carlo; Bisetto, Elena; Li, Mengxia et al. (2011) Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function. Mol Biol Cell 22:3887-901

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