Isolation of a small molecule inhibitor of DNA base excision repair

Madhusudan, Srinivasan; Smart, Fiona; Shrimpton, Paul; Parsons, Jason L.; Gardiner, Laurence; Houlbrook, S.; Talbot, Denis; Hammonds, Timothy R.; Freemont, Paul A.; Sternberg, Michael J.E.; Dianov, Grigory L.; Hickson, Ian D.

doi:10.1093/nar/gki781

Cited by 209 publications

(217 citation statements)

References 44 publications

(52 reference statements)

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“…Several small molecules have previously been identified as potential inhibitors of APE1's AP endonuclease activity (42,43,44). Cytotoxicity of these molecules has been evaluated by clonogenic survival, which does not illuminate the mechanism by which the molecules are cytotoxic.…”

Section: Resultsmentioning

confidence: 99%

Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

240

284

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With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24-and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.base excision repair | comet assay | DNA repair D NA damage is a critical risk factor for cancer, aging, and heritable diseases (1-3), and it is the underlying basis for most frontline cancer therapies (4). Increased knowledge about DNA damage and repair would facilitate disease prevention, identification of individuals at increased risk of cancer, discovery of novel therapeutics, and better drug safety testing. Despite their obvious translational impact, most DNA damage assays have not changed significantly in more than two decades, and thus are not compatible with modern high throughput screening technologies. To better assess the biological impact of damage, we need data that reflect the integrated effect of multiple DNA repair pathways and subpathways, in response to a range of DNA lesions, measured in an accurate and high throughput fashion.The single cell gel electrophoresis or "comet" assay is based on the principle that relaxed loops (induced by single strand breaks) and DNA fragments migrate farther in an agarose gel than undamaged DNA (5-8). The alkali comet assay sensitively detects a range of DNA lesions, including strand breaks (single and double), as well as alkali sensitive sites. Although most base lesions are not directly detected, base adducts can be detected when converted to abasic sites or single strand breaks with the addition of purified DNA repair enzymes (7-11). The comet assay has been used in a variety of applications, including genotoxicity testing, human biomonitoring and epidemiology, environmental healt...

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Section: Resultsmentioning

confidence: 99%

Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

240

284

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“…The ''low affinity'' attribute may stem from the fact that small molecules are unable to replicate the ''high affinity'' binding contacts seen in the APE1/AP-DNA complex, which spans roughly 7-8 bp in length. Indeed, in an extensive structure-function activity relationship study, the introduced modifications only slightly increased the effectiveness of the analogs against (7,131,142,167), confirming that the nuclease function(s) of the protein plays a critical role in repairing alkylative DNA damage, presumably abasic sites. Future studies will need to examine the utility of the most promising compounds in both combinatorial and synthetic lethal treatment paradigms, and may need to employ covalently linked, small molecules that bind simultaneously multiple functional sites of APE1.…”

Section: Small-molecule Inhibitorsmentioning

confidence: 88%

Human Apurinic/Apyrimidinic Endonuclease 1

2014

Antioxidants & Redox Signaling

224

221

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Significance: Human apurinic/apyrimidinic endonuclease 1 (APE1, also known as REF-1) was isolated based on its ability to cleave at AP sites in DNA or activate the DNA binding activity of certain transcription factors. We review herein topics related to this multi-functional DNA repair and stress-response protein. Recent Advances: APE1 displays homology to Escherichia coli exonuclease III and is a member of the divalent metal-dependent a/b fold-containing phosphoesterase superfamily of enzymes. APE1 has acquired distinct active site and loop elements that dictate substrate selectivity, and a unique N-terminus which at minimum imparts nuclear targeting and interaction specificity. Additional activities ascribed to APE1 include 3¢-5¢ exonuclease, 3¢-repair diesterase, nucleotide incision repair, damaged or site-specific RNA cleavage, and multiple transcription regulatory roles. Critical Issues: APE1 is essential for mouse embryogenesis and contributes to cell viability in a genetic background-dependent manner. Haploinsufficient APE1 +/ -mice exhibit reduced survival, increased cancer formation, and cellular/tissue hyper-sensitivity to oxidative stress, supporting the notion that impaired APE1 function associates with disease susceptibility. Although abnormal APE1 expression/localization has been seen in cancer and neuropathologies, and impaired-function variants have been described, a causal link between an APE1 defect and human disease remains elusive. Future Directions: Ongoing efforts aim at delineating the biological role(s) of the different APE1 activities, as well as the regulatory mechanisms for its intra-cellular distribution and participation in diverse molecular pathways. The determination of whether APE1 defects contribute to human disease, particularly pathologies that involve oxidative stress, and whether APE1 small-molecule regulators have clinical utility, is central to future investigations. Antioxid. Redox Signal. 20,

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“…[17][18][19][20][21][22][23][24]. These findings reveal a prominent role for APE1 in the repair of specific oxidative, alkylation, and enzymatic DNA intermediates, and identify this protein as a potential target for certain therapeutic paradigms.…”

Section: Introductionmentioning

confidence: 99%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

109

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Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3 ¶ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu 96 (changed to Gln) and Asp 210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue B-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.

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Isolation of a small molecule inhibitor of DNA base excision repair

Cited by 209 publications

References 44 publications

Single cell trapping and DNA damage analysis using microwell arrays

Single cell trapping and DNA damage analysis using microwell arrays

Human Apurinic/Apyrimidinic Endonuclease 1

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

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