Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein–DNA Interaction

Gavande, Navnath; VanderVere-Carozza, Pamela S.; Mishra, Akaash K.; Vernon, Tyler; Pawelczak, Katherine S.; Turchi, John J.

doi:10.1021/acs.jmedchem.7b00780

Cited by 12 publications

(13 citation statements)

References 66 publications

(176 reference statements)

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“…To confirm the mechanism of inhibitions via compound binding to the target protein and not via binding to the DNA substrates, we conducted a fluorescent intercalator displacement (FID) assay as described previously. 32 The displacement of a DNA binding dye and decrease in fluorescence is indicative of the compound's Figure 2 demonstrate as expected that positive control, doxorubicin a known non-covalent DNA binding chemotherapeutic, resulted in a concentration dependent reduction in fluorescence. As expected, minimal DNA binding activity was observed for any of the novel RPA inhibitors (19, 23, 26, 27, 43 and 45).…”

supporting

confidence: 55%

Structure-Guided Optimization of Replication Protein A (RPA)–DNA Interaction Inhibitors

Gavande

VanderVere-Carozza

Pawelczak

et al. 2020

ACS Med. Chem. Lett.

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Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination and the DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility and excellent cellular uptake. Among a series of analogues, compound 43, 44, 45 and 46 hold promise for further development of novel anti-cancer agents.

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supporting

confidence: 55%

Structure-Guided Optimization of Replication Protein A (RPA)–DNA Interaction Inhibitors

Gavande

VanderVere-Carozza

Pawelczak

et al. 2020

ACS Med. Chem. Lett.

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“…The analysis of DNA binding of the Pt-PyED compound was performed as described previously (16,17). Briefly, a competitive DNA intercalation assay was performed using SYBR Green I (Sigma-Aldrich, St. Louis, MO) and sonicated salmon sperm DNA (8.3 ng/μl)(Fisher) in 25 mM MOPS (pH 6.5).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells

Keller¹,

Porter

Garrett³

et al. 2018

Radiation Research

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Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

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“…While the development of X80 derivatives focused on optimizing direct interactions with the Ku proteins, the mechanism could be a function of the compounds binding to DNA and inhibiting the binding of Ku. We first assessed binding to DNA using a fluorescence displacement assay (FDA) as we have described for the previous X80 derivatives ( 21 ). Similar to previous results, the data obtained with the newer derivatives show minimal direct DNA interaction up to concentrations 10× the calculated IC 50 ’s for DNA-PK kinase activity ( Supplementary Figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

“…Reactions were incubated for 30 min at 25°C and products separated by electrophoresis on a 6% non-denaturing polyacrylamide gel. Gels were imaged using a PhosphorImager and quantified by ImageQuant (Molecular Dynamics) as we have described previously ( 20 , 21 ).…”

Section: Methodsmentioning

confidence: 99%

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku–DNA interaction

Gavande

VanderVere-Carozza

Pawelczak

et al. 2020

Nucleic Acids Research

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DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku–DNA interaction. We have developed a series of highly potent and specific Ku–DNA binding inhibitors (Ku-DBi’s) that block the Ku–DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi’s directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi’s cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi’s. The utility of Ku-DBi’s was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi’s, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

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Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein–DNA Interaction

Cited by 12 publications

References 66 publications

Structure-Guided Optimization of Replication Protein A (RPA)–DNA Interaction Inhibitors

Structure-Guided Optimization of Replication Protein A (RPA)–DNA Interaction Inhibitors

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku–DNA interaction

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