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Journal of the American Chemical Society Mar 2012A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined....
A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.
Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Microscopy, Fluorescence; Nitrogen Oxides; Oxidative Stress; Photons; Spectrophotometry, Ultraviolet
PubMed: 22380794
DOI: 10.1021/ja210315x -
European Journal of Medicinal Chemistry Sep 2017As bacterial biofilms display extreme tolerance to conventional antibiotic treatments, it has become imperative to develop new antibacterial strategies with alternative...
As bacterial biofilms display extreme tolerance to conventional antibiotic treatments, it has become imperative to develop new antibacterial strategies with alternative mechanisms of action. Herein, we report the synthesis of a series of ciprofloxacin-nitroxide conjugates and their corresponding methoxyamine derivatives in high yield. This was achieved by linking various nitroxides or methoxyamines to the secondary amine of the piperazine ring of ciprofloxacin using amide bond coupling. Biological evaluation of the prepared compounds on preformed P. aeruginosa biofilms in flow cells revealed substantial dispersal with ciprofloxacin-nitroxide hybrid 25, and virtually complete killing and removal (94%) of established biofilms in the presence of ciprofloxacin-nitroxide hybrid 27. Compounds 25-28 were shown to be non-toxic in both human embryonic kidney 293 (HEK 293) cells and human muscle rhabdomyosarcoma (RD) cells at concentrations up to 40 μM. Significantly, these hybrids demonstrate the potential of antimicrobial-nitroxide agents to overcome the resistance of biofilms to antimicrobials via stimulation of biofilm dispersal or through direct cell killing.
Topics: Anti-Bacterial Agents; Biofilms; Cell Line, Tumor; Ciprofloxacin; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Microbial Sensitivity Tests; Molecular Structure; Nitrogen Oxides; Pseudomonas aeruginosa; Structure-Activity Relationship
PubMed: 28709125
DOI: 10.1016/j.ejmech.2017.06.058 -
Journal of Chromatography. B,... Jul 2012Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC-MS/MS method for...
Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC-MS/MS method for quantitative determination of MX in human plasma. In this method, MX and its stable isotope methoxyl-d(3)-amine (MX-d3 as internal standard) were directly derivatized in human plasma with 4-(N,N-diethylamino)benzaldehyde. The derivatized MX and IS were extracted by methyl-tert-butyl ether, and separated isocratically on a Xterra C18 column (2.1 mm × 100 mm) using an aqueous mobile phase containing 45% acetonitrile and 0.4% formic acid at a flow rate of 0.200 ml/min. Quantitation of MX was carried out by multiple-reaction-monitoring (MRM) mode of positive turbo-ion-spray tandem mass spectrometry. This method has been validated according to FDA guidelines for bioanalytical method. The linear calibration range for MX was 1.25-500 ng/ml in human plasma with a correlation coefficient≥0.9993. The intra- and inter-assay precision (%CV) at three concentration levels (3.50, 45.0 and 450 ng/ml) ranged 0.9-1% and 0.8-3%, respectively. The stability studies showed that MX met the acceptable criteria under all tested conditions. The method developed had been applied to the determination of plasma MX concentrations in the first-in-human phase I clinical trial, and PK data were presented.
Topics: Antineoplastic Combined Chemotherapy Protocols; Benzaldehydes; Chromatography, Liquid; Clinical Trials, Phase I as Topic; Dacarbazine; Drug Stability; Humans; Hydroxylamines; Linear Models; Liquid-Liquid Extraction; Neoplasms; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Temozolomide
PubMed: 22743337
DOI: 10.1016/j.jchromb.2012.05.036 -
Oncotarget Nov 2020TRC102 inhibits base excision repair by binding abasic sites and preventing AP endonuclease processing; it potentiates the activity of alkylating agents, including...
BACKGROUND
TRC102 inhibits base excision repair by binding abasic sites and preventing AP endonuclease processing; it potentiates the activity of alkylating agents, including temozolomide, in murine models. In published xenograft studies, TRC102 enhanced the antitumor effect of temozolomide regardless of cell line genetic characteristics, e.g., O6-methylguanine DNA methyltransferase (MGMT), mismatch repair (MMR), or p53 status.
MATERIALS AND METHODS
We conducted a phase 1 trial of TRC102 with temozolomide given orally on days 1-5 of 28-day cycles in adult patients with refractory solid tumors that had progressed on standard therapy. Tumor induction of nuclear biomarkers of DNA damage response (DDR) γH2AX, pNBs1, and Rad51 was assessed in the context of MGMT and MMR protein expression for expansion cohort patients.
RESULTS
Fifty-two patients were enrolled (37 escalation, 15 expansion) with 51 evaluable for response. The recommended phase 2 dose was 125 mg TRC102, 150 mg/m temozolomide QDx5. Common adverse events (grade 3/4) included anemia (19%), lymphopenia (12%), and neutropenia (10%). Four patients achieved partial responses (1 non-small cell lung cancer, 2 granulosa cell ovarian cancer, and 1 colon cancer) and 13 patients had a best response of stable disease. Retrospective analysis of 15 expansion cohort patients did not demonstrate a correlation between low tumor MGMT expression and patient response, but treatment induced nuclear Rad51 responses in 6 of 12 patients.
CONCLUSIONS
The combination of TRC 102 with temozolomide is active, with 4 of 51 patients experiencing a partial response and 13 of 51 experiencing stable disease, and the side effect profile is manageable.
PubMed: 33216844
DOI: 10.18632/oncotarget.27784 -
Metabolites Dec 2021Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and...
Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL -Methyl--trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.
PubMed: 34940646
DOI: 10.3390/metabo11120888 -
Biochemistry Nov 2016The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine...
The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N-HB-dA), 1,N-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N-HMHP-dA), and N,N-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N,N-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N-HB-dA, (R,S)-1,N-HMHP-dA, and (R,R)-N,N-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N-HB-dA > (R,R)-N,N-DHB-dA > (R,S)-1,N-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells.
Topics: Animals; Butadienes; Cell Line; Cell Line, Tumor; Cricetinae; Cricetulus; DNA Repair; Deoxyadenosines; Humans
PubMed: 27552084
DOI: 10.1021/acs.biochem.6b00553 -
RSC Advances Mar 2019Tyrosine kinase inhibitors (TKIs) are very efficient for the treatment of EGFR-mutated lung cancer and show improved therapeutic efficacy. However, treatment with both...
Tyrosine kinase inhibitors (TKIs) are very efficient for the treatment of EGFR-mutated lung cancer and show improved therapeutic efficacy. However, treatment with both first- and second-generation TKIs results in acquired resistance and is related to various toxicities; the EGFR T790M mutation has been associated with this resistance. Naquotinib (ASP8273, NQT) is a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor that has been shown to be more potent than osimertinib in the management of L858R plus T790M mutations. However, its bioactivation may occur and promote the formation of reactive electrophiles that are toxic. We hypothesize that these reactive intermediates are potentially involved in the side effects of NQT. Reactive metabolites are often formed by phase I metabolic reactions and cannot be characterized directly as they are transient in nature. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we screened for metabolites of NQT formed during incubation with human liver microsomes and evaluated the generation of reactive electrophiles using capturing agents, such as methoxyamine and potassium cyanide, as nucleophiles that form stable adducts for identification by LC-MS/MS. Eight NQT phase I metabolites were found that had been formed by -demethylation, oxidation, hydroxylation, and reduction. In addition, three reactive electrophiles, two aldehydes, and one iminium ion were identified, and the corresponding bioactivation mechanisms were proposed. The reported side effects of NQT may be related to the generation of reactive metabolites. Based on a literature review, this may be the first study of phase I metabolites, detailed structural characterizations, and NQT reactive intermediates.
PubMed: 35520926
DOI: 10.1039/c9ra00224c -
The Journal of Biological Chemistry May 1985This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease...
This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.
Topics: Animals; Cattle; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Endodeoxyribonucleases; Hydroxylamines; N-Glycosyl Hydrolases; Rats; Uracil; Uracil-DNA Glycosidase
PubMed: 2580833
DOI: No ID Found -
ACS Omega Oct 2022The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links...
Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.
The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBHCN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.
PubMed: 36278095
DOI: 10.1021/acsomega.2c05736 -
Frontiers in Cell and Developmental... 2021The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that...
The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that are involved in gene expression control. In mammals, Apurinic-apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is the main AP endonuclease of the base excision repair (BER) pathway and is involved in active demethylation processes. In addition, APE1/Ref-1, through its redox function, regulates several transcriptional factors. However, the transcriptional control targets of each APE1 function are not completely known. In this study, a transcriptomic approach was used to investigate the effects of chemical inhibition of APE1/Ref-1 redox or DNA repair functions by E3330 or methoxyamine (MX) in an inflammatory cellular model. Under lipopolysaccharide (LPS) stimulation, both E3330 and MX reduced the expression of some cytokines and chemokines. Interestingly, E3330 treatment reduced cell viability after 48 h of the treatment. Genes related to inflammatory response and mitochondrial processes were downregulated in both treatments. In the E3330 treatment, RNA processing and ribosome biogenesis genes were downregulated, while they were upregulated in the MX treatment. Furthermore, in the E3330 treatment, the cellular stress response was the main upregulated process, while the cellular macromolecule metabolic process was observed in MX-upregulated genes. Nuclear respiratory factor 1 (NRF1) was predicted to be a master regulator of the downregulated genes in both treatments, while the ETS transcription factor ELK1 (ELK1) was predicted to be a master regulator only for E3330 treatment. Decreased expression of ELK1 and its target genes and a reduced 28S/18S ratio were observed, suggesting impaired rRNA processing. In addition, both redox and repair functions can affect the expression of NRF1 and GABPA target genes. The master regulators predicted for upregulated genes were YY1 and FLI1 for the E3330 and MX treatments, respectively. In summary, the chemical inhibition of APE1/Ref-1 affects gene expression regulated mainly by transcriptional factors of the ETS family, showing partial overlap of APE1 redox and DNA repair functions, suggesting that these activities are not entirely independent. This work provides a new perspective on the interaction between APE1 redox and DNA repair activity in inflammatory response modulation and transcription.
PubMed: 34616737
DOI: 10.3389/fcell.2021.731588