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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 -
Nucleic Acids Research Oct 1991In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic...
In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.
Topics: Base Sequence; Binding Sites; DNA; DNA Damage; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Genetic Techniques; HeLa Cells; Humans; Hydroxylamines; Molecular Sequence Data; Oligonucleotides; Substrate Specificity
PubMed: 1719478
DOI: 10.1093/nar/19.20.5569 -
Journal of the American Society For... May 2014Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important...
Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MS(n)), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.
Topics: Analytic Sample Preparation Methods; Animals; Female; Gas Chromatography-Mass Spectrometry; Hydrocortisone; Hydroxylamines; Indicators and Reagents; Mice; Molecular Structure; Molecular Weight; Pregnenes; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 24658800
DOI: 10.1007/s13361-014-0836-9 -
DNA Repair Nov 2009Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular...
Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H(2)O(2). However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage.
Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Hydrogen Peroxide; Hydroxylamines; Neuroblastoma; Oxidative Stress
PubMed: 19726241
DOI: 10.1016/j.dnarep.2009.08.003 -
PloS One 2015DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER) is a major repair pathway that often leads to...
DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER) is a major repair pathway that often leads to drug resistance in DNA-targeted cancer chemotherapy. In order to measure BER, we have developed a near infrared (NIR) fluorescent probe. This probe binds to a key intermediate, termed apurinic/apyrimidinic (AP) site, in the BER pathway where DNA damage and repair occurs. We have developed an assay to show the efficacy of the probe binding to AP sites and have shown that it can distinguish AP sites in DNA extract from chemotherapy treated cells. This probe has potential application in monitoring patient response to chemotherapy and evaluating new drugs in development.
Topics: Animals; Base Pairing; Base Sequence; Binding Sites; Cattle; Cell Line, Tumor; Colonic Neoplasms; DNA; DNA Breaks, Single-Stranded; DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Floxuridine; Fluorescent Dyes; Humans; Hydroxylamines; Methyl Methanesulfonate; Molecular Sequence Data; Spectrometry, Fluorescence; Time Factors
PubMed: 26309022
DOI: 10.1371/journal.pone.0131330 -
Carcinogenesis May 2008Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of...
Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.
Topics: Animals; CHO Cells; Cell Survival; Cricetinae; Cricetulus; DNA; DNA Repair; Hypoxanthine Phosphoribosyltransferase; Mammals; Mutagenesis; Mutagens
PubMed: 18332048
DOI: 10.1093/carcin/bgn058 -
European Journal of Biochemistry Dec 1979To refine the secondary structure model of the 5' end of the bacteriophage MS2 genome, 32P-labeled MS2 RNA was partially digested with T1 RNase or with Cm-RNase and the...
To refine the secondary structure model of the 5' end of the bacteriophage MS2 genome, 32P-labeled MS2 RNA was partially digested with T1 RNase or with Cm-RNase and the 5'-end fragment was isolated, renatured and submitted to treatment with methoxyamine or kethoxal. The resulting modified RNA was digested with T1 RNase and the products were separated by minifingerprinting. Methoxyamine-induced modification of exposed cytidines was detected by differential mobility of modified oligonucleotides, while kethoxal-induced alteration of exposed guanosines was monitored by resistance to T1 ribonuclease digestion. The positions of the modified residues are discussed in terms of an improved secondary structure model proposed for the 5' end of the viral RNA. The structure itself is discussed in relation to sequence conservation and biological function.
Topics: Aldehydes; Base Sequence; Butanones; Coliphages; Escherichia coli; Genes, Viral; Hydroxylamines; Nucleic Acid Conformation; Oligoribonucleotides; RNA, Viral; Ribonuclease T1; Ribonucleases
PubMed: 118878
DOI: 10.1111/j.1432-1033.1979.tb04277.x -
Langmuir : the ACS Journal of Surfaces... Feb 2011Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies...
Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies provide site-specific attachment of the biomolecules. In this study, oxime chemistry was exploited to conjugate proteins onto nanometer-sized features. Poly(Boc-aminooxy tetra(ethylene glycol) methacrylate) was synthesized by free radical polymerization. The polymer was patterned onto silicon wafers using an electron beam writer. Trifluoroacetic acid removal of the Boc groups provided the desired aminooxy functionality. In this manner, patterns of concentric squares and contiguous bowtie shapes were fabricated with 150-170-nm wide features. Ubiquitin modified at the N-terminus with an α-ketoamide group and N(ε)-levulinyl lysine-modified bovine serum albumin were subsequently conjugated to the polymer nanopatterns. Protein immobilization was confirmed by fluorescence microscopy. Control studies on protected surfaces and using proteins presaturated with O-methoxyamine indicated that attachment occurred via oxime bond formation.
Topics: Magnetic Resonance Spectroscopy; Microscopy, Atomic Force; Microscopy, Fluorescence; Molecular Structure; Nanostructures; Oximes; Polymers; Proteins
PubMed: 21192671
DOI: 10.1021/la103978x -
Chemical Research in Toxicology Jul 2009Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and...
Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and ionizing radiation have been shown to cause oxidative DNA sugar damage. Detection of DNA lesions is important for understanding the toxicological or therapeutic consequences associated with such agents. C4'-oxidized abasic sites (C4-AP) are produced by the antitumor drug bleomycin and ionizing radiation. The currently available methods for the detection of C4-AP cannot provide both structural and sequence information. We have developed an LC-ESI-MS-based approach for specific detection and mapping of C4-AP from a mixture of lesions. We show using Fe-bleomycin-damaged DNA that C4-AP can be detected at cytosine and thymine sites by direct MS analysis. Our results reveal that collision-induced dissociation of C4-AP-containing oligonucleotides results in preferential fragmentation at C4-AP sites with the formation of the unique a* ions (18 amu more than the a-B ions) that allow mapping of the C4-AP sites. Various chemical modification strategies (e.g., reduction with NaBH4 and NaBD4 and derivatization with methoxyamine and hydrazine, followed by LC-MS analysis) were also used for unambiguous detection of C4-AP sites. Finally, we show that the methods described here can detect the presence of C4-AP at specific sites in a complex sample such as hydroxyl radical-damaged DNA. The LC-MS approach was also used for the simultaneous detection of the other C4'-oxidation end product, 3'-phosphoglycolate, at a specific site in hydroxyl radical-damaged DNA. Thus, LC-MS provides a rapid and direct approach for the detection and mapping of oxidative DNA lesions.
Topics: Antineoplastic Agents; Base Sequence; Bleomycin; Chromatography, High Pressure Liquid; DNA; DNA Damage; Hydroxyl Radical; Ions; Oligonucleotides; Oxidation-Reduction; Radiation, Ionizing; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 19496605
DOI: 10.1021/tx900115z -
RSC Advances Oct 2019Sapitinib (AZD8931, SAP) is an epidermal growth factor receptor (EGFR) family (pan-erbB) tyrosine kinase inhibitor. In multiple tumor cell lines, SAP has been shown to...
Sapitinib (AZD8931, SAP) is an epidermal growth factor receptor (EGFR) family (pan-erbB) tyrosine kinase inhibitor. In multiple tumor cell lines, SAP has been shown to be a much more potent inhibitor of EGF-driven cellular proliferation than gefitinib. In this metabolic study, we tested the generation of reactive intermediates from SAP using human liver microsomes and a capturing agent (potassium cyanide) to trap the iminium reactive intermediates. The same metabolic reaction was further repeated in the presence of methoxyamine to trap aldehyde intermediates. The identification of SAP metabolites revealed that the hydroxylation metabolic reaction represents the major metabolic pathway occurring at the piperidine moiety. We characterized six phase I metabolites in addition to three reactive intermediates (, two iminiums and one aldehyde), therefore suggesting two probable SAP-bioactivation pathways. We hypothesized that the piperidine ring nitrogen (cyclic tertiary amine) activated the two adjacent α-carbons within the ring. The oxidative dealkylation of the -acetamide group led to an unstable aldehyde that was trapped using methoxyamine, generating an oxime adduct that was detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To the best of our knowledge, this is the first study presenting the structural characterization of SAP reactive intermediates.
PubMed: 35529145
DOI: 10.1039/c9ra03926k