-
Chemical Research in Toxicology Aug 2023Pexidartinib (PEX, TURALIO), a selective and potent inhibitor of the macrophage colony-stimulating factor-1 receptor, has been approved for the treatment of tenosynovial...
Pexidartinib (PEX, TURALIO), a selective and potent inhibitor of the macrophage colony-stimulating factor-1 receptor, has been approved for the treatment of tenosynovial giant cell tumor. However, frequent and severe adverse effects have been reported in the clinic, resulting in a boxed warning on PEX for its risk of liver injury. The mechanisms underlying PEX-related hepatotoxicity, particularly metabolism-related toxicity, remain unknown. In the current study, the metabolic activation of PEX was investigated in human/mouse liver microsomes (HLM/MLM) and primary human hepatocytes (PHH) using glutathione (GSH) and methoxyamine (NHOMe) as trapping reagents. A total of 11 PEX-GSH and 7 PEX-NHOMe adducts were identified in HLM/MLM using an LC-MS-based metabolomics approach. Additionally, 4 PEX-GSH adducts were detected in the PHH. CYP3A4 and CYP3A5 were identified as the primary enzymes responsible for the formation of these adducts using recombinant human P450s and CYP3A chemical inhibitor ketoconazole. Overall, our studies suggested that PEX metabolism can produce reactive metabolites mediated by CYP3A, and the association of the reactive metabolites with PEX hepatotoxicity needs to be further studied.
Topics: Mice; Humans; Animals; Cytochrome P-450 CYP3A; Chromatography, Liquid; Tyrosine Kinase Inhibitors; Tandem Mass Spectrometry; Protein Kinase Inhibitors; Cytochrome P-450 CYP3A Inhibitors; Microsomes, Liver; Metabolomics; Chemical and Drug Induced Liver Injury; Glutathione
PubMed: 37531179
DOI: 10.1021/acs.chemrestox.3c00164 -
Acta Pharmaceutica (Zagreb, Croatia) Jun 2023Methoxamine (Mox) is a well-known α1-adrenoceptor agonist, clinically used as a longer-acting analogue of epinephrine. 1,2-Mox (NRL001) has been also undergoing...
Methoxamine (Mox) is a well-known α1-adrenoceptor agonist, clinically used as a longer-acting analogue of epinephrine. 1,2-Mox (NRL001) has been also undergoing clinical testing to increase the canal resting pressure in patients with bowel incontinence. Here we show, that Mox hydrochloride acts as an inhibitor of base excision repair (BER). The effect is mediated by the inhibition of apurinic/apyrimidinic endonuclease APE1. We link this observation to our previous report showing the biologically relevant effect of Mox on BER - prevention of converting oxidative DNA base damage to double-stranded breaks. We demonstrate that its effect is weaker, but still significant when compared to a known BER inhibitor methoxyamine (MX). We further determined Mox's relative at 19 mmol L, demonstrating a significant effect of Mox on APE1 activity in clinically relevant concentrations.
Topics: Humans; Methoxamine; DNA Repair; Epinephrine; Receptors, Adrenergic; Endonucleases
PubMed: 37307375
DOI: 10.2478/acph-2023-0012 -
Molecules (Basel, Switzerland) May 2023Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and...
Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3()-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.
Topics: Rats; Animals; Chromatography, Liquid; Chromatography, High Pressure Liquid; Tandem Mass Spectrometry; Piperazines; Pyridones; Glutathione; Cyanides; Aldehydes; Microsomes, Liver
PubMed: 37241965
DOI: 10.3390/molecules28104225 -
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 -
Metabolites Aug 2022Preterm birth (PTB) is a social problem that adversely affects not only the survival rate of the fetus, but also the premature babies and families, so there is an urgent...
Preterm birth (PTB) is a social problem that adversely affects not only the survival rate of the fetus, but also the premature babies and families, so there is an urgent need to find accurate biomarkers. We noted that among causes, eubiosis of the vaginal microbial community to dysbiosis leads to changes in metabolite composition. In this study, short chain fatty acids (SCFAs) representing dysbiosis were derivatized using (-butyldimethylsilyl--methyltrifluoroacetamide, MTBSTFA) and targeted analysis was conducted in extracted organic phases of cervicovaginal fluid (CVF). In residual aqueous CVF, polar metabolites produced biochemistry process were derivatized using methoxyamine and ,-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and non-targeted analysis were conducted. Nine SCFAs were quantified, and 58 polar metabolites were detected in 90 clinical samples using gas chromatography/mass spectrometry (GC/MS). The criteria of statistical analysis and detection rate of clinical sample for development of PTB biomarkers were presented, and 19 biomarkers were selected based on it, consisting of 1 SCFA, 2 organic acids, 4 amine compounds, and 12 amino acids. In addition, the model was evaluated as a suitable indicator for predicting PTB without distinction between sample collection time. We hope that the developed biomarkers based on microbiota-derived metabolites could provide useful diagnostic biomarkers for actual patients and pre-pregnancy.
PubMed: 36005605
DOI: 10.3390/metabo12080734 -
Mikrochimica Acta Mar 2022Glyconanoparticles (G-NPs), biofunctional nanomaterials that can fully combine the unique properties of nanoparticles (NPs) with the bioactivities of carbohydrates, have...
Glyconanoparticles (G-NPs), biofunctional nanomaterials that can fully combine the unique properties of nanoparticles (NPs) with the bioactivities of carbohydrates, have become an appealing nanoplatform in analytical chemistry and biomedical research. However, there is currently a lack of an efficient and universal method for facile immobilization of reducing carbohydrates on NPs while maintaining their structure integrity, greatly limiting the preparation and application of G-NPs. Herein, a new and universal strategy for preparing carbohydrate-functionalized gold nanoclusters (Au NCs) was developed by using S-(3-(methoxyamino)propyl) thioacetate (MPTA) as a new bifunctional linker. MPTA with an N-methoxyamine group (-NHOMe) and a thioacetyl group (-SAc) was synthesized by a two-step strategy and then grafted onto Au NCs by an efficient click reaction. Subsequently, reducing carbohydrates could be readily immobilized onto MPTA-functionalized Au NCs (MPTA-Au NCs) by a reducing end ring-closure reaction under mild conditions. The obtained G-NPs showed average size of 1.9 ± 0.42 nm and strong fluorescence at 610 nm. Carbohydrates grafted on G-NPs still retained their structure integrity and specific recognition ability toward their receptor proteins. Notably, the affinity between G-NPs and proteins was increased by 1300 times compared with free carbohydrates with an association constant of (1.47 ± 0.356) × 10 M. The prepared fluorescent G-NPs were also successfully applied to lectin sensing and targeted breast cancer cell imaging with good performance. These results indicated that the intact immobilization of reducing carbohydrates (whether naturally or chemically accessed) on NPs could be easily achieved using MPTA, providing a simple, efficient, and universal strategy for G-NP preparation.
Topics: Carbohydrates; Gold; Lectins; Metal Nanoparticles; Spectrometry, Fluorescence
PubMed: 35332420
DOI: 10.1007/s00604-022-05220-w -
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 -
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 -
Molekuliarnaia Biologiia 2021Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a...
Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.
Topics: Amino Acid Sequence; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Escherichia coli; Escherichia coli Proteins; Mycobacterium tuberculosis
PubMed: 33871439
DOI: 10.31857/S002689842102004X -
Journal of Fungi (Basel, Switzerland) Feb 2021Pathogenic microbes are exposed to a number of potential DNA-damaging stimuli during interaction with the host immune system. Microbial survival in this situation...
Pathogenic microbes are exposed to a number of potential DNA-damaging stimuli during interaction with the host immune system. Microbial survival in this situation depends on a fine balance between the maintenance of DNA integrity and the adaptability provided by mutations. In this study, we investigated the association of the DNA repair response with the virulence of , a basidiomycete that causes life-threatening meningoencephalitis in immunocompromised individuals. We focused on the characterization of and putative genes, aiming to evaluate a possible role of the predicted Apurinic/apyrimidinic (AP) endonucleases 1 and 2 of the base excision repair (BER) pathway on response to stress conditions and virulence. Our results demonstrated the involvement of the putative AP-endonucleases Apn1 and Apn2 in the cellular response to DNA damage induced by alkylation and by UV radiation, in melanin production, in tolerance to drugs and in virulence of in vivo. We also pointed out the potential use of DNA repair inhibitor methoxy-amine in combination with conventional antifungal drugs, for the development of new therapeutic approaches against this human fungal pathogen. This work provides new information about the DNA damage response of the highly important pathogenic fungus .
PubMed: 33673204
DOI: 10.3390/jof7020133