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Chemical Research in Toxicology Feb 2022Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block...
Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.
Topics: DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Directed DNA Polymerase; Humans; Hydroxylamines
PubMed: 35089032
DOI: 10.1021/acs.chemrestox.1c00359 -
Investigational New Drugs Feb 2021Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by...
Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by binding to apurinic and apyrimidinic (AP) sites after removal of N-methyladenine and N-methylguanine, inhibiting site recognition of AP endonuclease. We conducted a phase I trial to determine the maximum tolerated dose and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Patients with advanced solid tumors and progression on standard treatment were enrolled to a standard 3 + 3 dose escalation trial assessing escalating doses of TMZ and MX. Tumor response was assessed per RECIST and adverse events (AEs) by CTCAEv3. Pharmacokinetics (PK) of MX and COMET assays on peripheral blood mononuclear cells were performed. 38 patients were enrolled-median age 59.5 years (38-76), mean number of cycles 2.9 [1-13]. No DLTs were observed. Cycle 1 grade 3 AEs included fatigue, lymphopenia, anemia, INR, leukopenia, neutropenia, allergic reaction, constipation, psychosis and paranoia. Cycle 2-13 grade 4 AEs included thrombocytopenia and confusion. A partial response was seen in 1 patient with a pancreatic neuroendocrine tumor (PNET) and six additional patients, each with different tumor types, demonstrated prolonged stable disease. MX PK was linear with dose and was not affected by concomitant TMZ. TMZ 200 mg/m daily × 5 may be safely administered with MX 150 mg/m intravenously once on day 1 with minimal toxicity. Further studies assessing this drug combination in select tumor types where temozolomide has activity may be warranted.
Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; DNA Repair; Dose-Response Relationship, Drug; Drug Synergism; Female; Half-Life; Humans; Hydroxylamines; Male; Maximum Tolerated Dose; Metabolic Clearance Rate; Middle Aged; Neoplasms; Temozolomide
PubMed: 32556884
DOI: 10.1007/s10637-020-00962-x -
European Journal of Pharmaceutical... Oct 2020Atomoxetine (ATX), a selective and potent inhibitor of the presynaptic norepinephrine transporter, is used mainly to treat attention-deficit hyperactivity disorder....
Atomoxetine (ATX), a selective and potent inhibitor of the presynaptic norepinephrine transporter, is used mainly to treat attention-deficit hyperactivity disorder. Although multiple adverse effects associated with ATX have been reported including severe liver injuries, the mechanisms of ATX-related toxicity remain largely unknown. Metabolism frequently contributes to adverse effects of a drug through reactive metabolites, and the bioactivation status of ATX is still not investigated yet. Here, we systematically investigated ATX metabolism, bioactivation, species difference in human, mouse, and rat liver microsomes (HLM, MLM, and RLM) and in mice using metabolomic approaches as mice and rats are commonly used animal models for the studies of drug toxicity. We identified thirty one ATX metabolites and adducts in LMs and mice, 16 of which are novel. In LMs, we uncovered two methoxyamine-trapped aldehydes, two cyclization metabolites, detoluene-ATX, and ATX-N-hydroxylation for the first time. Detoluene-ATX and one cyclization metabolite were also observed in mice. Using chemical inhibitors and recombinant CYP enzymes, we demonstrated that CYP2C8 and CYP2B6 mainly contribute to the formation of aldehyde; CYP2D6 is the dominant enzyme for the formation of ATX cyclization and detoluene-ATX; CYP3A4 is major enzyme responsible for the hydroxylamine formation. The findings concerning aldehydes should be very useful to further elucidate the mechanistic aspects of adverse effects associated with ATX from metabolic angles. Additionally, the species differences for each metabolite should be helpful to investigate the contribution of specific metabolites to ATX toxicity and possible drug-drug interactions in suitable models.
Topics: Animals; Atomoxetine Hydrochloride; Attention Deficit Disorder with Hyperactivity; Metabolomics; Mice; Microsomes, Liver; Norepinephrine; Rats
PubMed: 32712217
DOI: 10.1016/j.ejps.2020.105488 -
Molecular Pharmaceutics Jul 2023Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are...
Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are implicated in persistent and difficult to treat infections. With a diminishing antibiotic pipeline, new treatments are urgently required to combat biofilm infections. An emerging strategy to develop new treatments is the hybridization of antibiotics. The benefit of this approach is the extension of the useful lifetime of existing antibiotics. The oxazolidinones, which include the last resort antibiotic linezolid, are an attractive target for improving antibiofilm efficacy as they present one of the most recently discovered classes of antibiotics. A key step in the synthesis of new 3-aryl-2-oxazolidinone derivatives is the challenging formation of the oxazolidinone ring. Herein we report a direct synthetic route to the piperazinyl functionalized 3-aryl-2-oxazolidinone . We also demonstrate an application of these piperazine molecules by functionalizing them with a nitroxide moiety as a strategy to extend the useful lifetime of oxazolidinones and improve their potency against Methicillin-resistant (MRSA) biofilms. The antimicrobial susceptibility of the linezolid-nitroxide conjugate and its corresponding methoxyamine derivative (a control for biofilm dispersal) was assessed against planktonic cells and biofilms of MRSA. In comparison to linezolid and our lead compound (a piperazinyl oxazolidinone derivative), the linezolid-nitroxide conjugate displayed a minimum inhibitory concentration that was 4-16-fold higher. The opposite effect was seen in biofilms where the linezolid-nitroxide hybrid was >2-fold more effective (160 μg/mL versus >320 μg/mL) in eradicating MRSA biofilms. The methoxyamine derivative performed on par with linezolid. The drug-likeness of the compounds was also assessed, and all compounds were predicted to have good oral bioavailability. Our piperazinyl oxazolidinone derivative was confirmed to be lead-like and would be a good lead candidate for future functionalized oxazolidinones. The modification of antibiotics with a dispersal agent appears to be a promising approach for eradicating MRSA biofilms and overcoming the antibiotic resistance associated with the biofilm mode of growth.
Topics: Oxazolidinones; Linezolid; Methicillin-Resistant Staphylococcus aureus; Oxindoles; Anti-Bacterial Agents; Microbial Sensitivity Tests; Biofilms
PubMed: 37289102
DOI: 10.1021/acs.molpharmaceut.3c00095 -
ChemSusChem May 2020An alternative synthetic route towards the widely employed electroactive poly(TEMPO methacrylate) (PTMA) via a thermally robust methoxyamine-protecting group is...
An alternative synthetic route towards the widely employed electroactive poly(TEMPO methacrylate) (PTMA) via a thermally robust methoxyamine-protecting group is demonstrated herein. Protection of the radical moiety of hydroxy-TEMPO with a methyl functionality and subsequent esterification with methacrylic anhydride allows the high-yielding formation of the novel monomer methyl-TEMPO methacrylate (MTMA). The polymerization of MTMA to poly(MTMA) (PMTMA) is investigated via free radical polymerization and reversible addition-fragmentation chain-transfer polymerization (RAFT), a reversible-deactivation radical polymerization technique. Cleavage of the temperature-stable methoxyamine functionality by oxidative treatment of PMTMA with meta-chloroperbenzoic acid (mCPBA) releases the electroactive PTMA. The redox activity of PTMA was confirmed by cyclic voltammetry in lithium-ion coin cells.
PubMed: 32202387
DOI: 10.1002/cssc.201903529 -
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 -
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 -
International Journal of Molecular... May 2020Starting from dansyl-chloride, in reaction with 1,1-diphenylhydrazine and methoxyamine, two new fluorescent derivatives and were obtained and characterized by NMR, IR,...
Starting from dansyl-chloride, in reaction with 1,1-diphenylhydrazine and methoxyamine, two new fluorescent derivatives and were obtained and characterized by NMR, IR, UV-Vis, HR-MS, and fluorescence spectroscopy. The single-crystal X-ray structure was obtained for compound . Both compounds generate free radicals by oxidation, as demonstrated by ESR spectroscopy. Compound generates the corresponding hydrazyl-persistent free radical, evidenced directly by ESR spectroscopy, while compound generates in the first instance the methoxyaminyl short-lived free radical, which decomposes rapidly with the formation of the methoxy radical, evidenced by the ESR spin-trapping technique. By oxidation of compounds and , their fluorescence is quenched.
Topics: Dansyl Compounds; Electron Spin Resonance Spectroscopy; Free Radicals; Hydroxylamines; Phenylhydrazines; Spin Trapping
PubMed: 32443620
DOI: 10.3390/ijms21103559 -
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 -
Journal of Colloid and Interface Science Sep 2023Recently, quasi two-dimensional (Q-2D) perovskites with alternating cations in the interlayer space (ACI) have attracted more attentions owing to their elevated...
Recently, quasi two-dimensional (Q-2D) perovskites with alternating cations in the interlayer space (ACI) have attracted more attentions owing to their elevated stability compared with three-dimensional (3D) analogs. While the efficiency of the devices derived from Q-2D perovskites is much smaller than that based on 3D perovskites. Here, we utilized urea and methoxyamine hydrochloride (MOAH) dual additives to acquire high quality Q-2D ACI perovskite GA(MA)PbI (GA = guanidinium, MA = methylammonium) films. The efficiency of the perovskite solar cells (PSCs) derived from the Q-2D perovskite films induced by the synergistic effect of urea and MOAH dual additives increases to 20.32% from 17.21% for the devices without additive. This efficiency enhancement could be attributed to the enlarged grain size, improved crystallinity, optimized quantum well thickness distribution, and reduced trap states of the perovskite films. Moreover, the solar cells with dual additives present improved stability. The efficiency of devices with dual additives holds 95% of the original value after storage for 1600 h in ambient air. These results prove that the synergistic effect of urea and MOAH is an effective method to achieve highly efficient and stable Q-2D PSCs.
PubMed: 37235937
DOI: 10.1016/j.jcis.2023.05.132