RESUMO
PURPOSE: We describe the clinical pharmacology characterization of giredestrant in a first-in-human study. EXPERIMENTAL DESIGN: This phase Ia/Ib dose-escalation/-expansion study (NCT03332797) evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of giredestrant in estrogen receptor-positive HER2-negative locally advanced/metastatic breast cancer. The single-agent dose-escalation stage evaluated giredestrant 10, 30, 90, or 250 mg once daily. The dose-expansion stage evaluated single-agent giredestrant at 30, 100, and 250 mg once daily. Dose-escalation and -expansion phases also evaluated giredestrant 100 mg combined with palbociclib 125 mg. RESULTS: Following single-dose oral administration, giredestrant was rapidly absorbed and generally showed a dose-proportional increase in exposure at doses ranging from 10 to 250 mg. At the 30 mg clinical dose, maximum plasma concentration was 266 ng/mL (50.1%) and area under the concentration-time curve from 0 to 24 hours at steady state was 4,320 ng·hour/mL (59.4%). Minimal giredestrant concentrations were detected in urine, indicating that renal excretion is unlikely to be a major elimination route for giredestrant. Mean concentration of 4beta-hydroxycholesterol showed no apparent increase over time at both the clinical dose (30 mg) and a supratherapeutic dose (90 mg), suggesting that giredestrant may have low CYP3A induction potential in humans. No clinically relevant drug-drug interaction was observed between giredestrant and palbociclib. Giredestrant exposure was not affected by food and was generally consistent between White and Asian patients. CONCLUSIONS: This study illustrates how the integration of clinical pharmacology considerations into early-phase clinical trials can inform the design of pivotal studies and accelerate oncology drug development. SIGNIFICANCE: This work illustrates how comprehensive clinical pharmacology characterization can be integrated into first-in-human studies in oncology. It also demonstrates the value of understanding clinical pharmacology attributes to inform eligibility, concomitant medications, and combination dosing and to directly influence late-stage trial design and accelerate development.
Assuntos
Neoplasias da Mama , Farmacologia Clínica , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Interações MedicamentosasRESUMO
Microsampling technology for dried blood-derived samples provides an advantageous alternative to conventional venous blood for drug quantitation. Unlike conventional whole blood microsampling techniques, Noviplex is a novel, card-based technology for rapid dried plasma spot collection that retains the benefits of microsampling during collection and transportation, while avoiding the disadvantages of using whole blood samples. Giredestrant is a promising small-molecule therapeutic agent under development by Genentech to treat patients with estrogen receptor-positive breast cancer. In this study, we investigated the feasibility of using Noviplex cards for pharmacokinetic analysis of giredestrant levels in human plasma, including optimizing extraction recovery, evaluating in-card stability, and assessing batch precision and accuracy. We found that while the Noviplex card demonstrated levels of sensitivity, extraction recovery, and stability at ambient temperature that meet the requirements of pharmacokinetic analysis for clinical studies, further optimization of the filtration layers within the Noviplex card is necessary to improve filtration efficiency and consistency. This study reveals the possibilities as well as the limitations of the Noviplex card and provides a better understanding of the capabilities and risks of using the Noviplex card for drug quantitation in plasma.
Assuntos
Teste em Amostras de Sangue Seco , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodosRESUMO
Volumetric absorption microsampling devices offer minimally invasive and user-friendly collection of capillary blood in volumes as low as 10 µl. Herein we describe the assay validation for determination of the selective estrogen receptor degrader giredestrant (GDC-9545) in dried human whole blood collected using the Mitra® and Tasso-M20 devices. Both LC-MS/MS assays met validation acceptance criteria for the linear range 1-1000 ng/ml giredestrant. Mitra and Tasso-M20 samples were stable for 84 and 28 days at ambient conditions, respectively, and for 7-9 days at 40 and -70°C. Blood hematocrit, hyperlipidemia and anticoagulant did not impact quantitation of giredestrant. These validated assays are suitable for the determination of giredestrant in dried blood samples collected using Mitra and Tasso-M20 microsampling devices.
Assuntos
Coleta de Amostras Sanguíneas , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Hematócrito , Teste em Amostras de Sangue SecoRESUMO
Ipatasertib is a highly selective small-molecule pan-Akt inhibitor in clinical development. Ipatasertib is predominantly eliminated by the liver, and therefore, the effect of hepatic impairment on ipatasertib pharmacokinetics (PK) was evaluated. In this phase 1 open-label, parallel group study, the PK of ipatasertib were evaluated in subjects with hepatic impairment based on both the Child-Pugh and the National Cancer Institute Organ Dysfunction Working Group classification for hepatic impairment. A single dose of ipatasertib at 100 mg was administered and the PK was characterized in healthy subjects with normal hepatic function or mild, moderate, and severe hepatic impairment. Based on Child-Pugh classification, subjects with moderate and severe hepatic impairment had an ≈2- and 3-fold increase in systemic exposure (area under the plasma concentration-time curve from time 0 to infinity [AUC0-∞ ]) to ipatasertib, respectively, compared to subjects with normal hepatic function. Systemic exposure (AUC0-∞ ) to ipatasertib in subjects with mild hepatic impairment was comparable to that in subjects with normal hepatic function. In accordance with reduced clearance capacity, subjects with mild to severe hepatic impairment showed lower systemic exposure (AUC0-∞ ) of ipatasertib metabolite M1 (G-037720). Overall results were comparable between Child-Pugh and National Cancer Institute Organ Dysfunction Working Group classification criteria. Based on the results from this study, no dosage adjustment is required for ipatasertib when treating patients with mild hepatic impairment, whereas a dose reduction would be recommended for subjects with moderate or severe hepatic impairment. Based on real-world data analysis, ≈2% of the intended patient population is expected to need a modified dose due to moderate or severe hepatic impairment.
Assuntos
Antineoplásicos/farmacocinética , Falência Hepática/epidemiologia , Falência Hepática/metabolismo , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Adulto , Idoso , Área Sob a Curva , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Gravidade do PacienteRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. Although anti-fibrotic treatments, such as pirfenidone, are available that reduce the rate of disease progression, these medications have limitations in tolerability, and IPF patients still have poor prognoses. GDC-3280, an orally available small molecule that was designed to improve upon pirfenidone's activity, has anti-fibrotic activity in animal models. This first-in-human, phase 1 trial evaluated GDC-3280 to determine its safety, tolerability, and pharmacokinetics (PK). METHODS: Single and multiple ascending-doses of GDC-3280 were administered to healthy volunteers in two parts. Part A consisted of 6 treatment groups, each receiving a single, oral dose of GDC-3280 (25-1600 mg) or placebo in the fasted state. Part A also assessed the effect of food and coadministration of a proton pump inhibitor (rabeprazole) on the tolerability and PK of single doses of 400- and 800-mg GDC-3280. Part B consisted of 3 treatment groups who received either 200- or 275-mg GDC-3280 twice daily or 525-mg once daily after a low-fat meal for 7 days. The trial monitored treatment-emergent adverse events (TEAEs) and assessed the pharmacokinetics of GDC-3280 in blood and urine samples. RESULTS: Fifty-six subjects (42 GDC-3280, 14 placebo) in Part A and 24 subjects (18 GDC-3280, 6 placebo) in Part B received treatment. No deaths, serious adverse events, or dose-limiting adverse events occurred, and no subjects withdrew due to a TEAE. In both Parts A and B, most TEAEs were mild. The most frequent TEAEs in Part A were headache and nausea. TEAEs occurred more often when GDC-3280 was administered with food. Pretreatment and coadministration with rabeprazole had no effect on GDC-3280 tolerability. In Part B, the most frequent TEAEs were nausea, dizziness, nasal congestion, and cough. Transient, treatment-related increases in serum creatinine occurred at doses greater than 400 mg in Part A (12%-18% from baseline) and after multiple doses in each group in Part B (20%-34% from baseline). GDC-3280 was generally readily absorbed with a median tmax < 4.0 h following single- or repeat-dose oral administration. In Part A, less-than-dose-proportional increases in systemic exposure occurred, and in Part B, dose-proportional increases occurred within the dose range tested. At doses of 200 mg or lower, more than 50%-70% of orally administered doses were recovered in urine as unchanged GDC-3280 when subjects received a single dose of GDC-3280, suggesting renal excretion is one of the major routes of elimination. Administration of single doses of 400- and 800-mg GDC-3280 after a meal caused statistically significant increases in exposure due to increased rates of absorption compared to the fasted state. Pretreatment and coadministration of rabeprazole dosing led to decreases in exposure compared to GDC-3280 alone, indicating a weak drug-drug interaction. Following repeat dose administration, steady-state plasma concentrations of GDC-3280 were achieved within 2 days with an apparent terminal half-life (t1/2) between 5 and 6 h. CONCLUSIONS: Single and multiple doses of GDC-3280 were generally well tolerated, with acceptable safety and pharmacokinetic profiles that support twice-daily, oral administration with food in future clinical trials.
Assuntos
Interações Alimento-Droga , Administração Oral , Área Sob a Curva , Relação Dose-Resposta a Droga , Método Duplo-Cego , Voluntários Saudáveis , HumanosRESUMO
Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.
Assuntos
Alquilação/fisiologia , Antineoplásicos Alquilantes/metabolismo , DNA/metabolismo , Duocarmicinas/metabolismo , Adenina/metabolismo , Alquilantes/metabolismo , Adutos de DNA/metabolismoRESUMO
A specific and robust LC-MS/MS method was developed and validated for the quantitative determination of GDC-3280 in human plasma and urine. The nonspecific binding associated with urine samples was overcome by the addition of CHAPS. The sample volume was 25 µL for either matrix, and supported liquid extraction was employed for analyte extraction. d6-GDC-3280 was used as the internal standard. Linear standard curves (R2 > 0.9956) were established from 5.00 to 5000 ng/mL in both matrices with quantitation extended to 50,000 ng/mL through dilution. In plasma matrix, the precision (RSD) ranged from 1.5 to 9.9% (intra-run) and from 2.4 to 7.2% (inter-run); the accuracy (RE) ranged from 96.1 to 107% (intra-run) and from 96.7 to 104% (inter-run). Similarly, in urine the precision was 1.5-6.2% (intra-run) and 1.9-6.1% (inter-run); the accuracy was 83.1-99.3% (intra-run) and 87.1-98.3% (inter-run). Good recovery (>94%) and negligible matrix effect were achieved in both matrices. Long-term matrix stability was established for at least 703 days in plasma and 477 days in urine. Bench-top stability of 25 h and five freeze-thaw cycles were also confirmed in both matrices. The method was successfully implemented in GDC-3280's first-in-human trial for assessing its pharmacokinetic profiles.
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Cromatografia Líquida/métodos , Piridonas/sangue , Piridonas/urina , Espectrometria de Massas em Tandem/métodos , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Piridonas/química , Reprodutibilidade dos TestesRESUMO
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been a golden standard for high throughput small molecule bioanalysis in drug discovery for decades. Supercritical fluid chromatography (SFC) has caught more attention in recent decade due to its advantages of greener mobile phase, lower backpressure and higher separation power. For the first time, we evaluated supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) as a high throughput technique for bioanalysis of small molecule drug candidates and compared it to reversed phase LC-MS/MS. Twenty five compounds with diversified structures were evaluated using combination of 6 achiral columns and 4 different mobile phase compositions. To be able to make direct comparison between SFC and HPLC, same type of mass spectrometer was used as the detector. Extracted biological samples were injected to an SFC-MS/MS system and then re-injected to an LC-MS/MS system. It was found that the success rate of the SFC-MS/MS method development was more than 95% if using certain combinations of achiral column and mobile phase compositions without the time-consuming method scouting process. Sensitivity was established between 0.1 to 5.0 ng/mL for both SFC-MS/MS and LC-MS/MS with better sensitivity on SFC-MS/MS. Data from application studies correlated very well between the data produced by SFC-MS/MS and LC-MS/MS. Approximately 95% samples tested had ≤25% difference between SFC-MS/MS and LC-MS/MS data. It was demonstrated that SFC-MS/MS was comparable to golden standard LC-MS/MS and was an alternative choice for routine high throughput bioanalysis of small molecule drugs.
Assuntos
Cromatografia com Fluido Supercrítico/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia com Fluido Supercrítico/instrumentação , Descoberta de Drogas/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
INTRODUCTION: Pirfenidone film-coated tablets were developed to offer an alternative to the marketed capsule formulation. This study assessed the bioequivalence of the tablet and capsule formulations under fed and fasted states. METHODS: A Phase I, open-label, randomized, four-treatment-period, four-sequence, crossover pharmacokinetics study (NCT02525484) was conducted. Each subject received an 801-mg single dose of pirfenidone as three 267-mg capsules or one 801-mg tablet under fasted and fed conditions. Pirfenidone plasma C max, AUC0-t and AUC0-∞ were used to assess bioequivalence. RESULTS: Forty-four subjects were randomized to treatment. The 801-mg tablet in the fasted state met bioequivalence criteria [90% confidence intervals (CI) 80.00-125.00%] for the GLSM ratios of natural log-transformed C max, AUC0-t and AUC0-∞. Under fed conditions, the 801-mg tablet met the bioequivalence criteria for AUC0-t and AUC0-∞, but slightly exceeded the bioequivalence criteria for the C max (90% CI of 108.26-125.60%). The tablet C max was approximately 17% higher than that of the capsules. In the fed state, the tablet C max, and both AUC0-t and AUC0-∞ were reduced by 39% and 17%, respectively, relative to the fasted state. The tablet and capsules had acceptable tolerability profiles. CONCLUSIONS: The pirfenidone 801-mg tablet met bioequivalence criteria when compared with three 267-mg capsules in the fasted state. The tablet C max was slightly higher relative to capsules in the fed state, but this is not expected to have a clinically meaningful impact on the benefit-risk profile of pirfenidone. FUNDING: This work was supported by F. Hoffmann-La Roche Ltd.
Assuntos
Cápsulas/administração & dosagem , Cápsulas/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Piridonas/farmacologia , Comprimidos/administração & dosagem , Comprimidos/farmacocinética , Adolescente , Adulto , Área Sob a Curva , Disponibilidade Biológica , Estudos Cross-Over , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Equivalência Terapêutica , Adulto JovemRESUMO
PURPOSE: The exposure of G2917 decreased by four-fold at oral doses of 100 mg/kg twice daily for seven days in cynomolgus monkeys. Additional investigative work was conducted to understand: (1) the causes for the significant reduction in G2917 exposure in monkeys; (2) the extrapolation of in vitro induction data to in vivo findings in monkeys, and (3) the relevance of this pre-clinical finding to humans at the projected human efficacious dose. METHODS: Pharmacokinetic and induction potency (in vitro and in vivo) of G2917 in monkeys, and the in vitro human induction potency were studied. The hepatic CYP3A biomarkers 4ß-hydroxycholesterol (4ß-HC) and 6ß-hydroxycortisol/cortisol ratio (6ß-OHC/C) were monitored in in vivo studies. The static mechanistic model was used to quantitatively understand the in vitro-in vivo extrapolation (IVIVE) on the magnitude of induction retrospectively. Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and induction-based drug-drug interactions (DDI). RESULTS: All in vitro and in vivo data indicate that the significant reduction in exposure of G2917 in monkeys is caused by auto-induction of CYP3A. The mechanistic understanding of IVIVE of G2917 induction in monkey provides higher confidence in the induction risk prediction in human using the PBPK modeling. PBPK model analysis predicted minimum auto-induction and DDI liability in humans at the predicted efficacious dose. CONCLUSIONS: The learning of this example provided a strategy to address the human CYP3A induction risk prospectively when there is an auto-induction finding in preclinical toxicology study.
Assuntos
Simulação por Computador , Citocromo P-450 CYP3A/biossíntese , Fígado/efeitos dos fármacos , Farmacocinética , Administração Oral , Animais , Descoberta de Drogas , Interações Medicamentosas , Indução Enzimática , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/metabolismo , Hidroxicolesteróis/metabolismo , Fígado/metabolismo , Macaca fascicularis , Midazolam/farmacologia , Modelos Biológicos , RNA Mensageiro/biossíntese , Rifampina/farmacologiaRESUMO
Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of TâA (81%), TâC (68%) and GâA (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.
Assuntos
Reparo do DNA , Replicação do DNA , DNA/genética , Desoxiadenosinas/metabolismo , Desoxicitidina/análogos & derivados , Timidina/análogos & derivados , Sequência de Bases , Sistemas CRISPR-Cas , DNA/metabolismo , Adutos de DNA/genética , Adutos de DNA/metabolismo , Dano ao DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxicitidina/metabolismo , Edição de Genes , Células HEK293 , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Timidina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.
Assuntos
Meios de Cultura/química , Doenças Respiratórias/sangue , Ureia/sangue , Soluções Tampão , Radioisótopos de Carbono/análise , Radioisótopos de Carbono/sangue , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/sangue , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Ureia/análiseRESUMO
Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS(3)) method for the simultaneous quantification of O(6)-carboxymethyl-2'-deoxyguanosine (O(6)-CMdG), O(6)-methyl-2'-deoxyguanosine (O(6)-MedG), and N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of O(6)-CMdG and O(6)-MedG increased when the dose of azaserine was increased from 0 to 450 µM. We, however, did not observe an apparent dose-dependent induction of N(6)-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS(3) technique for the simultaneous quantification of O(6)-CMdG, O(6)-MedG, and N(6)-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis.
Assuntos
Azasserina/análise , Nanotecnologia , Azasserina/farmacologia , Células Cultivadas , Cromatografia Líquida , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Técnicas de Diluição do Indicador , Metilação/efeitos dos fármacos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Adutos de DNA/metabolismo , DNA Glicosilases/metabolismo , Alquilação , Animais , Linhagem Celular , Adutos de DNA/química , Adutos de DNA/genética , Reparo do DNA , Humanos , Camundongos , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Timidina/metabolismoRESUMO
N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O(4)-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O(4)-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O(4)-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells.
Assuntos
Adutos de DNA/química , Mutagênese , Timidina/análogos & derivados , Transcrição Gênica , Linhagem Celular , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Humanos , Timidina/químicaRESUMO
Reactive oxygen species can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells. Previous in vitro biochemical assays have suggested a role for human polymerase (Pol) η in the insertion step of translesion synthesis (TLS) across the (5'S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol ι and a two-subunit yeast Pol ζ complex (REV3/REV7) could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Pol η, Pol ι, and Pol ζ, but not Pol κ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol ι carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Desoxiadenosinas/metabolismo , Desoxiguanosina/análogos & derivados , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Adutos de DNA , Replicação do DNA , Desoxiadenosinas/química , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Cinética , Dados de Sequência Molecular , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , DNA Polimerase iotaRESUMO
O(2)- and O(4)-methylthymidine (O(2)-MdT and O(4)-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O(2)-MdT and O(4)-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf(-)), human DNA polymerase κ (pol κ), and Saccharomyces cerevisiae DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf(-) and pol η preferentially incorporated the correct nucleotide (dAMP) opposite O(2)-MdT, while O(4)-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite O(2)-MdT and O(4)-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if left unrepaired, O-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents.
Assuntos
Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Oxigênio/farmacologia , Timidina/análogos & derivados , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Humanos , Cinética , Inibidores da Síntese de Ácido Nucleico , Oxigênio/química , Saccharomyces cerevisiae/enzimologia , Timidina/química , Timidina/farmacologiaRESUMO
Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N(2)-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.
Assuntos
Dano ao DNA , Transcrição Gênica , Sequência de Bases , Cetrimônio , Compostos de Cetrimônio/química , Adutos de DNA , Primers do DNA , Humanos , Reação em Cadeia da PolimeraseRESUMO
Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5' nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/química , Desoxiguanosina/análogos & derivados , Saccharomyces cerevisiae/enzimologia , Pareamento de Bases , DNA/biossíntese , Adutos de DNA/química , Adutos de DNA/metabolismo , Primers do DNA/metabolismo , Replicação do DNA , Desoxiadenosinas/metabolismo , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Humanos , CinéticaRESUMO
Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.