RESUMEN
Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in ArchaeaIMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.
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Citocromos/metabolismo , Transporte de Electrón/fisiología , Membranas/metabolismo , Methanosarcina/metabolismo , Acetatos/metabolismo , Antraquinonas/farmacología , Citocromos/genética , Transporte de Electrón/genética , Electrones , Compuestos Férricos/metabolismo , Regulación de la Expresión Génica Arqueal , Bacterias Gramnegativas/metabolismo , Mesna/análogos & derivados , Metano/metabolismo , Metanol/metabolismo , Methanosarcina/efectos de los fármacos , Methanosarcina/genética , Methanosarcina/crecimiento & desarrollo , Oxidación-Reducción , Oxidorreductasas/metabolismo , TranscriptomaRESUMEN
Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 µM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.
Asunto(s)
Mercaptopurina/análogos & derivados , Methanosarcina/enzimología , Metiltransferasas/metabolismo , Catálisis , Mercaptopurina/metabolismo , Mesna/análogos & derivados , Mesna/metabolismo , Methanosarcina/genética , Metilación , Metiltransferasas/genética , Vitamina B 12/metabolismoRESUMEN
BACKGROUND: Transitional cell carcinoma is the most common bladder cancer of dogs. Cisplatin combined with piroxicam provides superior response rates, but unacceptable rates of nephrotoxicity. Tavocept is a chemoprotectant that has mitigated cisplatin toxicity and decreased the required infusion/diuresis volume in clinical trials in humans. HYPOTHESIS/OBJECTIVES: We hypothesized that Tavocept would decrease diuresis volume and time and facilitate safe administration of a cisplatin/piroxicam protocol to dogs with bladder cancer. Secondary objectives were to compare response rate and survival times to an historical comparator group treated without Tavocept. ANIMALS: Fourteen client-owned dogs were prospectively enrolled. METHODS: Tumor volume was measured by computed tomography at days 0, 42, and 84. Dogs received combination Tavocept/cisplatin with a shortened diuresis protocol. A total of 4 doses was planned, with concurrent administration of piroxicam. Serial biochemical analyses were evaluated for azotemia. RESULTS: A 90-minute infusion/diuresis time was used for all dogs. Three dogs (21%) had concurrent increases in serum creatinine (>2.0 mg/dL) and BUN (>42 mg/dL) concentrations; 2 of these dogs were isosthenuric. This frequency of nephrotoxicity is significantly less (P = 0.0406) than that of an historical control group treated without Tavocept. Overall response rate was 27%. Median survival time was comparable to historical controls (253 vs. 246 days). CONCLUSIONS AND CLINICAL IMPORTANCE: Tavocept decreased the required diuresis time with cisplatin from > 6 hours to 90 minutes, while also decreasing occurrence of azotemia. Survival time was comparable, but the response rate was inferior to an historical comparator group. Further evaluation in other tumors susceptible to platinum agents is warranted.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/veterinaria , Cisplatino/uso terapéutico , Diuresis/efectos de los fármacos , Enfermedades de los Perros/tratamiento farmacológico , Mesna/análogos & derivados , Neoplasias de la Vejiga Urinaria/veterinaria , Animales , Antineoplásicos/efectos adversos , Nitrógeno de la Urea Sanguínea , Carcinoma de Células Transicionales/tratamiento farmacológico , Cisplatino/efectos adversos , Creatinina/sangre , Perros , Quimioterapia Combinada , Mesna/uso terapéutico , Piroxicam/uso terapéutico , Estudios Prospectivos , Insuficiencia Renal/inducido químicamente , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Here I report on the identification of 1-mercaptoethanesulfonic acid (1-MES), an analogue of 2-mercaptoethanesulfonic acid (coenzyme M, HSCoM). 1-MES and HSCoM were both present in the growth media of eight different methanogens at concentrations ranging from â¼1 to 100 µM. In an effort to determine a chemical origin of 1-MES, several plausible chemical routes were examined each assuming that HSCoM was the precursor. In all examined routes, no 1-MES was formed. However, 1-MES was formed when a solution of vinylsulfonic acid and sulfide were exposed to ultraviolet light. On the basis of these results, I conclude 1-MES is formed enzymatically. This was confirmed by growing a culture of Methanococcus maripaludis S2 in the presence of [1,1',2,2'-2H4]HSCoM and measuring the incorporation of deuterium into 1-MES. 1-MES incorporated three of the four deuteriums from the fed HSCoM. This result is consistent with the abstraction of a C-2 deuterium of the HSCoM, likely by a 5'-dAdoCH2⢠radical, followed by a radical rearrangement in which the sulfonic acid moves to position C-1, followed by abstraction of a H⢠likely from 5'-dAdoCH2D. At present, the reason for the production of 1-MES is not clear. This is the first report of the occurrence of 1-MES in Nature.
Asunto(s)
Vías Biosintéticas , Mesna/análogos & derivados , Mesna/metabolismo , Methanococcus/metabolismo , Etilenos/metabolismo , Methanococcus/enzimología , Methanococcus/crecimiento & desarrollo , Sulfuros/metabolismo , Ácidos Sulfónicos/metabolismoRESUMEN
Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243-1249, 2017.
Asunto(s)
Mesna/análogos & derivados , Metanol/metabolismo , Methanosarcina barkeri/enzimología , Methanosarcina barkeri/genética , Reactores Biológicos/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Mesna/metabolismo , Metano/metabolismo , Modelos Moleculares , Oxidación-Reducción , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismoRESUMEN
Low pH is an important environmental stressor of plant root cells. Understanding the mechanisms of stress and tolerance to acidity is critical; however, there is no widely accepted pH buffer for studies of plant cells at low pH. Such a buffer might also benefit studies of Al toxicity, in which buffering at low pH is also important. The challenge is to find a buffer with minimal cellular effects. We examined the cytotoxicity and possible metabolic disturbances of four buffers that have adequate pKa values and potential use for studies in the pH range of 4.0-5.0. These were homopipes (homopiperazine-1,4-bis (2-ethanesulfonic acid); pKa1 4.4), 3,3-dimethylglutaric acid (pKa1 3.73), ß-alanine (pKa1 3.70) and potassium biphthalate (pKa1 2.95; pKa2 5.41). First, tobacco BY-2 cells were grown in a rich medium containing 10 mM of each buffer or MES (2-(N-morpholino) ethanesulfonic acid) as a control, with the pH initially adjusted to 5.7. ß-alanine was clearly toxic and dimethylgluturate and biphthalate were found to be cytostatic, in which no culture growth occurred but cell viability was either unaffected or decreased only after 5 days. Only homopipes allowed normal culture growth and cell viability. Homopipes (10 mM) was then tested in cell cultures with an initial pH of 4.3 ± 0.17 in minimal medium to examine whether its undissociated species (H2A) displayed any cellular effects and no cytotoxic effects were observed. It is possible to conclude that among tested buffers, homopipes is the most suitable for studies at low pH, and may be especially useful for aluminum toxicity experiments.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/química , Mesna , Nicotiana/citología , Tampones (Química) , Proliferación Celular/efectos de los fármacos , Mesna/análogos & derivados , Piperazinas , Factores de TiempoRESUMEN
Find your path: Methyl-coenzymeâ M reductase (MCR, turquoise) reversibly catalyzes the reduction of methyl-coenzymeâ M (methyl-S-CoM) with coenzyme B (CoB-SH) to form methane and the heterodisulfide. Recently, spectroscopic methods were used to detect trapped intermediates in a stopped-flow system, and CoM-S-NiII was identified after half a turnover of the MCR reaction (F430 =nickel porphinoid). This finding supports a methyl-radical catalytic mechanism.
Asunto(s)
Mesna/análogos & derivados , Metano/metabolismo , Fosfotreonina/análogos & derivados , Espectroscopía de Resonancia por Spin del Electrón , Mesna/metabolismo , Metano/química , Modelos Moleculares , Estructura Molecular , Fosfotreonina/metabolismoRESUMEN
INTRODUCTION: It is important for sarcoma patients to receive the correct dose of Mesna as an adjuvant with ifosfamide to reduce the risk of hemorrhagic cystitis. This paper describes a study conducted to evaluate the physicochemical stability of Mesna for injection formulation over 14 days. METHODS: Mesna samples (n = 4, 20 mg/ml) were incubated in glass vials at 37 + 0.5ºC. Mesna concentrations were determined by liquid chromatography-mass spectrometry (LC-MS/MS), and nuclear magnetic resonance spectroscopy (NMR) was used to detect degradation products. Evaporative losses and pH were also monitored. RESULTS: Our results differed from those published in existing literature. Both LC-MS/MS and NMR indicated that Mesna was unstable. The mean percentage decrease in Mesna concentration was 40% by day 14 of the analysis. The presence of Mesna's dimer Dimesna was detected on day 0 and its concentration increased over time. Dimesna was the only by-product identified. CONCLUSION: Both LC-MS/MS and NMR analyses confirmed the instability of Mesna and its conversion into Dimesna.
Asunto(s)
Cromatografía Liquida/métodos , Espectroscopía de Resonancia Magnética/métodos , Mesna/análisis , Espectrometría de Masas en Tándem/métodos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Concentración de Iones de Hidrógeno , Inyecciones , Mesna/análogos & derivados , Mesna/químicaRESUMEN
Glutaredoxin (Grx), peroxiredoxin (Prx), and thioredoxin (Trx) are redoxin family proteins that catalyze different types of chemical reactions that impact cell growth and survival through functionally distinct intracellular pathways. Much research is focused on understanding the roles of these redoxin proteins in the development and/or progression of human diseases. Grx and Prx are overexpressed in human cancers, including human lung cancers. BNP7787 is a novel investigational agent that has been evaluated in previous clinical studies, including non-small cell lung cancer (NSCLC) studies. Herein, data from activity assays, mass spectrometry analyses, and X-ray crystallographic studies indicate that BNP7787 forms mixed disulfides with select cysteine residues on Grx and Prx and modulates their function. Studies of interactions between BNP7787 and Trx have been conducted and reported separately. Despite the fact that Trx, Grx, and Prx are functionally distinct proteins that impact oxidative stress, cell proliferation and disease processes through different intracellular pathways, BNP7787 can modify each protein and appears to modulate function through mechanisms that are unique to each target protein. Tumor cells are often genomically heterogeneous containing subpopulations of cancer cells that often express different tumor-promoting proteins or that have multiple dysregulated signaling pathways modulating cell proliferation and drug resistance. A multi-targeted agent that simultaneously modulates activity of proteins important in mediating cell proliferation by functionally distinct intracellular pathways could have many potentially useful therapeutic applications.
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Antineoplásicos/farmacocinética , Cisteína/metabolismo , Glutarredoxinas/química , Mesna/análogos & derivados , Peroxirredoxinas/química , Sitios de Unión , Cristalografía por Rayos X , Glutarredoxinas/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Espectrometría de Masas , Mesna/farmacocinética , Modelos Moleculares , Peroxirredoxinas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The chemical reduction of the disulfide homodimer dimesna to its constituent mesna moieties is essential for its mitigation of nephrotoxicity associated with cisplatin and ifosfamide anticancer therapies and enhancement of dialytic clearance of the cardiovascular risk factor homocysteine. The objective of this study was to investigate potential enzymatic and non-enzymatic mechanisms of intracellular dimesna reduction. Similar to endogenous intracellular disulfides, dimesna undergoes thiol-disulfide exchange with thiolate anion-forming sulfhydryl groups via the two-step SN2 reaction. Determination of equilibrium constants of dimesna reduction when mixed with cysteine or glutathione provided a mechanistic explanation for dramatic cysteine and homocysteine depletion, but sparing of the endogenous antioxidant glutathione, previously observed during mesna therapy. Dimesna was reduced by recombinant enzymes of the thioredoxin system; however, oxidation of NADPH by the glutaredoxin system was only observed in the presence of combined dimesna and reduced glutathione, suggesting formation of oxidized glutathione following an initial non-enzymatic reduction of dimesna. Production of mesna by enzymatic and non-enzymatic mechanisms in HeLa cell lysate following dimesna incubation was demonstrated by a loss in mesna production following protein denaturation and prediction of residual non-enzymatic mesna production by mathematical modeling of thiol-disulfide exchange reactions. Reaction modeling also revealed that mixed disulfides make up a significant proportion of intracellular thiols, supporting their role in providing additional nephroprotection, independent of direct platinum conjugation.
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Cisteína/metabolismo , Glutatión/metabolismo , Homocisteína/metabolismo , Riñón/enzimología , Hígado/enzimología , Mesna/análogos & derivados , Animales , Línea Celular , Femenino , Humanos , Mesna/farmacocinética , Mesna/farmacología , Ratones , Oxidación-Reducción/efectos de los fármacosRESUMEN
Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide- and cisplatin-induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P-glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady-state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug-drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide- and cisplatin-induced toxicities.
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Riñón/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mesna/farmacocinética , Sustancias Protectoras/farmacocinética , Adulto , Femenino , Tasa de Filtración Glomerular , Células HeLa , Humanos , Masculino , Mesna/análogos & derivados , Persona de Mediana Edad , Transportadores de Anión Orgánico/metabolismo , Probenecid/farmacología , Distribución Tisular , Adulto JovenRESUMEN
Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M in methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for methyl-coenzyme M reductase from Methanothermobacter marburgensis is described. The procedure is an accumulation of almost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). Component C of the methylreductase system of Methanobacterium. J. Biol. Chem.256, 4259-4262). To provide a context for this procedure, some background information is provided, including a description of whole cell experiments that provided much of our knowledge of the behavior and properties of methyl-coenzyme M reductase.
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Methanobacteriaceae/enzimología , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Mesna/análogos & derivados , Mesna/química , Mesna/metabolismo , Methanobacteriaceae/crecimiento & desarrollo , Fosfotreonina/análogos & derivados , Fosfotreonina/química , Fosfotreonina/metabolismoRESUMEN
BNP7787, an investigational drug undergoing global Phase III development, appears to have potential advantages over other cytoprotective compounds that have been evaluated for preventing and mitigating cisplatin-induced nephrotoxicity. Herein, we characterized the in vitro accumulation of BNP7787 in human renal proximal tubule cells (HK-2) in which cisplatin is known to be taken up and accumulate. HK-2 cells were incubated with pharmacological concentrations of BNP7787 for varying times. Temperature-dependent accumulation of BNP7787 in cells was observed and the BNP7787-derived metabolite, mesna, formed intracellularly was directly monitored. The peak level of BNP7787-derived mesna measured in HK-2 cells was approximately 0.6 nmol/10(6) cells; this is pharmacologically similar to reported platinum concentrations in kidney cells and may be sufficient to afford nephroprotection. Therefore, in addition to previously suggested glomerular filtration, the cellular accumulation of BNP7787 by HK-2 cells is a plausible newly identified mechanism by which BNP7787 may accumulate in renal tubular cells, where it can exert its pharmacological effects to protect against cisplatin-induced nephrotoxicity by direct covalent conjugation of mesna with cisplatin, or by the formation of BNP7787-derived mesna-disulfide heteroconjugates that exert nephroprotective effects by inhibition of the key toxification enzyme targets γ-glutamyltranspeptidase and aminopeptidase N.
Asunto(s)
Túbulos Renales Proximales/metabolismo , Mesna/análogos & derivados , Línea Celular , Cromatografía Líquida de Alta Presión , Electroquímica , Humanos , Túbulos Renales Proximales/citología , Mesna/farmacocinéticaRESUMEN
The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been determined in a state in which CO(2) is observed providing insights into the mechanism of carboxylation. In the substrate encapsulated state of the enzyme, CO(2) is bound at the base of a narrow hydrophobic substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic to hydrophilic environment where CO(2) is located in position for attack on the carbanion of the ketopropyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively discriminates against H(2)O and prevents protonation of the ketopropyl leaving group.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/metabolismo , Xanthobacter/enzimología , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Descarboxilación , Interacciones Hidrofóbicas e Hidrofílicas , Mesna/análogos & derivados , Mesna/química , Mesna/metabolismo , Conformación ProteicaRESUMEN
PURPOSE: We conducted a phase I trial of BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate, Tavocept™), a novel chemoprotective and antitumor enhancing agent administered in combination with paclitaxel and cisplatin. The primary aim was to determine a safe and potentially efficacious BNP7787 dose for preventing and mitigating paclitaxel- and cisplatin-induced toxicities and to evaluate for preliminary evidence of efficacy of treatment. PATIENTS AND METHODS: Twenty-two patients with stage IIIB/IV non-small cell lung cancer (NSCLC) received BNP7787 alone 1 week before co-administration of BNP7787 with paclitaxel followed by cisplatin. Twenty-one patients were treated with BNP7787 in escalating doses of 4.1-41.0 g/m² concurrently with paclitaxel 175 mg/m² and cisplatin 75 mg/m² every 3 weeks. RESULTS: The appropriate dose was determined to be 18.4 g/m² of BNP7787 although no dose-limiting toxicity was observed up to 41.0 g/m². Mild intravenous site discomfort, thirst, and nausea were the most common toxicities. One patient developed grade 2 skin rash, which was severe enough to preclude further study treatment. The AUC(0-inf) of the metabolite mesna was approximately 6.3% of the AUC(0-inf) of BNP7787. Co-administration of paclitaxel and cisplatin did not appear to influence the pharmacokinetics of BNP7787 and mesna. The overall response rate was encouraging; 43% including 11 patients with prior chemotherapy. CONCLUSIONS: The recommended dose for phase II/III studies is 18.4 mg/m² of BNP7787 in combination with paclitaxel and cisplatin. Further studies are warranted to assess whether BNP7787 prevents and mitigates common and serious paclitaxel- and cisplatin-related side effects and enhances the efficacy of paclitaxel and cisplatin in advanced NSCLC patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Mesna/análogos & derivados , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Área Bajo la Curva , Carcinoma de Pulmón de Células no Pequeñas/patología , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Mesna/efectos adversos , Mesna/farmacocinética , Mesna/farmacología , Persona de Mediana Edad , Estadificación de Neoplasias , Paclitaxel/administración & dosificación , Resultado del TratamientoRESUMEN
PURPOSE: Previous studies from our laboratory have identified a role for gamma-glutamyl transpeptidase (GGT) in BNP7787 (disodium 2,2'-dithio-bis ethane sulfonate, dimesna, Tavocept™)-mediated cisplatin nephroprotection. Dekant has proposed that gamma-glutamyl transpeptidase (GGT), aminopeptidase N (APN) and cysteine-conjugate-ß-lyase (CCBL) comprise a multi-enzyme pathway that acts on xenobiotic-glutathione conjugates converting them to nephrotoxic metabolites. We report modulation of APN activity within this pathway by BNP7787-derived mesna-disulfide heteroconjugates. METHODS: A fluorimetric assay was used to determine the effect of BNP7787, BNP7787-derived mesna-disulfide heteroconjugates, and the BNP7787 metabolite, mesna (sodium 2-mercaptoethane sulfonate), on the initial velocity and overall progress curve of the human APN reaction in vitro. RESULTS: Neither BNP7787 nor mesna-cysteinyl-glutamate inhibited human APN. Select BNP7787-derived mesna-disulfide heteroconjugates (mesna-cysteine, mesna-glutathione, mesna-cysteinyl-glycine) and high concentrations of mesna inhibited APN activity. Allosteric effects on the enzyme progress curve outside of the linear initial velocity region were observed for mesna-cysteinyl-glycine, mesna-glutathione and mesna-cysteinyl-glutamate and appeared to be a function of having both mesna and di- or tri-peptide functionalities in one molecule. In situ-generated mesna-cisplatin conjugates were not a substrate for human APN. CONCLUSIONS: BNP7787-mediated prevention or mitigation of cisplatin-induced nephrotoxicity may involve APN inhibition by certain BNP7787-derived mesna-disulfide heteroconjugates and appears correlated to the presence of a glycinate moiety and/or an anionic group. Two general mechanisms for BNP7787-mediated nephroprotection of cisplatin-induced nephrotoxicity involving the GGT, APN and CCBL nephrotoxigenic pathway are proposed which acting in a concerted and/or synergistic manner, and thereby prevent or mitigate cisplatin-induced renal toxicity.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Cisplatino/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/prevención & control , Mesna/análogos & derivados , Sustancias Protectoras/farmacología , Regulación Alostérica , Biocatálisis/efectos de los fármacos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Cisplatino/análogos & derivados , Cisplatino/metabolismo , Cisplatino/farmacología , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacología , Dipéptidos/farmacología , Glutatión/análogos & derivados , Glutatión/metabolismo , Glutatión/farmacología , Glicina/análogos & derivados , Glicina/metabolismo , Glicina/farmacología , Humanos , Cinética , Mesna/metabolismo , Mesna/farmacología , Modelos Biológicos , Sustancias Protectoras/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.
Asunto(s)
Mesna/análogos & derivados , Methanobacteriaceae/enzimología , Oxidorreductasas/metabolismo , Fosfotreonina/análogos & derivados , Acetilación , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Mesna/metabolismo , Metano/metabolismo , Methanobacteriaceae/metabolismo , Fosfotreonina/química , Fosfotreonina/metabolismo , Espectrofotometría , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Taxane and platinum drugs are important agents in the treatment of cancer and have shown activity against a variety of tumors, including ovarian, breast, and lung cancer, either as single agents or in combination with other chemotherapy drugs. However, a serious and prevalent side effect of taxane (docetaxel and all formulations/derivatives of paclitaxel) and platinum (cisplatin, carboplatin, and oxaliplatin) agents is dose-limiting chemotherapy-induced peripheral neuropathy (CIPN). CIPN can result in treatment delays, dose modifications, and, in severe cases, discontinuation of chemotherapy. Consequently, effective treatments for CIPN are needed. Dimesna (BNP7787; Tavocept; disodium 2,2'-dithio-bis-ethanesulfonate) is an investigational drug that is undergoing international clinical development as a treatment that is coadministered with first-line taxane and platinum combination chemotherapy in patients with inoperable advanced primary adenocarcinoma of the lung. BNP7787 is currently being developed with the objective of increasing the survival of cancer patients receiving taxane- and/or cisplatin-based chemotherapy. Additional data indicate that BNP7787 may also protect against common and serious chemotherapy-induced toxicities, including chemotherapy-induced anemia, nausea, emesis, nephrotoxicity, and neuropathy, without interfering with antitumor activity of the chemotherapeutic agent(s). Studies herein show that BNP7787 prevents aberrant microtubule protein (MTP) polymerization that is caused by exposure of MTP to paclitaxel or cisplatin. BNP7787 modulates paclitaxel-induced hyperpolymerization of MTP in a dose-dependent manner, and mesna, an in vivo metabolite of BNP7787, protects against time-dependent cisplatin-induced inactivation of MTP. We propose that interactions between BNP7787 and MTP may play a role in BNP7787-mediated protection against CIPN.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Mesna/análogos & derivados , Proteínas de Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Paclitaxel/farmacología , Animales , Bovinos , Interacciones Farmacológicas , Mesna/farmacología , Microtúbulos/patología , Polimerizacion/efectos de los fármacosRESUMEN
Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.
Asunto(s)
Methanobacteriaceae/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Fosfotreonina/análogos & derivados , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Mesna/análogos & derivados , Mesna/química , Mesna/metabolismo , Metano/química , Metano/metabolismo , Methanobacteriaceae/metabolismo , Modelos Moleculares , Fosfotreonina/química , Fosfotreonina/metabolismo , Conformación ProteicaRESUMEN
The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys(82)) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys(87) was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys(82). These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.