RESUMEN
AIMS: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. RESULTS: [Mn(CO)4S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. INNOVATION: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. CONCLUSIONS: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration. Rebound Track: This work was rejected during standard peer review and rescued by rebound peer review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Miguel Aon, Giancarlo Biagini, James Imlay, and Nigel Robinson. Antioxid. Redox Signal. 28, 1286-1308.
Asunto(s)
Antibacterianos/farmacología , Monóxido de Carbono/química , Complejos de Coordinación/química , Escherichia coli K12/efectos de los fármacos , Manganeso/química , Tiocarbamatos/química , Antibacterianos/química , Monóxido de Carbono/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Complejos de Coordinación/metabolismo , Escherichia coli K12/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Modelos Estadísticos , Solubilidad , Transcriptoma , Agua/químicaRESUMEN
Carbon monoxide (CO) is generated by heme oxygenase-1 (HO-1) and displays important signaling, anti-apoptotic and anti-inflammatory activities, indicating that pharmacological agents mimicking its action may have therapeutic benefit. This study examined the biochemical and pharmacological properties of CORM-401, a recently described CO-releasing molecule containing manganese as a metal center. We used in vitro approaches, ex-vivo rat aortic rings and the EA.hy926 endothelial cell line in culture to address how CORM-401 releases CO and whether the compound modulates vascular tone and pro-angiogenic activities, respectively. We found that CORM-401 released up to three CO/mole of compound depending on the concentration of the acceptor myoglobin. Oxidants such as H2O2, tert-butyl hydroperoxide or hypochlorous acid increased the CO liberated by CORM-401. CORM-401 also relaxed pre-contracted aortic rings and vasorelaxation was enhanced in combination with H2O2. Consistent with the release of multiple CO molecules, CORM-401-induced vasodilation was three times higher than that elicited by CORM-A1, which exhibits a similar half-life to CORM-401 but liberates only one CO/mole of compound. Furthermore, endothelial cells exposed to CORM-401 accumulated CO intracellularly, accelerated migration in vitro and increased VEGF and IL-8 levels. Studies using pharmacological inhibitors revealed HO-1 and p38 MAP kinase as two independent and parallel mechanisms involved in stimulating migration. We conclude that the ability of CORM-401 to release multiple CO, its sensitivity to oxidants which increase CO release, and its vascular and pro-angiogenic properties highlight new advances in the design of CO-releasing molecules that can be tailored for the treatment of inflammatory and oxidative stress-mediated pathologies.
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Inductores de la Angiogénesis/farmacología , Monóxido de Carbono/metabolismo , Endotelio Vascular/metabolismo , Oxidantes/farmacología , Inductores de la Angiogénesis/química , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Monóxido de Carbono/agonistas , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Humanos , Masculino , Oxidantes/química , Ratas , Ratas WistarRESUMEN
AIMS: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools. RESULTS: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3. INNOVATION: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects. CONCLUSION: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.
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Monóxido de Carbono/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Mutación , Compuestos Organometálicos/metabolismo , Membrana Celular/metabolismo , Escherichia coli K12/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Hemo/genética , Hemo/metabolismo , Lactococcus lactis/metabolismo , Modelos Teóricos , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , TranscriptomaRESUMEN
CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O2 to the ion nitrate (NO3(-)). The protoheme of Hmp binds not only O2 and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)(3)Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo.
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Monóxido de Carbono/metabolismo , Dihidropteridina Reductasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hemoproteínas/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/metabolismo , Compuestos Organometálicos/metabolismo , Anaerobiosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Espacio Intracelular/metabolismo , Hierro/metabolismo , Solubilidad , Sulfatos/farmacología , SuspensionesRESUMEN
Carbon monoxide (CO) is a toxic gas that binds to haems, but also plays critical signalling and cytoprotective roles in mammalian systems; despite problems associated with systemic delivery by inhalation of the gas, it may be employed therapeutically. CO delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas; CO-RMs are also attractive candidates as novel antimicrobial agents. Salmonella enterica serovar Typhimurium is an enteropathogen causing gastroenteritis in humans. Recent studies have implicated haem oxygenase-1 (HO-1), the protein that catalyses the degradation of haem into biliverdin, free iron and CO, in the host immune response to Salmonella infection. In several studies, CO administration via CO-RMs elicited many of the protective roles of HO-1 induction and so we investigated the effects of a well-characterized water-soluble CO-RM, Ru(CO)3Cl(glycinate) (CORM-3), on Salmonella. CORM-3 exhibits toxic effects at concentrations significantly lower than those reported to cause toxicity to RAW 264.7 macrophages. We demonstrated here, through oxyhaemoglobin assays, that CORM-3 did not release CO spontaneously in phosphate buffer, buffered minimal medium or very rich medium. CORM-3 was, however, accumulated to high levels intracellularly (as shown by inductively coupled plasma MS) and released CO inside cells. Using growing Salmonella cultures without prior concentration, we showed for the first time that sensitive dual-beam integrating cavity absorption spectrophotometry can detect directly the CO released from CORM-3 binding in real-time to haems of the bacterial electron transport chain. The toxic effects of CO-RMs suggested potential applications as adjuvants to antibiotics in antimicrobial therapy.
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Antibacterianos/metabolismo , Monóxido de Carbono/metabolismo , Compuestos Organometálicos/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Hemo/metabolismo , Humanos , Unión Proteica , EspectrofotometríaRESUMEN
BACKGROUND: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO - a critical gasotransmitter - in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. METHODS: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli - cytochrome bd-I, cytochrome bd-II and cytochrome bo', to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24µM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. RESULTS: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo'. Cytochromes bo' and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. CONCLUSIONS: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. GENERAL SIGNIFICANCE: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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Acetilcisteína/farmacología , Monóxido de Carbono/metabolismo , Respiración de la Célula/efectos de los fármacos , Citocromos/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/metabolismo , Compuestos Organometálicos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Consumo de Oxígeno/efectos de los fármacos , Antioxidantes/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Grupo Citocromo b , Citocromos/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Proteínas de Escherichia coli/metabolismo , Hemo/metabolismo , Oxigenoterapia Hiperbárica , Leghemoglobina/metabolismo , Oxidorreductasas/metabolismo , Rutenio/farmacologíaRESUMEN
AIMS: Carbon monoxide (CO)-releasing molecules (CO-RMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically. One such application is antimicrobial activity; therefore, we aimed to characterize and compare the effects of the CO-RM, CORM-3, and its inactivated counterpart, where all labile CO has been removed, at the transcriptomic and cellular level. RESULTS: We found that both compounds are able to penetrate the cell, but the inactive form is not inhibitory to bacterial growth under conditions where CORM-3 is. Transcriptomic analyses revealed that the bacterial response to inactivated CORM-3 (iCORM-3) is much lower than to the active compound and that a wide range of processes appear to be affected by CORM-3 and to a lesser extent iCORM-3, including energy metabolism, membrane transport, motility, and the metabolism of many sulfur-containing species, including cysteine and methionine. INNOVATION: This work has demonstrated that both CORM-3 and its inactivated counterpart react with cellular functions to yield a complex response at the transcriptomic level. A full understanding of the actions of both compounds is vital to understand the toxic effects of CO-RMs. CONCLUSION: This work has furthered our understanding of how CORM-3 behaves at the cellular level and identifies the responses that occur when the host is exposed to the Ru compound as well as those that result from the released CO. This is a vital step in laying the groundwork for future development of optimized CO-RMs for eventual use in antimicrobial therapy.
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Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Compuestos Organometálicos/farmacología , Compuestos de Sulfhidrilo/metabolismo , Transcriptoma/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Anaerobiosis , Evaluación Preclínica de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Redes y Vías Metabólicas/efectos de los fármacos , Consumo de Oxígeno , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.
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Monóxido de Carbono/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Compuestos Organometálicos/farmacología , Vasodilatadores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Monóxido de Carbono/química , Línea Celular , Técnicas In Vitro , Hierro/química , Macrófagos , Masculino , Ratones , Compuestos Organometálicos/química , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatadores/químicaRESUMEN
AIMS: Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here, we established its mode of action. RESULTS: CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H(+)/O quotients, i.e., H(+) extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true "uncoupling," that is, dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K(+) (or Na(+)), causing a stimulation of respiration and H(+) pumping to compensate for the transient drop in membrane potential (ΔΨ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). INNOVATION: This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport, and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. CONCLUSION: CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases, and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis.
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Antiinfecciosos/química , Antiinfecciosos/farmacología , Monóxido de Carbono/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacologíaRESUMEN
Carbon monoxide-releasing molecules (CO-RMs) emulate the beneficial (e.g., anti-inflammatory) effects of CO in biology. CO release from CO-RMs is routinely determined in the presence of reduced deoxy-myoglobin by measuring the formation of carboxy-myoglobin (Mb-CO). Previous studies have highlighted discrepancies between the apparent CO release rates of some CO-RMs established using this assay versus other experimental data where a slower or more complex mechanism of release is suggested. It has been hypothesized that some CO-RMs require a CO acceptor, believed to be reduced myoglobin in Mb-CO assays, in order to facilitate the release of CO. Here, we show, for the first time, that CO is not liberated from the ruthenium (Ru)-based [Ru(CO)(3)Cl(2)](2) (CORM-2) and [Ru(CO)(3)Cl(glycinate)] (CORM-3) at an appreciable rate in the presence of reduced myoglobin alone. Rather, we confirm that it is the reducing agent sodium dithionite that facilitates release of CO from these CO-RMs. Other sulfite compounds, namely sodium sulfite and potassium metabisulfite, also promote the liberation of CO from CORM-3. We describe an alternative oxy-hemoglobin assay that eliminates dithionite and suggest that the efficacy of CO-RMs results from intracellular interactions with anions that facilitate CO delivery to therapeutic targets.
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Monóxido de Carbono/análisis , Compuestos Organometálicos/química , Oxihemoglobinas/química , Animales , Monóxido de Carbono/química , Catálisis , Bovinos , Ditionita/química , Mioglobina/sangre , Mioglobina/química , Sulfitos/químicaRESUMEN
AIMS: Carbon monoxide (CO) delivered in a controlled manner to cells and organisms mediates a variety of pharmacological effects to the extent that CO-releasing molecules (CO-RMs) are being developed for therapeutic purposes. Recently, ruthenium-based CO-RMs have been shown to posses important bactericidal activity. Here we assessed the effect of fast CO releasers containing ruthenium (Ru(CO)(3)Cl(glycinate) [CORM-3] and tricarbonyldichlororuthenium(II) dimer [CORM-2]) and a novel slow manganese-based CO releaser ([Me(4)N][Mn(CO)(4)(thioacetate)(2)] [CORM-371]) on O(2) consumption and growth of Pseudomonas aeruginosa (PAO1). We then compared these effects with the action elicited by sodium boranocarbonate (CORM-A1), which lacks a transition metal but liberates CO with a rate similar to CORM-371. RESULTS: CORM-2, CORM-3, and, to a lesser extent, CORM-371 exerted a significant bactericidal effect and decreased O(2) consumption in PAO1 in vitro. The effect appeared to be independent of reactive oxygen species production, but in the case of metal-containing compounds it was prevented by the thiol donor N-acetylcysteine. In contrast, CORM-A1 was bacteriostatic rather than bactericidal in vitro eliciting only a moderate and transient decrease in O(2) consumption. INNOVATION: None of the tested CO-RMs was toxic to murine macrophages or human fibroblasts at the concentration impairing PA01 growth but only ruthenium-containing CO-RMs showed potential therapeutic properties by increasing the survival of mice infected with PA01. CONCLUSION: CO carriers inhibit bacterial growth and O(2) consumption in vitro, but transition metal carbonyls appear more powerful than compounds spontaneously liberating CO. The nature of the metal in CO-RMs also modulates the anti-bacterial effect, with ruthenium-based CO-RMs being efficacious both in vitro and in vivo.
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Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Acetilcisteína/farmacología , Animales , Monóxido de Carbono/metabolismo , Células Cultivadas , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Oxígeno/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
[Fe(L-cysteinate)(2)(CO)(2)](2-) is a CO releasing molecule which has low cytotoxicity to RAW264.7 macrophages. It provides an example of CO binding using ligands available to ion channels which use CO as a signalling molecule in the absence of heme. Previous work has shown that this compound consists of five isomers and it was proposed that the two isomers with trans-dicarbonyls are dominant. In this work the isomers are re-assigned and shown to be capable of releasing CO, albeit too slowly to act as a signalling receptor. It is shown that by linking the two L-cysteines together to form [Fe(SCH(2)CH{CO(2)H}NHCH(2))(2)(CO)(2)], only one isomer is isolated.
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Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Cisteína/análogos & derivados , Cisteína/química , Compuestos Ferrosos/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína/toxicidad , Relación Dosis-Respuesta a Droga , Compuestos Ferrosos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Modelos Moleculares , Conformación Molecular , Unión ProteicaRESUMEN
Carbon monoxide (CO) is a classical respiratory inhibitor, but CO-releasing molecules (CO-RMs) have therapeutic value, increasing phagocytosis, and reducing sepsis-induced lethality. CORM-3, Ru(CO)(3) Cl(glycinate), a ruthenium-based carbonyl that liberates CO under physiological conditions, has previously been shown to inhibit bacterial growth and respiration, even at high concentrations of oxygen. Here, we report the effects of CORM-3 on the microaerophilic foodborne pathogen Campylobacter jejuni. Even at CO-RM (i.e., CO) concentrations that exceed dissolved oxygen levels, CORM-3 does not inhibit microaerobic growth. This insensitivity is not due to failure of CORM-3 to penetrate cells, as revealed by assay with extracellular myoglobin and by the ability of CO from externally added CORM-3 to bind intracellular membrane-associated respiratory oxidases. Even at almost 200 µ M oxygen, CORM-3 inhibits formate-dependent respiration and leads to generation of hydrogen peroxide. This work shows that CO-RMs have valuable properties as antimicrobial agents; however, growth inhibition does not always accompany inhibition of respiration, even when ambient oxygen concentrations are low.
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Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/metabolismo , Respiración de la Célula/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Compuestos Organometálicos/farmacología , Oxidorreductasas/metabolismo , Animales , Respiración de la Célula/fisiología , Mioglobina/metabolismo , Oxidantes/metabolismo , Oxidorreductasas/antagonistas & inhibidoresRESUMEN
[Mn(CO)(4){S(2)CNMe(CH(2)CO(2)H)}], 1, is shown to be a CO releasing molecule providing at least three moles CO per mole of compound. The mechanism of CO loss is dissociative and reversible and was investigated using Gaussian 09 calculations. The reversible binding of CO results in a relatively stable solution of the compound, while in the presence of a CO receptor or a ligand to prevent the rebinding of CO, the CO is lost rapidly. The X-ray structure was determined.
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Monóxido de Carbono/química , Manganeso/química , Compuestos Organometálicos/química , Agua/química , Animales , Monóxido de Carbono/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Cinética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Compuestos Organometálicos/farmacología , SolubilidadRESUMEN
New CO-releasing molecules, CO-RMs, based on indenyl iron carbonyls have been identified. Half-lives for carbon monoxide (CO) release, (1)H NMR, (13)C NMR, IR, mass spectra, elemental analysis and biological data have been determined for the compounds. Limited correlations have been made between half-lives and ΔG() for CO release and spectroscopic parameters, ν(CO) and δ((13)CO). X-ray structures have been determined for [Fe(η(5)-C(9)H(7))(CO)(2)L][BF(4)] where L is CO, NCMe, PPh(3), P(OPh)(3), NC(5)H(5) or 1-methylimidazole. Improved preparations of [Fe(η(5)-C(9)H(7))(CO)(2)](2) and [Fe(η(5)-C(9)H(7))(CO)(3)][BF(4)] are reported.
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Monóxido de Carbono/química , Compuestos de Hierro Carbonilo/química , Animales , Monóxido de Carbono/toxicidad , Línea Celular , Supervivencia Celular , Cristalografía por Rayos X , RatonesRESUMEN
Carbon monoxide (CO) liberated by a water-soluble carbon monoxide-releasing molecule (CORM-3) induces a positive inotropic effect with a negative chronotropic effect in normal rat hearts. However, the efficacy of CORM-3 under conditions of chronic cardiac insufficiency is unknown. In a rat model of doxorubicin-induced cardiomyopathy, CORM-3 (20 microg/min) produced a positive inotropic effect as demonstrated by significant increases in systolic pressure (P < 0.05) and pressure derivative (dp/dt max) over time (P < 0.05). A similar dose of CO-depleted negative control (inactive CORM-3) failed to cause any change in these parameters. When the inotrope dobutamine was added at a dose of 10 microM following CORM-3, there was no additional increase in systolic pressure or dp/dt max. However, significant rises in systolic pressure and dp/dt max were observed after dobutamine administration to the hearts previously treated with inactive CORM-3. These results suggest that CORM-3 produces a positive inotropic effect in doxorubicin cardiomyopathy rat hearts, similar to that reported previously in normal hearts. The inotropic effect produced by CO in the doxorubicin cardiomyopathy heart was mimicked by a classical inotrope (dobutamine), suggesting that either a maximal inotropic effect is achieved at this dose of CORM-3 or both drugs utilize shared signaling pathways in cardiac muscle.
Asunto(s)
Monóxido de Carbono/uso terapéutico , Cardiomiopatías/inducido químicamente , Cardiomiopatías/tratamiento farmacológico , Cardiotónicos/uso terapéutico , Doxorrubicina/toxicidad , Compuestos Organometálicos/uso terapéutico , Animales , Monóxido de Carbono/farmacocinética , Cardiomiopatías/metabolismo , Cardiotónicos/farmacocinética , Cardiotoxinas/toxicidad , Técnicas In Vitro , Masculino , Compuestos Organometálicos/farmacocinética , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.
Asunto(s)
Monóxido de Carbono/metabolismo , Células Endoteliales/efectos de los fármacos , Compuestos Organometálicos/farmacología , Trasplante Heterólogo , Animales , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Concanavalina A/farmacología , Células Endoteliales/citología , Humanos , Interleucina-1beta/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Macaca fascicularis , Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sus scrofa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Carbon monoxide (CO) is a colorless, odorless gas with a reputation for being an anthropogenic poison; there is extensive documentation of the modes of human exposure, toxicokinetics, and health effects. However, CO is also generated endogenously by heme oxygenases (HOs) in mammals and microbes, and its extraordinary biological activities are now recognized and increasingly utilized in medicine and physiology. This review introduces recent advances in CO biology and chemistry and illustrates the exciting possibilities that exist for a deeper understanding of its biological consequences. However, the microbiological literature is scant and is currently restricted to: 1) CO-metabolizing bacteria, CO oxidation by CO dehydrogenase (CODH) and the CO-sensing mechanisms that enable CO oxidation; 2) the use of CO as a heme ligand in microbial biochemistry; and 3) very limited information on how microbes respond to CO toxicity. We demonstrate how our horizons in CO biology have been extended by intense research activity in recent years in mammalian and human physiology and biochemistry. CO is one of several "new" small gas molecules that are increasingly recognized for their profound and often beneficial biological activities, the others being nitric oxide (NO) and hydrogen sulfide (H2S). The chemistry of CO and other heme ligands (oxygen, NO, H2S and cyanide) and the implications for biological interactions are briefly presented. An important advance in recent years has been the development of CO-releasing molecules (CO-RMs) for aiding experimental administration of CO as an alternative to the use of CO gas. The chemical principles of CO-RM design and mechanisms of CO release from CO-RMs (dissociation, association, reduction and oxidation, photolysis, and acidification) are reviewed and we present a survey of the most commonly used CO-RMs. Amongst the most important new applications of CO in mammalian physiology and medicine are its vasoactive properties and the therapeutic potentials of CO-RMs in vascular disease, anti-inflammatory effects, CO-mediated cell signaling in apoptosis, applications in organ preservation, and the effects of CO on mitochondrial function. The very limited literature on microbial growth responses to CO and CO-RMs in vitro, and the transcriptomic and physiological consequences of microbial exposure to CO and CO-RMs are reviewed. There is current interest in CO and CO-RMs as antimicrobial agents, particularly in the control of bacterial infections. Future prospects are suggested and unanswered questions posed.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Monóxido de Carbono/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Complejos Multienzimáticos/metabolismo , Antiinfecciosos/uso terapéutico , Apoptosis/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Monóxido de Carbono/toxicidad , Humanos , Sulfuro de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Enfermedades VascularesRESUMEN
Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.
Asunto(s)
Antibacterianos/farmacología , Monóxido de Carbono/farmacología , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/biosíntesis , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Compuestos Organometálicos/farmacología , Factores de Transcripción/biosíntesis , Anaerobiosis/efectos de los fármacos , Anaerobiosis/fisiología , Antibacterianos/metabolismo , Monóxido de Carbono/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos Organometálicos/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Carbon monoxide-releasing molecules (CO-RMs) are pharmacologically active as they protect against cardiac graft rejection and cold ischemia-mediated renal dysfunction. We investigated the cardioprotective role of carbon monoxide (CO) released from CORM-3 against cold ischemia-mediated injury in the heart and evaluated its potential application in the clinical setting of cardiac transplantation. METHODS: Isolated rat hearts underwent cold ischemic storage for 4 or 6 hours using St Thomas Hospital solution that was supplemented with either CORM-3 (50 mumol/liter) or its inactive counterpart (iCORM-3), which does not release CO. Hearts were then reperfused. Both functional parameters and release of cardiac enzymes were assessed. RESULTS: Addition of CORM-3 to the preservation solution resulted in a significant improvement in systolic and diastolic function as well as coronary flow when compared with hearts treated with iCORM-3. In addition, lower levels of the cardiac enzymes creatine kinase and lactate dehydrogenase were measured in the perfusate of hearts stored with CORM-3. CONCLUSIONS: The improved functional recovery and reduced enzyme release after cardiac cold storage with CORM-3, but not iCORM-3, indicate that CO is the main mediator of myocardial protection. Thus, CO-RMs can be used as adjuvants to improve the preservation of hearts for transplantation.