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1
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Oza PP, Kashfi K. The Triple Crown: NO, CO, and H 2S in cancer cell biology. Pharmacol Ther 2023; 249:108502. [PMID: 37517510 PMCID: PMC10529678 DOI: 10.1016/j.pharmthera.2023.108502] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 07/16/2023] [Accepted: 07/19/2023] [Indexed: 08/01/2023]
Abstract
Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are three endogenously produced gases with important functions in the vasculature, immune defense, and inflammation. It is increasingly apparent that, far from working in isolation, these three exert many effects by modulating each other's activity. Each gas is produced by three enzymes, which have some tissue specificities and can also be non-enzymatically produced by redox reactions of various substrates. Both NO and CO share similar properties, such as activating soluble guanylate cyclase (sGC) to increase cyclic guanosine monophosphate (cGMP) levels. At the same time, H2S both inhibits phosphodiesterase 5A (PDE5A), an enzyme that metabolizes sGC and exerts redox regulation on sGC. The role of NO, CO, and H2S in the setting of cancer has been quite perplexing, as there is evidence for both tumor-promoting and pro-inflammatory effects and anti-tumor and anti-inflammatory activities. Each gasotransmitter has been found to have dual effects on different aspects of cancer biology, including cancer cell proliferation and apoptosis, invasion and metastasis, angiogenesis, and immunomodulation. These seemingly contradictory actions may relate to each gas having a dual effect dependent on its local flux. In this review, we discuss the major roles of NO, CO, and H2S in the context of cancer, with an effort to highlight the dual nature of each gas in different events occurring during cancer progression.
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Affiliation(s)
- Palak P Oza
- Department of Molecular, Cellular and Biomedical Sciences, Sophie Davis School of Biomedical Education, City University of New York School of Medicine, New York, NY 10031, USA
| | - Khosrow Kashfi
- Department of Molecular, Cellular and Biomedical Sciences, Sophie Davis School of Biomedical Education, City University of New York School of Medicine, New York, NY 10031, USA; Graduate Program in Biology, City University of New York Graduate Center, New York 10091, USA.
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2
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Frei A, Verderosa AD, Elliott AG, Zuegg J, Blaskovich MAT. Metals to combat antimicrobial resistance. Nat Rev Chem 2023; 7:202-224. [PMID: 37117903 PMCID: PMC9907218 DOI: 10.1038/s41570-023-00463-4] [Citation(s) in RCA: 203] [Impact Index Per Article: 101.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/06/2023] [Indexed: 02/10/2023]
Abstract
Bacteria, similar to most organisms, have a love-hate relationship with metals: a specific metal may be essential for survival yet toxic in certain forms and concentrations. Metal ions have a long history of antimicrobial activity and have received increasing attention in recent years owing to the rise of antimicrobial resistance. The search for antibacterial agents now encompasses metal ions, nanoparticles and metal complexes with antimicrobial activity ('metalloantibiotics'). Although yet to be advanced to the clinic, metalloantibiotics are a vast and underexplored group of compounds that could lead to a much-needed new class of antibiotics. This Review summarizes recent developments in this growing field, focusing on advances in the development of metalloantibiotics, in particular, those for which the mechanism of action has been investigated. We also provide an overview of alternative uses of metal complexes to combat bacterial infections, including antimicrobial photodynamic therapy and radionuclide diagnosis of bacterial infections.
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Affiliation(s)
- Angelo Frei
- Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.
- Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland.
| | - Anthony D Verderosa
- Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia
| | - Alysha G Elliott
- Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia
| | - Johannes Zuegg
- Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia
| | - Mark A T Blaskovich
- Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.
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3
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CORM-3 induces DNA damage through Ru(II) binding to DNA. Biochem J 2022; 479:1429-1439. [PMID: 35726678 PMCID: PMC9342897 DOI: 10.1042/bcj20220254] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 06/20/2022] [Accepted: 06/21/2022] [Indexed: 12/02/2022]
Abstract
When the ‘CO-releasing molecule-3’, CORM-3 (Ru(CO)3Cl(glycinate)), is dissolved in water it forms a range of ruthenium complexes. These are taken up by cells and bind to intracellular ligands, notably thiols such as cysteine and glutathione, where the Ru(II) reaches high intracellular concentrations. Here, we show that the Ru(II) ion also binds to DNA, at exposed guanosine N7 positions. It therefore has a similar cellular target to the anticancer drug cisplatin, but not identical, because Ru(II) shows no evidence of forming intramolecular crossbridges in the DNA. The reaction is slow, and with excess Ru, intermolecular DNA crossbridges are formed. The addition of CORM-3 to human colorectal cancer cells leads to strand breaks in the DNA, as assessed by the alkaline comet assay. DNA damage is inhibited by growth media containing amino acids, which bind to extracellular Ru and prevent its entry into cells. We conclude that the cytotoxicity of Ru(II) is different from that of platinum, making it a promising development target for cancer therapeutics.
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Mansour AM, Khaled RM, Khaled E, Ahmed SK, Ismael OS, Zeinhom A, Magdy H, Ibrahim SS, Abdelfatah M. Ruthenium(II) carbon monoxide releasing molecules: Structural perspective, antimicrobial and anti-inflammatory properties. Biochem Pharmacol 2022; 199:114991. [DOI: 10.1016/j.bcp.2022.114991] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 03/05/2022] [Accepted: 03/07/2022] [Indexed: 01/12/2023]
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5
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Cavazza C, Collin-Faure V, Pérard J, Diemer H, Cianférani S, Rabilloud T, Darrouzet E. Proteomic analysis of Rhodospirillum rubrum after carbon monoxide exposure reveals an important effect on metallic cofactor biosynthesis. J Proteomics 2022; 250:104389. [PMID: 34601154 DOI: 10.1016/j.jprot.2021.104389] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 09/16/2021] [Accepted: 09/20/2021] [Indexed: 12/14/2022]
Abstract
Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H2O → CO2 + H2). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions. SIGNIFICANCE: This proteomic study highlights the fact that even in a microorganism able to handle carbon monoxide and in some way detoxifying it via the intrinsic action of the carbon monoxide dehydrogenase (CODH), CO has important effects on metal homeostasis, metal cofactors and metalloproteins. These effects are direct or indirect via transcription regulation, and amplified by the high interdependency of cofactors biosynthesis.
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Affiliation(s)
- Christine Cavazza
- Univ. Grenoble Alpes, CEA, CNRS, IRIG, CBM, F-38000 Grenoble, France.
| | | | - Julien Pérard
- Univ. Grenoble Alpes, CEA, CNRS, IRIG, CBM, F-38000 Grenoble, France.
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France; Infrastructure Nationale de Protéomique ProFI - FR2048 (CNRS-CEA), 67087 Strasbourg, France.
| | - Sarah Cianférani
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France; Infrastructure Nationale de Protéomique ProFI - FR2048 (CNRS-CEA), 67087 Strasbourg, France.
| | - Thierry Rabilloud
- Univ. Grenoble Alpes, CEA, CNRS, IRIG, CBM, F-38000 Grenoble, France.
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6
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Cheng J, Hu J. Recent Advances on Carbon Monoxide Releasing Molecules for Antibacterial Applications. ChemMedChem 2021; 16:3628-3634. [PMID: 34613654 DOI: 10.1002/cmdc.202100555] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Revised: 09/21/2021] [Indexed: 12/26/2022]
Abstract
Carbon monoxide (CO) has been known as an endogenous signaling molecule in addition to an air pollutant. It plays a critical role in many physiological and pathological processes. Therefore, CO has been recognized as a potent therapeutic agent for the treatment of numerous diseases such as cancers, rheumatoid arthritis, and so on. Instead of direct CO inhalation, two main categories of CO-releasing molecules (CORMs) (i. e., metal carbonyls and nonmetallic CO donors) have been developed to safely and locally deliver CO to target tissues. In this minireview, we summarize the recent achievements of CORMs on antibacterial applications. It appears that the antibacterial activity of CORMs is different from CO gas, which is tightly correlated to not only the types of CORMs applied but also the tested bacterial strains. In some circumstances, the antibacterial mechanisms are debated and need to be clarified. We hope more attention can be paid to this emerging field and new antibacterial agents with a low risk of drug resistance can be developed.
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Affiliation(s)
- Jian Cheng
- Department of Polymer Science and Engineering, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, 96 Jinzhai Road, Anhui 230026, Hefei, China
| | - Jinming Hu
- Department of Polymer Science and Engineering, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, 96 Jinzhai Road, Anhui 230026, Hefei, China
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7
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Munteanu AC, Uivarosi V. Ruthenium Complexes in the Fight against Pathogenic Microorganisms. An Extensive Review. Pharmaceutics 2021; 13:874. [PMID: 34199283 PMCID: PMC8232020 DOI: 10.3390/pharmaceutics13060874] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Revised: 06/07/2021] [Accepted: 06/09/2021] [Indexed: 12/13/2022] Open
Abstract
The widespread use of antibiotics has resulted in the emergence of drug-resistant populations of microorganisms. Clearly, one can see the need to develop new, more effective, antimicrobial agents that go beyond the explored 'chemical space'. In this regard, their unique modes of action (e.g., reactive oxygen species (ROS) generation, redox activation, ligand exchange, depletion of substrates involved in vital cellular processes) render metal complexes as promising drug candidates. Several Ru (II/III) complexes have been included in, or are currently undergoing, clinical trials as anticancer agents. Based on the in-depth knowledge of their chemical properties and biological behavior, the interest in developing new ruthenium compounds as antibiotic, antifungal, antiparasitic, or antiviral drugs has risen. This review will discuss the advantages and disadvantages of Ru (II/III) frameworks as antimicrobial agents. Some aspects regarding the relationship between their chemical structure and mechanism of action, cellular localization, and/or metabolism of the ruthenium complexes in bacterial and eukaryotic cells are discussed as well. Regarding the antiviral activity, in light of current events related to the Covid-19 pandemic, the Ru (II/III) compounds used against SARS-CoV-2 (e.g., BOLD-100) are also reviewed herein.
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Affiliation(s)
- Alexandra-Cristina Munteanu
- Department of General and Inorganic Chemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 020956 Bucharest, Romania
| | - Valentina Uivarosi
- Department of General and Inorganic Chemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 020956 Bucharest, Romania
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8
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Southam HM, Williamson MP, Chapman JA, Lyon RL, Trevitt CR, Henderson PJF, Poole RK. 'Carbon-Monoxide-Releasing Molecule-2 (CORM-2)' Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects. Antioxidants (Basel) 2021; 10:antiox10060915. [PMID: 34198746 PMCID: PMC8227206 DOI: 10.3390/antiox10060915] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Revised: 05/31/2021] [Accepted: 06/02/2021] [Indexed: 02/06/2023] Open
Abstract
Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.
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Affiliation(s)
- Hannah M. Southam
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
| | - Michael P. Williamson
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
| | - Jonathan A. Chapman
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
- Centre for Bacterial Cell Biology, Medical School, Newcastle University, Newcastle upon Tyne NE2 4AX, UK
| | - Rhiannon L. Lyon
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
| | - Clare R. Trevitt
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
| | - Peter J. F. Henderson
- School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK;
| | - Robert K. Poole
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (H.M.S.); (M.P.W.); (J.A.C.); (R.L.L.); (C.R.T.)
- Correspondence:
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9
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Cheng J, Gan G, Shen Z, Gao L, Zhang G, Hu J. Red Light‐Triggered Intracellular Carbon Monoxide Release Enables Selective Eradication of MRSA Infection. Angew Chem Int Ed Engl 2021. [DOI: 10.1002/ange.202104024] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Affiliation(s)
- Jian Cheng
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
| | - Guihai Gan
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
| | - Zhiqiang Shen
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
| | - Lei Gao
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
| | - Guoying Zhang
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
| | - Jinming Hu
- CAS Key Laboratory of Soft Matter Chemistry Hefei National Laboratory for Physical Science at the Microscale Department of Polymer Science and Engineering University of Science and Technology of China, Hefei 230026 Anhui China
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10
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Cheng J, Gan G, Shen Z, Gao L, Zhang G, Hu J. Red Light-Triggered Intracellular Carbon Monoxide Release Enables Selective Eradication of MRSA Infection. Angew Chem Int Ed Engl 2021; 60:13513-13520. [PMID: 33829616 DOI: 10.1002/anie.202104024] [Citation(s) in RCA: 57] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Indexed: 12/23/2022]
Abstract
Carbon monoxide (CO) is an important gaseous signaling molecule. The use of CO-releasing molecules such as metal carbonyls enables the elucidation of the pleiotropic functions of CO. Although metal carbonyls show a broad-spectrum antimicrobial activity, it remains unclear whether the bactericidal property originates from the transition metals or the released CO. Here, we develop nonmetallic CO-releasing micelles via a photooxygenation mechanism of 3-hydroxyflavone derivatives, enabling CO release under red light irradiation (e.g., 650 nm). Unlike metal carbonyls that non-specifically internalize into both Gram-positive and Gram-negative bacteria, the nonmetallic micelles are selectively taken up by S. aureus instead of E. coli cells, exerting a selective bactericidal effect. Further, we demonstrate that the CO-releasing micelles can cure methicillin-resistant S. aureus (MRSA)-infected wounds, simultaneously eradicating MRSA pathogens and accelerating wound healing.
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Affiliation(s)
- Jian Cheng
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
| | - Guihai Gan
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
| | - Zhiqiang Shen
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
| | - Lei Gao
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
| | - Guoying Zhang
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
| | - Jinming Hu
- CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, China
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11
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Varney AM, Smitten KL, Thomas JA, McLean S. Transcriptomic Analysis of the Activity and Mechanism of Action of a Ruthenium(II)-Based Antimicrobial That Induces Minimal Evolution of Pathogen Resistance. ACS Pharmacol Transl Sci 2021; 4:168-178. [PMID: 33615170 PMCID: PMC7887750 DOI: 10.1021/acsptsci.0c00159] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Indexed: 01/30/2023]
Abstract
Increasing concern over rising levels of antibiotic resistance among pathogenic bacteria has prompted significant research into developing efficacious alternatives to antibiotic treatment. Previously, we have reported on the therapeutic activity of a dinuclear ruthenium(II) complex against pathogenic, multi-drug-resistant bacterial pathogens. Herein, we report that the solubility properties of this lead are comparable to those exhibited by orally available therapeutics that in comparison to clinically relevant antibiotics it induces very slow evolution of resistance in the uropathogenic, therapeutically resistant, E. coli strain EC958, and this resistance was lost when exposure to the compound was temporarily removed. With the aim of further investigating the mechanism of action of this compound, the regulation of nine target genes relating to the membrane, DNA damage, and other stress responses provoked by exposure to the compound was also studied. This analysis confirmed that the compound causes a significant transcriptional downregulation of genes involved in membrane transport and the tricarboxylic acid cycle. By contrast, expression of the chaperone protein-coding gene, spy, was significantly increased suggesting a requirement for repair of damaged proteins in the region of the outer membrane. The complex was also found to display activity comparable to that in E. coli in a range of other therapeutically relevant Gram-negative pathogens.
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Affiliation(s)
- Adam M. Varney
- School
of Science and Technology, Nottingham Trent
University, Nottingham NG11 8NS, United Kingdom
| | - Kirsty L. Smitten
- Department
of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, United
Kingdom
| | - Jim A. Thomas
- Department
of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, United
Kingdom
| | - Samantha McLean
- School
of Science and Technology, Nottingham Trent
University, Nottingham NG11 8NS, United Kingdom
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12
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Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major Target. Antimicrob Agents Chemother 2019; 63:AAC.00643-19. [PMID: 31332064 DOI: 10.1128/aac.00643-19] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 07/12/2019] [Indexed: 12/23/2022] Open
Abstract
In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.
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13
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Reactivity of CORM [RuII(CO)3Cl2{N-(N1-methylbenzimidazole)}] with aminoacids. Synthesis, and analytical and structural study for the new binuclear cis-[RuI(CO)2(N-MBI)(μ2-O,O-BAL)]2 sawhorse complex at solid state and in solution. J Mol Struct 2019. [DOI: 10.1016/j.molstruc.2019.02.051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
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14
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Southam HM, Smith TW, Lyon RL, Liao C, Trevitt CR, Middlemiss LA, Cox FL, Chapman JA, El-Khamisy SF, Hippler M, Williamson MP, Henderson PJF, Poole RK. A thiol-reactive Ru(II) ion, not CO release, underlies the potent antimicrobial and cytotoxic properties of CO-releasing molecule-3. Redox Biol 2018; 18:114-123. [PMID: 30007887 PMCID: PMC6067063 DOI: 10.1016/j.redox.2018.06.008] [Citation(s) in RCA: 73] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Accepted: 06/23/2018] [Indexed: 12/25/2022] Open
Abstract
Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological ‘gasotransmitter’, in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media – such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) – protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 μM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 μM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs.
Carbon monoxide-releasing molecules (CORMs) are used for experimental CO delivery. CORM-3 is a potent antimicrobial, but is reportedly beneficial to mammalian cells. We demonstrate CORM-3 releases little CO in buffers and cell culture media. Redox-active Ru2+ is the biological agent, binding tightly to metabolites e.g. thiol. These results necessitate a major reappraisal of the biological effects of CORMs.
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Affiliation(s)
- Hannah M Southam
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Thomas W Smith
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, UK
| | - Rhiannon L Lyon
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Chunyan Liao
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Clare R Trevitt
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Laurence A Middlemiss
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, UK
| | - Francesca L Cox
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, UK
| | - Jonathan A Chapman
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, UK
| | - Sherif F El-Khamisy
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Michael Hippler
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S3 7HF, UK
| | - Michael P Williamson
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
| | - Peter J F Henderson
- School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
| | - Robert K Poole
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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15
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Wareham LK, McLean S, Begg R, Rana N, Ali S, Kendall JJ, Sanguinetti G, Mann BE, Poole RK. The Broad-Spectrum Antimicrobial Potential of [Mn(CO) 4(S 2CNMe(CH 2CO 2H))], a Water-Soluble CO-Releasing Molecule (CORM-401): Intracellular Accumulation, Transcriptomic and Statistical Analyses, and Membrane Polarization. Antioxid Redox Signal 2018; 28:1286-1308. [PMID: 28816060 PMCID: PMC5905950 DOI: 10.1089/ars.2017.7239] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
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.
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Affiliation(s)
- Lauren K Wareham
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Samantha McLean
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom .,2 School of Science and Technology , Nottingham Trent University, Nottingham, United Kingdom
| | - Ronald Begg
- 3 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Namrata Rana
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Salar Ali
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - John J Kendall
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Guido Sanguinetti
- 3 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Brian E Mann
- 4 Department of Chemistry, The University of Sheffield , Sheffield, United Kingdom
| | - Robert K Poole
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
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16
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Rana N, Jesse HE, Tinajero-Trejo M, Butler JA, Tarlit JD, von Und Zur Muhlen ML, Nagel C, Schatzschneider U, Poole RK. A manganese photosensitive tricarbonyl molecule [Mn(CO)3(tpa-κ 3N)]Br enhances antibiotic efficacy in a multi-drug-resistant Escherichia coli. MICROBIOLOGY-SGM 2017; 163:1477-1489. [PMID: 28954688 PMCID: PMC5845575 DOI: 10.1099/mic.0.000526] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Carbon monoxide-releasing molecules (CORMs) are a promising class of new antimicrobials, with multiple modes of action that are distinct from those of standard antibiotics. The relentless increase in antimicrobial resistance, exacerbated by a lack of new antibiotics, necessitates a better understanding of how such novel agents act and might be used synergistically with established antibiotics. This work aimed to understand the mechanism(s) underlying synergy between a manganese-based photoactivated carbon monoxide-releasing molecule (PhotoCORM), [Mn(CO)3(tpa-κ3N)]Br [tpa=tris(2-pyridylmethyl)amine], and various classes of antibiotics in their activities towards Escherichia coli EC958, a multi-drug-resistant uropathogen. The title compound acts synergistically with polymyxins [polymyxin B and colistin (polymyxin E)] by damaging the bacterial cytoplasmic membrane. [Mn(CO)3(tpa-κ3N)]Br also potentiates the action of doxycycline, resulting in reduced expression of tetA, which encodes a tetracycline efflux pump. We show that, like tetracyclines, the breakdown products of [Mn(CO)3(tpa-κ3N)]Br activation chelate iron and trigger an iron starvation response, which we propose to be a further basis for the synergies observed. Conversely, media supplemented with excess iron abrogated the inhibition of growth by doxycycline and the title compound. In conclusion, multiple factors contribute to the ability of this PhotoCORM to increase the efficacy of antibiotics in the polymyxin and tetracycline families. We propose that light-activated carbon monoxide release is not the sole basis of the antimicrobial activities of [Mn(CO)3(tpa-κ3N)]Br.
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Affiliation(s)
- Namrata Rana
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
| | - Helen E Jesse
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
| | - Mariana Tinajero-Trejo
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK.,Present address: Cell Biology Program, The Hospital for Sick Children, Toronto, Canada
| | - Jonathan A Butler
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK.,Present address: School of Healthcare Science, Manchester Metropolitan University, UK
| | - John D Tarlit
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
| | | | - Christoph Nagel
- Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
| | - Ulrich Schatzschneider
- Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
| | - Robert K Poole
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
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17
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Sahlberg Bang C, Demirel I, Kruse R, Persson K. Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli. PLoS One 2017; 12:e0178541. [PMID: 28591134 PMCID: PMC5462378 DOI: 10.1371/journal.pone.0178541] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Accepted: 05/15/2017] [Indexed: 12/02/2022] Open
Abstract
Treatment of urinary tract infections is today a challenge due to the increasing prevalence of multidrug-resistant ESBL-producing uropathogenic Escherichia coli (UPEC). There is an urgent need for new treatment strategies for multidrug-resistant UPEC and preferably with targets that have low potential for development of resistance. Carbon monoxide-releasing molecules (CORMs) are novel and potent antibacterial agents. The present study examines the transcriptomic targets of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate in response to a single exposure to CORM-2 and after repeated exposure to CORM-2. The bacterial viability and minimal inhibitory concentration (MIC) were also examined after repeated exposure to CORM-2. Microarray analysis revealed that a wide range of processes were affected by CORM-2, including a general trend of down-regulation in energy metabolism and biosynthesis pathways and up-regulation of the SOS response and DNA repair. Several genes involved in virulence (ibpB), antibiotic resistance (marAB, mdtABC) and biofilm formation (bhsA, yfgF) were up-regulated, while some genes involved in virulence (kpsC, fepCEG, entABE), antibiotic resistance (evgA) and biofilm formation (artIP) were down-regulated. Repeated exposure to CORM-2 did not alter the gene expression patterns, the growth inhibitory response to CORM-2 or the MIC values for CORM-2, cefotaxime, ciprofloxacin and trimethoprim. This study identifies several enriched gene ontologies, modified pathways and single genes that are targeted by CORM-2 in a multidrug-resistant UPEC isolate. Repeated exposure to CORM-2 did not change the gene expression patterns or fold changes and the susceptibility to CORM-2 remained after repeated exposure.
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Affiliation(s)
- Charlotte Sahlberg Bang
- School of Medical Sciences, Faculty of Medicine and Health, iRiSC—Inflammatory Response and Infection Susceptibility Centre, Örebro University, Örebro, Sweden
- * E-mail:
| | - Isak Demirel
- School of Medical Sciences, Faculty of Medicine and Health, iRiSC—Inflammatory Response and Infection Susceptibility Centre, Örebro University, Örebro, Sweden
| | - Robert Kruse
- School of Medical Sciences, Faculty of Medicine and Health, iRiSC—Inflammatory Response and Infection Susceptibility Centre, Örebro University, Örebro, Sweden
| | - Katarina Persson
- School of Medical Sciences, Faculty of Medicine and Health, iRiSC—Inflammatory Response and Infection Susceptibility Centre, Örebro University, Örebro, Sweden
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18
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Takeda TA, Sasai M, Adachi Y, Ohnishi K, Fujisawa JI, Izawa S, Taketani S. Potential role of heme metabolism in the inducible expression of heme oxygenase-1. Biochim Biophys Acta Gen Subj 2017; 1861:1813-1824. [PMID: 28347842 DOI: 10.1016/j.bbagen.2017.03.018] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2016] [Revised: 03/06/2017] [Accepted: 03/23/2017] [Indexed: 01/17/2023]
Abstract
BACKGROUND The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. METHODS We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. RESULTS The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. CONCLUSIONS We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. GENERAL SIGNIFICANCE The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.
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Affiliation(s)
- Taka-Aki Takeda
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan
| | - Machiko Sasai
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan
| | - Yuka Adachi
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan
| | - Keiko Ohnishi
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan
| | - Jun-Ichi Fujisawa
- Department of Microbiology, Kansai Medical University, Hirakata, Osaka 573-8510, Japan
| | - Shingo Izawa
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan
| | - Shigeru Taketani
- Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8510, Japan; Unit of Research Complex, Kansai Medical University, Hirakata, Osaka 573-8510, Japan.
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19
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Abstract
Ruthenium is seldom mentioned in microbiology texts, due to the fact that this metal has no known, essential roles in biological systems, nor is it generally considered toxic. Since the fortuitous discovery of cisplatin, first as an antimicrobial agent and then later employed widely as an anticancer agent, complexes of other platinum group metals, such as ruthenium, have attracted interest for their medicinal properties. Here, we review at length how ruthenium complexes have been investigated as potential antimicrobial, antiparasitic and chemotherapeutic agents, in addition to their long and well-established roles as biological stains and inhibitors of calcium channels. Ruthenium complexes are also employed in a surprising number of biotechnological roles. It is in the employment of ruthenium complexes as antimicrobial agents and alternatives or adjuvants to more traditional antibiotics, that we expect to see the most striking developments in the future. Such novel contributions from organometallic chemistry are undoubtedly sorely needed to address the antimicrobial resistance crisis and the slow appearance on the market of new antibiotics.
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20
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Nacci C, Fanelli M, Potenza MA, Leo V, Montagnani M, De Salvia MA. Carbon monoxide contributes to the constipating effects of granisetron in rat colon. World J Gastroenterol 2016; 22:9333-9345. [PMID: 27895421 PMCID: PMC5107697 DOI: 10.3748/wjg.v22.i42.9333] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/24/2016] [Revised: 09/16/2016] [Accepted: 10/19/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the mechanisms underlying the potential contribution of the heme oxygenase/carbon monoxide (HO/CO) pathway in the constipating effects of granisetron.
METHODS For in vivo studies, gastrointestinal motility was evaluated in male rats acutely treated with granisetron [25, 50, 75 μg/kg/subcutaneous (sc)], zinc protoporphyrin IX [ZnPPIX, 50 μg/kg/intraperitoneal (ip)] and hemin (50 μmol/L/kg/ip), alone or in combination. For in vitro studies, the contractile neurogenic response to electrical field stimulation (EFS, 3, 5, 10 Hz, 14 V, 1 ms, pulse trains lasting 10 s), as well as the contractile myogenic response to acetylcholine (ACh, 0.1-100 μmol/L) were evaluated on colon specimens incubated with granisetron (3 μmol/L, 15 min), ZnPPIX (10 μmol/L, 60 min) or CO-releasing molecule-3 (CORM-3, 100, 200, 400 μmol/L) alone or in combination. These experiments were performed under co-treatment with or without atropine (3 μmol/L, a muscarinic receptor antagonist) or NG-nitro-L-Arginine (L-NNA, 100 μmol/L, a nitric oxide synthase inhibitor).
RESULTS Administration of granisetron (50, 75 μg/kg) in vivo significantly increased the time to first defecation (P = 0.045 vs vehicle-treated rats), clearly suggesting a constipating effect of this drug. Although administration of ZnPPIX or hemin alone had no effect on this gastrointestinal motility parameter, ZnPPIX co-administered with granisetron abolished the granisetron-induced constipation. On the other hand, co-administration of hemin and granisetron did not modify the increased constipation observed under granisetron alone. When administered in vitro, granisetron alone (3 μmol/L) did not significantly modify the colon’s contractile response to either EFS or ACh. Incubation with ZnPPIX alone (10 μmol/L) significantly reduced the colon’s contractile response to EFS (P = 0.016) but had no effect on contractile response to ACh. Co-administration of ZnPPIX and atropine (3 μmol/L) abolished the ZnPPIX-mediated decrease in contractile response to EFS. Conversely, incubation with CORM-3 (400 μmol/L) alone increased both the contractile response to EFS at 10 Hz (10 Hz: 71.02 ± 19.16 vs 116.25 ± 53.70, P = 0.01) and the contractile response to ACh (100 μmol/L) (P = 0.012). Co-administration of atropine abolished the CORM-3-mediated effects on the EFS-mediated response. When granisetron was co-incubated in vitro with ZnPPIX, the ZnPPIX-mediated decrease in colon contractile response to EFS was lost. On the other hand, co-incubation of granisetron and CORM-3 (400 μmol/L) further increased the colon’s contractile response to EFS (at 5 Hz: P = 0.007; at 10 Hz: P = 0.001) and to ACh (ACh 10 μmol/L: P = 0.001; ACh 100 μmol/L: P = 0.001) elicited by CORM-3 alone. L-NNA co-administered with granisetron and CORM-3 abolished the potentiating effect of CORM-3 on granisetron on both the EFS-induced and ACh-induced contractile response.
CONCLUSION Taken together, findings from in vivo and in vitro studies suggest that the HO/CO pathway is involved in the constipating effects of granisetron.
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21
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Wareham LK, Begg R, Jesse HE, Van Beilen JWA, Ali S, Svistunenko D, McLean S, Hellingwerf KJ, Sanguinetti G, Poole RK. Carbon Monoxide Gas Is Not Inert, but Global, in Its Consequences for Bacterial Gene Expression, Iron Acquisition, and Antibiotic Resistance. Antioxid Redox Signal 2016; 24:1013-28. [PMID: 26907100 PMCID: PMC4921903 DOI: 10.1089/ars.2015.6501] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
AIMS Carbon monoxide is a respiratory poison and gaseous signaling molecule. Although CO-releasing molecules (CORMs) deliver CO with temporal and spatial specificity in mammals, and are proven antimicrobial agents, we do not understand the modes of CO toxicity. Our aim was to explore the impact of CO gas per se, without intervention of CORMs, on bacterial physiology and gene expression. RESULTS We used tightly controlled chemostat conditions and integrated transcriptomic datasets with statistical modeling to reveal the global effects of CO. CO is known to inhibit bacterial respiration, and we found expression of genes encoding energy-transducing pathways to be significantly affected via the global regulators, Fnr, Arc, and PdhR. Aerobically, ArcA-the response regulator-is transiently phosphorylated and pyruvate accumulates, mimicking anaerobiosis. Genes implicated in iron acquisition, and the metabolism of sulfur amino acids and arginine, are all perturbed. The global iron-related changes, confirmed by modulation of activity of the transcription factor Fur, may underlie enhanced siderophore excretion, diminished intracellular iron pools, and the sensitivity of CO-challenged bacteria to metal chelators. Although CO gas (unlike H2S and NO) offers little protection from antibiotics, a ruthenium CORM is a potent adjuvant of antibiotic activity. INNOVATION This is the first detailed exploration of global bacterial responses to CO, revealing unexpected targets with implications for employing CORMs therapeutically. CONCLUSION This work reveals the complexity of bacterial responses to CO and provides a basis for understanding the impacts of CO from CORMs, heme oxygenase activity, or environmental sources. Antioxid. Redox Signal. 24, 1013-1028.
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Affiliation(s)
- Lauren K Wareham
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Ronald Begg
- 2 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Helen E Jesse
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Johan W A Van Beilen
- 3 Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands
| | - Salar Ali
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Dimitri Svistunenko
- 4 Biomedical EPR Facility, School of Biological Sciences, University of Essex , Colchester, United Kingdom
| | - Samantha McLean
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Klaas J Hellingwerf
- 3 Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands
| | - Guido Sanguinetti
- 2 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Robert K Poole
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
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22
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Tinajero-Trejo M, Rana N, Nagel C, Jesse HE, Smith TW, Wareham LK, Hippler M, Schatzschneider U, Poole RK. Antimicrobial Activity of the Manganese Photoactivated Carbon Monoxide-Releasing Molecule [Mn(CO)3(tpa-κ(3)N)](+) Against a Pathogenic Escherichia coli that Causes Urinary Infections. Antioxid Redox Signal 2016; 24:765-80. [PMID: 26842766 PMCID: PMC4876522 DOI: 10.1089/ars.2015.6484] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
AIMS We set out to investigate the antibacterial activity of a new Mn-based photoactivated carbon monoxide-releasing molecule (PhotoCORM, [Mn(CO)3(tpa-κ(3)N)](+)) against an antibiotic-resistant uropathogenic strain (EC958) of Escherichia coli. RESULTS Activated PhotoCORM inhibits growth and decreases viability of E. coli EC958, but non-illuminated carbon monoxide-releasing molecule (CORM) is without effect. NADH-supported respiration rates are significantly decreased by activated PhotoCORM, mimicking the effect of dissolved CO gas. CO from the PhotoCORM binds to intracellular targets, namely respiratory oxidases in strain EC958 and a bacterial globin heterologously expressed in strain K-12. However, unlike previously characterized CORMs, the PhotoCORM is not significantly accumulated in cells, as deduced from the cellular manganese content. Activated PhotoCORM reacts avidly with hydrogen peroxide producing hydroxyl radicals; the observed peroxide-enhanced toxicity of the PhotoCORM is ameliorated by thiourea. The PhotoCORM also potentiates the effect of the antibiotic, doxycycline. INNOVATION The present work investigates for the first time the antimicrobial activity of a light-activated PhotoCORM against an antibiotic-resistant pathogen. A comprehensive study of the effects of the PhotoCORM and its derivative molecules upon illumination is performed and mechanisms of toxicity of the activated PhotoCORM are investigated. CONCLUSION The PhotoCORM allows a site-specific and time-controlled release of CO in bacterial cultures and has the potential to provide much needed information on the generality of CORM activities in biology. Understanding the mechanism(s) of activated PhotoCORM toxicity will be key in exploring the potential of this and similar compounds as antimicrobial agents, perhaps in combinatorial therapies with other agents. Antioxid. Redox Signal. 24, 765-780.
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Affiliation(s)
- Mariana Tinajero-Trejo
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Namrata Rana
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Christoph Nagel
- 2 Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg , Würzburg, Germany
| | - Helen E Jesse
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Thomas W Smith
- 3 Department of Chemistry, The University of Sheffield , Sheffield, United Kingdom
| | - Lauren K Wareham
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Michael Hippler
- 3 Department of Chemistry, The University of Sheffield , Sheffield, United Kingdom
| | - Ulrich Schatzschneider
- 2 Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg , Würzburg, Germany
| | - Robert K Poole
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
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23
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Sahlberg Bang C, Kruse R, Johansson K, Persson K. Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization. BMC Microbiol 2016; 16:64. [PMID: 27067266 PMCID: PMC4828782 DOI: 10.1186/s12866-016-0678-7] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Accepted: 03/29/2016] [Indexed: 12/24/2022] Open
Abstract
Background Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. Results The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 μM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. Conclusion This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.
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Affiliation(s)
- Charlotte Sahlberg Bang
- Faculty of Medicine and Health, Örebro University, SE-701 82, Örebro, Sweden.,iRiSC - Inflammatory Responses and Infection Susceptibility Centre, Örebro University, SE-701 82, Örebro, Sweden
| | - Robert Kruse
- Faculty of Medicine and Health, Örebro University, SE-701 82, Örebro, Sweden.,iRiSC - Inflammatory Responses and Infection Susceptibility Centre, Örebro University, SE-701 82, Örebro, Sweden
| | - Kjell Johansson
- Faculty of Medicine and Health, Örebro University, SE-701 82, Örebro, Sweden
| | - Katarina Persson
- Faculty of Medicine and Health, Örebro University, SE-701 82, Örebro, Sweden. .,iRiSC - Inflammatory Responses and Infection Susceptibility Centre, Örebro University, SE-701 82, Örebro, Sweden.
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24
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Wilson JL, Wareham LK, McLean S, Begg R, Greaves S, Mann BE, Sanguinetti G, Poole RK. CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli. Antioxid Redox Signal 2015; 23:148-62. [PMID: 25811604 PMCID: PMC4492677 DOI: 10.1089/ars.2014.6151] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
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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Affiliation(s)
- Jayne Louise Wilson
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Lauren K Wareham
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Samantha McLean
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Ronald Begg
- 2 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Sarah Greaves
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
| | - Brian E Mann
- 3 Department of Chemistry, The University of Sheffield , Sheffield, United Kingdom
| | - Guido Sanguinetti
- 2 School of Informatics, The University of Edinburgh , Edinburgh, United Kingdom
| | - Robert K Poole
- 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom
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25
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Wareham LK, Poole RK, Tinajero-Trejo M. CO-releasing Metal Carbonyl Compounds as Antimicrobial Agents in the Post-antibiotic Era. J Biol Chem 2015; 290:18999-9007. [PMID: 26055702 PMCID: PMC4521022 DOI: 10.1074/jbc.r115.642926] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The possibility of a “post-antibiotic era” in the 21st century, in which common infections may kill, has prompted research into radically new antimicrobials. CO-releasing molecules (CORMs), mostly metal carbonyl compounds, originally developed for therapeutic CO delivery in animals, are potent antimicrobial agents. Certain CORMs inhibit growth and respiration, reduce viability, and release CO to intracellular hemes, as predicted, but their actions are more complex, as revealed by transcriptomic datasets and modeling. Progress is hindered by difficulties in detecting CO release intracellularly, limited understanding of the biological chemistry of CO reactions with non-heme targets, and the cytotoxicity of some CORMs to mammalian cells.
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Affiliation(s)
- Lauren K Wareham
- From the Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom
| | - Robert K Poole
- From the Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom
| | - Mariana Tinajero-Trejo
- From the Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom
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26
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Borirak O, Rolfe MD, de Koning LJ, Hoefsloot HCJ, Bekker M, Dekker HL, Roseboom W, Green J, de Koster CG, Hellingwerf KJ. Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2015; 1854:1269-79. [PMID: 26049081 DOI: 10.1016/j.bbapap.2015.05.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2015] [Revised: 05/12/2015] [Accepted: 05/28/2015] [Indexed: 10/23/2022]
Abstract
Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.
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Affiliation(s)
- Orawan Borirak
- Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Matthew D Rolfe
- Krebs Institute, Molecular Biology and Biotechnology, University of Sheffield, United Kingdom
| | - Leo J de Koning
- Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Huub C J Hoefsloot
- Biosystems Data Analysis, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Martijn Bekker
- Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Henk L Dekker
- Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Winfried Roseboom
- Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Jeffrey Green
- Krebs Institute, Molecular Biology and Biotechnology, University of Sheffield, United Kingdom
| | - Chris G de Koster
- Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
| | - Klaas J Hellingwerf
- Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands.
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27
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Nagel C, McLean S, Poole RK, Braunschweig H, Kramer T, Schatzschneider U. Introducing [Mn(CO)3(tpa-κ(3)N)](+) as a novel photoactivatable CO-releasing molecule with well-defined iCORM intermediates - synthesis, spectroscopy, and antibacterial activity. Dalton Trans 2015; 43:9986-97. [PMID: 24855638 DOI: 10.1039/c3dt51848e] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
[Mn(CO)3(tpa-κ(3)N)]Br was prepared as a novel photoactivatable CO-releasing molecule (PhotoCORM) from [MnBr(CO)5] and tris(2-pyridylmethyl)amine (tpa) for the delivery of carbon monoxide to biological systems, with the κ(3)N binding mode of the tetradentate tpa ligand demonstrated by X-ray crystallography. The title compound is a CORM prodrug stable in solution in the dark for up to 16 h. However, photoactivation at 365 nm leads to CO release from the metal coordination sphere and transfer to haem proteins, as demonstrated by the standard myoglobin assay. Different iCORM intermediates could be detected with solution IR spectroscopy and assigned using DFT vibrational calculations. The antibacterial activity of the complex was studied on Escherichia coli. No effects were observed when the cultures were either kept in the dark in the presence of PhotoCORM or illuminated in the absence of metal complex. However, photoactivation of [Mn(CO)3(tpa-κ(3)N)]Br at 365 nm led to the appearance of the spectral signatures of CO-coordinated haems in the terminal oxidases of the bacterial electron transport chain in whole-cell UV/Vis absorption spectra. Significant internalization of the PhotoCORM was demonstrated by ICP-MS measurement of the intracellular manganese concentration. In particular when using medium with succinate as the sole carbon source, a very pronounced and concentration-dependent decrease in the E. coli growth rate could be observed upon illumination in the presence of metal complex, which is attributed to the constrained energy metabolism under these conditions and a strong indicator of terminal oxidase inhibition by carbon monoxide delivered from the PhotoCORM.
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Affiliation(s)
- Christoph Nagel
- Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
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28
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Arrowsmith RL, Atkin AJ, Botchway SW, Fairlamb IJS, Lynam JM, Moir JWB, Pascu SI, Ward JS, Zhang WQ. Confocal and fluorescence lifetime imaging sheds light on the fate of a pyrene-tagged carbon monoxide-releasing Fischer carbene chromium complex. Dalton Trans 2015; 44:4957-62. [PMID: 25553721 DOI: 10.1039/c4dt03312d] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The synthesis of a new pyrene-containing Fischer carbene complex is described. The complex has a broad absorbance spectrum between 300 and 400 nm and, on excitation at 345 nm in CH2Cl2 solution, emission is observed at 395 and 415 nm. Emission is also observed in PBS buffer, but in this case the resulting spectra are much broader. Confocal and fluorescence lifetime imaging indicate that emission occurs on treating HeLa cells with the complex and co-localisation studies demonstrate that this is from the mitochondria and lipid-rich regions of the cell.
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Affiliation(s)
- Rory L Arrowsmith
- Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
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29
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Tinajero-Trejo M, Denby KJ, Sedelnikova SE, Hassoubah SA, Mann BE, Poole RK. Carbon monoxide-releasing molecule-3 (CORM-3; Ru(CO)3Cl(glycinate)) as a tool to study the concerted effects of carbon monoxide and nitric oxide on bacterial flavohemoglobin Hmp: applications and pitfalls. J Biol Chem 2014; 289:29471-82. [PMID: 25193663 PMCID: PMC4207967 DOI: 10.1074/jbc.m114.573444] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2014] [Revised: 09/01/2014] [Indexed: 11/06/2022] Open
Abstract
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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Affiliation(s)
| | - Katie J Denby
- From the Departments of Molecular Biology & Biotechnology and
| | | | | | - Brian E Mann
- Chemistry, The University of Sheffield, S10 2TN United Kingdom
| | - Robert K Poole
- From the Departments of Molecular Biology & Biotechnology and
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30
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Rana N, McLean S, Mann BE, Poole RK. Interaction of the carbon monoxide-releasing molecule Ru(CO)3Cl(glycinate) (CORM-3) with Salmonella enterica serovar Typhimurium: in situ measurements of carbon monoxide binding by integrating cavity dual-beam spectrophotometry. MICROBIOLOGY-SGM 2014; 160:2771-2779. [PMID: 25085864 DOI: 10.1099/mic.0.081042-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
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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Affiliation(s)
- Namrata Rana
- Department of Molecular Biology & Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Samantha McLean
- Department of Molecular Biology & Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Brian E Mann
- Department of Chemistry, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Robert K Poole
- Department of Molecular Biology & Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
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31
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Kohlenstoffmonoxid freisetzende Moleküle für die therapeutische CO-Verabreichung in vivo. Angew Chem Int Ed Engl 2014. [DOI: 10.1002/ange.201311225] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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32
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García-Gallego S, Bernardes GJL. Carbon-monoxide-releasing molecules for the delivery of therapeutic CO in vivo. Angew Chem Int Ed Engl 2014; 53:9712-21. [PMID: 25070185 DOI: 10.1002/anie.201311225] [Citation(s) in RCA: 256] [Impact Index Per Article: 23.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2013] [Revised: 02/17/2014] [Indexed: 11/07/2022]
Abstract
The development of carbon-monoxide-releasing molecules (CORMs) as pharmaceutical agents represents an attractive and safer alternative to administration of gaseous CO. Most CORMs developed to date are transition-metal carbonyl complexes. Although such CORMs have showed promising results in the treatment of a number of animal models of disease, they still lack the necessary attributes for clinical development. Described in this Minireview are the methods used for CORM selection, to date, and how new insights into the reactivity of metal-carbonyl complexes in vivo, together with advances in methods for live-cell CO detection, are driving the design and synthesis of new CORMs, CORMs that will enable controlled CO release in vivo in a spatial and temporal manner without affecting oxygen transport by hemoglobin.
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Affiliation(s)
- Sandra García-Gallego
- Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge (UK); Present Address: Polymer and Fibre Technology, KTH Royal Institute of Technology, 100 44 Stockholm (Sweden)
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33
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Tinajero-Trejo M, Jesse HE, Poole RK. Gasotransmitters, poisons, and antimicrobials: it's a gas, gas, gas! F1000PRIME REPORTS 2013; 5:28. [PMID: 23967379 PMCID: PMC3732073 DOI: 10.12703/p5-28] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We review recent examples of the burgeoning literature on three gases that have major impacts in biology and microbiology. NO, CO and H2S are now co-classified as endogenous gasotransmitters with profound effects on mammalian physiology and, potentially, major implications in therapeutic applications. All are well known to be toxic yet, at tiny concentrations in human and cell biology, play key signalling and regulatory functions. All may also be endogenously generated in microbes. NO and H2S share the property of being biochemically detoxified, yet are beneficial in resisting the bactericidal properties of antibiotics. The mechanism underlying this protection is currently under debate. CO, in contrast, is not readily removed; mounting evidence shows that CO, and especially organic donor compounds that release the gas in biological environments, are themselves effective, novel antimicrobial agents.
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34
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Jesse HE, Nye TL, McLean S, Green J, Mann BE, Poole RK. Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo'' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2013; 1834:1693-703. [PMID: 23624261 PMCID: PMC3787766 DOI: 10.1016/j.bbapap.2013.04.019] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/04/2012] [Revised: 04/18/2013] [Accepted: 04/19/2013] [Indexed: 11/04/2022]
Abstract
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 0.24 μM) 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.
Cytochrome bd-I is a CORM- insensitive heme-protein in E. coli. The oxygen affinity of the ‘third oxidase’, cytochrome bd-II is low (Km 0.24 μM). Non-thiol antioxidants do not prevent CO-RM-mediated inhibition of respiration. N-acetylcysteine reduces the uptake of CORM-2 and CORM-3 by E. coli.
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Affiliation(s)
- Helen E Jesse
- Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, S10 2TN, UK.
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