20 research outputs found

    Methyl-Hydroxylamine as an Efficacious Antibacterial Agent That Targets the Ribonucleotide Reductase Enzyme

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    The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme

    Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme

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    This work was supported by grants from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III - PI10/01438), the European Regional Development Fund (FEDER), and the Generalitat de Catalunya (2009SGR-108) to EJ, and grants from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III - PI081062), the European Regional Development Fund (FEDER),), the Spanish Ministerio de Ciencia e Innovación (BFU2011-24066), the ERA-NET PathoGenoMics, the Ramón y Cajal program and the Catalan and Spanish cystic fibrosis federation to ET.The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RN

    Hydroxylamine Derivatives as a New Paradigm in the Search of Antibacterial Agents

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    Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria

    Hydroxylamine derivatives as a new paradigm in the search of antibacterial agents

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    Altres ajuts: Catalan and Spanish cystic fibrosis federation, the EIT Health, and Obra Social "La Caixa"Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N -substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N -hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria

    New antimicrobial strategies against bacterial infections

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    [eng] Bacterial infections are a major health concern worldwide due to the mortality and comorbidity associated levels. Besides the development of drug-resistance mechanisms, most recalcitrant infections are caused by bacteria forming biofilms, which are bacterial communities that grow within an extracellular polymeric substance (EPS) matrix that protects bacterial cells from the action of antimicrobials and immune mechanisms. The frequent appearance and spreading of bacterial drug-resistant strains, together with the recalcitrance of infections caused by biofilms, has pointed out the urgent need to develop novel antibacterial agents that target essential bacterial processes and biofilm-forming bacteria. This thesis investigated the development of new antibacterial strategies by both targeting bacterial essential processes and biofilm-forming bacteria. Ribonucleotide reductase (RNR) are essential enzymes required by any cellular organism, since they catalyze the synthesis of deoxyribonucleotides, critical for both DNA synthesis and repair processes. Due to its key role in DNA replication, several RNR inhibitors have been developed as antiproliferative drugs in cancer and infectious diseases. Amongst RNR inhibitors, radical scavenger molecules have proved for long its inhibitory activity against the RNR enzyme, including some cytotoxic hydroxylamine derivatives such as hydroxyurea (HU). Here, the use of hydroxylamine derivative compounds as antibacterial agents specifically targeting the bacterial RNR enzyme was investigated. The results provided show that N-methyl-hydroxylamine (M-HA) molecule and some newly synthesized Nhydroxylamine derivative molecules (N-HA) inhibit bacterial RNR, displaying a wide range of antibacterial activity against different bacterial pathogens, together with low cytotoxicity to eukaryotic cells. Also, M-HA molecule shows intracellular antimycobacterial activity during macrophages infection, together with Pseudomonas aeruginosa in vitro antibiofilm activity. Several of the N-HA molecules display antibiofilm activity against P. aeruginosa, Staphylococcus aureus, and Escherichia coli biofilms, and we demonstrate the ability of such molecules to inhibit bacterial growth by radical scavenging of the RNR enzyme. Further, this thesis explores the use of a drug delivery system based on poly(lactic-co-glycolic) (PLGA) nanoparticles (NPs) to remove P. aeruginosa biofilms by combining the action of the antibiotic ciprofloxacin and the DNA-hydrolytic activity of the deoxyribonuclease I enzyme (DNase I). The synthesized biodegradable PLGA NPs, loaded with ciprofloxacin and functionalized with DNase I through poly(lysine) (PL) coating, synergistically improve the ciprofloxacin antibacterial efficacy by disassembling the extracellular DNA, one of the main components of the EPS matrix

    Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme

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    This work was supported by grants from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III - PI10/01438), the European Regional Development Fund (FEDER), and the Generalitat de Catalunya (2009SGR-108) to EJ, and grants from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III - PI081062), the European Regional Development Fund (FEDER),), the Spanish Ministerio de Ciencia e Innovación (BFU2011-24066), the ERA-NET PathoGenoMics, the Ramón y Cajal program and the Catalan and Spanish cystic fibrosis federation to ET.The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RN

    Nano-engineering stable contact-based antimicrobials: chemistry at the interface between nano-gold and bacteria.

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    Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications

    Confocal microscopy of <i>P</i>. <i>aeruginosa</i> biofilms grown on flow cell.

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    <p>The formed biofilms were treated for 24 h with HU, HA and M-HA at 40 <b>μ</b>g/ml final concentration. Each panel shows the maximum Z-projection and the orthogonal views for each stack.</p

    Disassembling the existing <i>P</i>. <i>aeruginosa</i> biofilms by adding HU, HA and M-HA.

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    <p><i>P</i>. <i>aeruginosa</i> bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of radical scavenger compounds were changed every 24 hours over three days (Days 1, 2 and 3). The percentage of biofilm biomass production is represented for each day. The results are the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, <i>P</i> < 0.05; versus non-treated biofilms). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl- hydroxylamine.</p

    <i>P</i>. <i>aeruginosa</i> viability according to the LIVE/DEAD assay after treating with HU, HA, and M-HA.

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    <p>The bacteria were grown with HU (7.6 μg/ml), HA (8.3 μg/ml), or M-HA (6.7 μg/ml) for 3 and 24 hours and stained with LIVE/DEAD assay. Live cells were green (SYTO 9 dye) and dead cells were red (propidium iodide dye) under a fluorescent microscope. Magnification, x 1000. HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.</p
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