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

Abstract

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.

Fig 1. P. aeruginosa viability according to the LIVE/DEAD assay after treating with HU, HA, and M-HA.

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.

Fig 2. Dose-response curves for HU, HA and M-HA treatments of murine macrophages.

The level of growth alteration for J774 macrophages that were treated with different doses of radical scavenger compounds at 72 hours post-treatment. Cell viability was measured by using an MTT assay. Values represent the means ± standard deviation (SD) of triplicate cultures. The data are representative of one of at least three independent experiments. (*, P < 0.05 vs. non-treated cells). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.

Fig 3. The intracellular BCG growth inhibition of macrophages treated with different doses of HU, HA and M-HA.

The effects of different doses of radical scavenger compounds on the intracellular viability of BCG at 72 hours post-infection. A) Compounds were added 3 hours after infection B) Compounds were renewed every 24 hours after infection. The results are expressed as the means ± standard deviation (SD) of triplicate wells in percentages of inhibition with respect to non-treated cells. The data are representative of one of at least two independent experiments where statistically significant differences between treated and untreated cells (P < 0.05) for all compounds and concentrations. HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.

Fig 4. TNF-α and IL-12 production as triggered by BCG-infected macrophages that were treated with different doses of HU, HA and M-HA.

J774 macrophages were infected with BCG and treated with different concentrations of radical scavenger compounds, and TNF-α and IL-12 levels were measured 24 hours post-infection. The results represent the means ± SD of triplicate preparations with one representative of two independent experiments. A Mann-Whitney test was performed (*, P < 0.01; versus non-treated macrophages (control)). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.

Fig 5. The inhibition of biofilm formation by different doses of HU, HA and M-HA.

The biofilm formation of P. aeruginosa PAO1 was quantified as the absorbance of crystal violet stain (A₅₇₀ nm) after being cultured for 24 h in 96-well plates in the presence and absence of the radical scavenger compounds. The values represent the percentages of biofilm biomass production. The results are expressed as the means ± SD of five replicates from one representative of three independent experiments. A Student's t-test was performed (**, P < 0.01; versus non-treated biofilms). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.

Fig 6. Disassembling the existing P. aeruginosa biofilms by adding HU, HA and M-HA.

P. aeruginosa 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 (*, P < 0.05; versus non-treated biofilms). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl- hydroxylamine.

Fig 7. Confocal microscopy of P. aeruginosa biofilms grown on flow cell.

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

Fig 8. The synergistic effect of ciprofloxacin and M-HA on the reduction in P. aeruginosa biofilm formation.

P. aeruginosa bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of ciprofloxacin with/without M-HA were added. Biofilm formation (crystal violet stain) was evaluated 24 hours later. The biofilm biomass production percentage is represented here. The results are the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, P < 0.05; **, P < 0.005 versus ciprofloxacin treated biofilms). CPX, ciprofloxacin; and M-HA, methyl-hydroxylamine.