Inhibition of Escherichia coli nitroreductase by the constituents in Syzygium aromaticum / 中国天然药物

Gut bacterial nitroreductases play an important role in reduction of various nitroaromatic compounds to the corresponding N-nitroso compounds, hydroxylamines or aromatic amines, most of which are carcinogenic and mutagenic agents. Inhibition of gut nitroreductases has been recognized as an attractive approach for reducing mutagen metabolites in the colon, so as to prevent colon diseases. In this study, the inhibitory effects of 55 herbal medicines against Escherichia coli(E. coli) nitroreductase (EcNfsA) were examined. Compared with other herbal extracts, Syzygium aromaticum extract showed superior inhibitory potency toward EcNfsA mediated nitrofurazone reduction. Then, the inhibitory effects of 22 major constituents in Syzygium aromaticum against EcNfsA were evaluted. Compared with other tested natural compounds, ellagic acid, corilagin, betulinic acid, oleanic acid, ursolic acid, urolithin M5 and isorhamnetin were found with strong to moderate inhibitory effect against EcNfsA, with IC50 values ranging from 0.67 to 28.98 mol·L-1. Furthermore, the inhibition kinetic analysis and docking simulation demonstrated that ellagic acid and betulinic acid potently inhibited EcNfsA (Ki < 2 μmol·L -1) in a competitively inhibitory manner, which created strong interactions with the catalytic triad of EcNfsA. In summary, our findings provide new scientific basis for explaining the anti-mutagenic activity of Syzygium aromaticum, where some newly identified EcNfsA inhibitors can be used for developing novel agents to reduce the toxicity induced by bacterial nitroreductase.

A novel nitroreductase-enhanced MRI contrast agent and its potential application in bacterial imaging

Nitroreductases (NTRs) are known to be able to metabolize nitro-substituted compounds in the presence of reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. NTRs are present in a wide range of bacterial genera and, to a lesser extent, in eukaryotes hypoxic tumour cells and tumorous tissues, which makes it an appropriate biomarker for an imaging target to detect the hypoxic status of cancer cells and potential bacterial infections. To evaluate the specific activation level of NTR, great efforts have been devoted to the development of fluorescent probes to detect NTR activities using fluorogenic methods to probe its behaviour in a cellular context; however, NTR-responsive MRI contrast agents are still by far underexplored. In this study, -nitrobenzyl substituted -weighted magnetic resonance imaging (MRI) contrast agent Gd-DOTA-PNB (probe ) has been designed and explored for the possible detection of NTR. Our experimental results show that probe could serve as an MRI-enhanced contrast agent for monitoring NTR activity. The response and mechanism of the NTR catalysed reduction of probe have been investigated through LC-MS and MRI. -nitrobenzyl substituted probe was catalytically reduced by NTR to the intermediate -aminobenzyl substituted probe which then underwent a rearrangement elimination reaction to Gd-DOTA, generating the enhanced -weighted MR imaging. Further, LC-MS and MRI studies of living have confirmed the NTR activity detection ability of probe at a cellular level. This method may potentially be used for the diagnosis of bacterial infections.

Identification of a type I nitroreductase gene in non-virulent Trypanosoma rangeli

ABSTRACT Trypanosomatid type I nitroreductases (NTRs), i.e., mitochondrial enzymes that metabolise nitroaromatic pro-drugs, are essential for parasite growth, infection, and survival. Here, a type I NTR of non-virulent protozoan Trypanosoma rangeli is described and compared to those of other trypanosomatids. The NTR gene was isolated from KP1(+) and KP1(-) strains, and its corresponding transcript and 5' untranslated region (5'UTR) were determined. Bioinformatics analyses and nitro-drug activation assays were also performed. The results indicated that the type I NTR gene is present in both KP1(-) and KP1(+) strains, with 98% identity. However, the predicted subcellular localisation of the protein differed among the strains (predicted as mitochondrial in the KP1(+) strain). Comparisons of the domains and 3D structures of the NTRs with those of orthologs demonstrated that the nitroreductase domain of T. rangeli NTR is conserved across all the strains, including the residues involved in the interaction with the FMN cofactor and in the tertiary structure characteristics of this oxidoreductase protein family. mRNA processing and expression were also observed. In addition, T. rangeli was shown to be sensitive to benznidazole and nifurtimox in a concentration-dependent manner. In summary, T. rangeli appears to have a newly discovered functional type I NTR.

Introducing nitazoxanide as a promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in clinical isolates

OBJECTIVE@#To identify the frequencies (F) of ferredoxin and nitroreductase mutations on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazole-resistant giardiasis.@*METHODS@#Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran. DNAs were extracted and amplified by targeting ferredoxin and GlNR genes. The amplicons were directly sequenced to determine gene mutations.@*RESULTS@#The various amino acid substitutions (F: 20%, Haplotype diversity: 0.891, Tajima's D: -0.44013) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates. Only two haplotypes (F: 5%, HD: 0.345; Tajima's D: 0.77815) characterized in metronidazole-resistant isolates of GlNR, however, no point mutations was found in symptomatic isolates.@*CONCLUSIONS@#Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity. This leads to decrease in metronidazole drug delivery into the cells. Mutations in these isolates may lead to their resistance to metronidazole. No to low synonymous mutations of GlNR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

Introducing nitazoxanide as a promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in clinical isolates

Objective To identify the frequencies (F) of ferredoxin and nitroreductase mutations on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazole-resistant giardiasis. Methods Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran. DNAs were extracted and amplified by targeting ferredoxin and GlNR genes. The amplicons were directly sequenced to determine gene mutations. Results The various amino acid substitutions (F: 20%, Haplotype diversity: 0.891, Tajima's D: −0.440 13) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates. Only two haplotypes (F: 5%, HD: 0.345; Tajima's D: 0.778 15) characterized in metronidazole-resistant isolates of GlNR, however, no point mutations was found in symptomatic isolates. Conclusions Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity. This leads to decrease in metronidazole drug delivery into the cells. Mutations in these isolates may lead to their resistance to metronidazole. No to low synonymous mutations of GlNR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.