Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB-BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Šfrom the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure-function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

An update on derivatisation and repurposing of clinical nitrofuran drugs.

5-nitrofurans (NFs) have been in clinical use for over 60 years. These affordable drugs are used for the treatment of a broad spectrum of diseases ranging from urinary tract infections to cancer. The anti-pathogenic effect of clinical NFs occurs following a step-wise process involving activation by azoreduction, followed by nitroreduction catalysed by azoreductases and nitroreductases (NTRs), respectively. Azoreduction yields stable metabolites that have the ability to covalently bind to cellular proteins. Nitroreduction, on the other hand, occurs by type I or II reduction of the nitro group in the presence of parasitic NADPH-cytochrome P450 reductases. Type I NTRs catalyse, under anaerobic conditions, the reduction of NFs to produce anti-pathogenic hydroxylamine. Under aerobic conditions, nitroreduction catalysed by type II NTRs produces reactive oxygen and nitrogen species (ROS/RNS), causing oxidative stress to pathogens and ultimate death. This multi-activity nature of NFs thus allows the repurposing of these drugs from agricultural chemicals and basic antibiotics to efficient therapies against human life-threatening diseases. Cases of NF resistance in pathogens are also limited likely due to this multi-activity, as well as effectivity under both aerobic and anaerobic conditions. However, multi-activity of these drugs can also infer toxicity. Molecular derivatisation is an effective strategy to improve efficacy, lower toxicity, diversify activity and address pathogen resistance associated with the use of these drugs.

Conditional Demyelination and Remyelination in a Transgenic Xenopus laevis.

Multiple sclerosis (MS) is the first cause of acquired disability progression in the young adult. Pathology of MS associates inflammation, demyelination, and neurodegeneration. The development of immunotherapies, by reducing the relapse rate, has profoundly impacted short-term prognosis and patients' quality of life. These anti-inflammatory medications, however, have not proven to be sufficient to prevent long-term disability progression, resulting from axonal transection and neuronal damage, consequences of prolonged demyelination. Promoting remyelination is therefore a key therapeutic strategy to limit handicap progression, and represent the major therapeutic challenge in MS. Here we present a simple, rapid, and cost-effective experimental model developed in Xenopus laevis to screen in vivo molecules promoting remyelination.

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

Hypoxia-triggered single molecule probe for high-contrast NIR II/PA tumor imaging and robust photothermal therapy.

Hypoxia is a common characteristic of solid tumors. This important feature is associated with resistance to radio-chemotherapy, which results in poor prognosis and probability of tumor recurrence. Taking advantage of background-free NIR II fluorescence imaging and deeper-penetrating photoacoustic (PA) imaging, we developed a hypoxia-triggered and nitroreductase (NTR) enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy (PTT), which will overcome cellular resistance during hypoxia. Methods: The single molecule probe IR1048-MZ was synthesized by conjugating a nitro imidazole group as a specific hypoxia trigger with an IR-1048 dye as a NIR II/PA signal reporter. We investigated the NIR II fluorescence, NIR absorbance and photothermal effect in different hypoxia conditions in vitro, and performed NIR II/PA tumor imaging and hypoxia-activated photothermal therapy in mice. Results: This versatile molecular probe IR1048-MZ not only realized high-contrast tumor visualization with a clear boundary by NIR II fluorescence imaging, but also afforded deep-tissue penetration at the centimeter level by 3D PA imaging. Moreover, after being activated by NTR that is overexpressed in hypoxic tumors, the probe exhibited a significant photothermal effect for curative tumor ablation with no recurrence. Conclusions: We have developed the first hypoxia-triggered and NTR enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy. By tracing the activity of NTR, IR1048-MZ may be a promising contrast agent and theranostic formulation for other hypoxia-related diseases (such as cancer, inflammation, stroke, and cardiac ischemia).

Evaluating 5-Nitrothiazoles as Trypanocidal Agents.

The growth-inhibitory properties of a 5-nitrothiazole series were evaluated against Trypanosoma brucei. A subset of related compounds displayed the greatest potency toward the parasite while exhibiting little cytotoxic effect on mammalian cells, with this antiparasitic activity dependent on expression of a type I nitroreductase by the trypanosome. We conclude that the 5-nitrothiazole class of nitroheterocyclic drugs may represent a new lead in the treatment of human African trypanosomiasis.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.

Noninvasive theranostic imaging of HSV1-sr39TK-NTR/GCV-CB1954 dual-prodrug therapy in metastatic lung lesions of MDA-MB-231 triple negative breast cancer in mice.

Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 µg/ml GCV and 10 µM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following two consecutive doses of a combination of GCV (40 mg/kg) and CB1954 (40 mg/kg) administered at 5 day intervals. In contrast, the respective treatment condition in animals expressing HSV1-sr39TK or NTR separately, showed minimal or no effect on tumor reduction as measured by bioluminescence (tumor mass) and [(18)F]-FHBG microPET (TK expression) imaging. These highlight the strong therapeutic effect of the dual fusion prodrug therapy and its use in theranostic imaging of tumor monitoring in living animals by multimodality molecular imaging.

Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis.

Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

Structure-activity relationships of antitubercular nitroimidazoles. 3. Exploration of the linker and lipophilic tail of ((s)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-amino PA-824).

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 µM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Antioxidant effects of the chestnut (Castanea crenata) inner shell extract in t-BHP-treated HepG2 cells, and CCl4- and high-fat diet-treated mice.

The antioxidant effects of chestnut inner shell extract (CISE) were investigated in a tert-butylhydroperoxide (t-BHP)-treated HepG2 cells, and in mice that were administered carbon tetrachloride (CCl(4)) and fed a high-fat diet (HFD). Pre-incubation with CISE significantly blocked the oxidative stress induced by t-BHP treatment in HepG2 cells (P<0.05) and preserved the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase compared to group treated with t-BHP only. Similarly, the CCl(4)- and HFD-induced reduction of antioxidant enzymes activities in liver was prevented by CISE treatment compared to control groups. Furthermore, hepatic lipid peroxidation were remarkably lower (P<0.05) in the CISE-treated groups with t-BHP or HFD. To determine the active compound of CISE, the fractionation of CISE has been conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the t-BHP-induced ROS generation and reduction in antioxidant enzyme activity in an in vitro model system. From these results, it was demonstrated that CISE has the ability to protect against damage from oxidative stressors such as t-BHP, CCl(4) and HFD in in vitro and in vivo models. The CISE might be useful for the prevention of oxidative damage in liver cells and tissues.

Dietary supplementation of resveratrol suppresses colonic tumour incidence in 1,2-dimethylhydrazine-treated rats by modulating biotransforming enzymes and aberrant crypt foci development.

Diet-induced changes in the activities of bacterial enzymes are known to play a role in colon cancer development. Resveratrol has been implicated as a protective agent in carcinogenesis. In the present study, the effect of resveratrol on the activities of faecal and colonic biotransforming enzymes such as beta-glucuronidase, beta-glucosidase, beta-galactosidase, mucinase, nitroreductase and faecal sulfatase activity was assessed. The total number of aberrant crypt foci and their distribution in the proximal, medial and distal colon were observed in 1,2-dimethylhydrazine (DMH)-induced rats (group 3) and other treatment groups (groups 4-6). DMH (0.02 g/kg body weight) was given subcutaneously once a week for 15 consecutive weeks, and the experiment was terminated at 30 weeks. DMH-treated rats showed elevated levels of cancer-associated bacterial enzyme activities, whereas on resveratrol supplementation in three different regimens, rats showed lowered activities. Resveratrol supplementation throughout the experimental period (group 6) exerted a more pronounced effect (P < 0.01) by modulating the development of aberrant crypt foci and the activities of bacterial enzymes than did the other treatment regimens (groups 4 and 5). Thus, the present results demonstrate the inhibitory effect of resveratrol on DMH-induced colon carcinogenesis in rats.

Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model.

A study was conducted to evaluate the effects of therapeutic and residual doses of ciprofloxacin on the human intestinal flora implanted into germ-free mice. Ciprofloxacin was administered daily via drinking water at concentrations to provide doses of 0, 0.125, 1.25, and 12.5mg/kg b.w. Changes in the intestinal flora composition, alteration in bacterial enzyme activities, fecal short chain fatty acid concentration and bacterial cellular fatty acid profiles, overgrowth of resistant bacteria, and disruption of the colonization barrier were the endpoints evaluated in the feces of human-flora-associated (HFA) mice. Ciprofloxacin at all tested doses decreased significantly the aerobic populations and particularly the population of Enterobacteriaceae. Selection of resistant Bacteroides fragilis group was noticed in HFA mice receiving 12.5mg/kg b.w. In mice challenged with a Salmonella strain, exogenous Salmonella persisted in the feces of all treated mice indicating that the flora responsible for the colonization barrier effect was disturbed by the antibiotic treatment. None of the studied metabolic parameters of the flora were affected by ciprofloxacin at any dose level. Under the experimental conditions of the study, the no-observed-effect level of ciprofloxacin was found to be less than 0.125 mg/kg b.w.

Investigation of alternative prodrugs for use with E. coli nitroreductase in 'suicide gene' approaches to cancer therapy.

The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system. We have examined the efficacy of an alternative approach utilising the E. coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs. V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells. In general other prodrugs which were found by HPLC to act as substrates for purified E. coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute. In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold). The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.

Metronidazole susceptibility factors in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a facultative anaerobic bacterium that displays moderate susceptibility to metronidazole and this study was undertaken to identify the factors involved. A. actinomycetemcomitans appeared two to four times less susceptible to metronidazole when grown in air supplemented with 5% CO2 than under anaerobic conditions. Ferredoxin-linked pyruvate:oxidoreductase activity was absent but each strain exhibited nitroreductase activity which corresponded directly with uptake of metronidazole and susceptibility to the drug under anaerobic conditions but not in air supplemented with 5% CO2. Nitroreductase activity therefore appears responsible for the susceptibility of A. actinomycetemcomitans to metronidazole.

Genotoxicities of nitropyrenes and their modulation by apigenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems.

Four naturally occurring compounds, indole-3-carbinol (I3C), apigenin (Api), ellagic acid (EA) and tannic acid (TA), were tested for their inhibitory effects against 1-nitropyrene- (1-NP) or 1,6-dinitropyrene (1,6-DNP)-induced genotoxicity in Salmonella tester strains and Chinese hamster ovary (CHO) cells. Api and TA strongly inhibited the bacterial mutagenesis induced by nitropyrenes, while I3C and EA had little or no effect. For example, in TA98, 0.2 mumole Api resulted in 48% and 56% inhibition of the mutagenicity induced by 4 nmole 1-NP and 0.035 nmole 1,6-DNP, respectively. With an equal dose, TA caused 46% and 50% reduction of the mutagenicity induced by 1-NP and 1,6-DNP, respectively. As expected, a good correlation was observed between the antimutagenicity of nitropyrenes and their inhibitory effect on nitroreductase activity. This indicated that one of the possible antimutagenic mechanisms of Api or TA was to inactivate the metabolism of nitropyrenes. Two biological end-points, cytotoxicity and sister-chromatid exchange (SCEs), were used to screen the antigenotoxic effects of these compounds in CHO cells. At the sub-cytotoxic dose, I3C, Api and TA all protected against the cytotoxicity induced by 1-NP and 1,6-DNP, but only TA and Api gave a significant reduction of the frequency of SCEs. Moreover, this reduction was found to be highly dose-dependent.

Changes in intestinal function of rats initiated with DMH and fed varying levels of butterfat, calcium, and magnesium.

The effects of dietary calcium, magnesium, and butterfat on intestinal function and flora in rats initiated with 1,2-dimethylhydrazine (DMH) were studied. Male weanling rats were assigned to six isocaloric diets that varied in their levels of calcium and magnesium (0.25% Ca with 0.05% Mg, 1.0% Ca with 0.05% Mg, or 0.625% Ca with 0.50% Mg) and butterfat (5% or 20%). One-half of the rats in each treatment were injected subcutaneously with DMH weekly for four weeks. This short-term exposure to DMH increased colonic ornithine decarboxylase (ODC) activity and the mass of cecal contents. Ingestion of the high levels of either calcium or magnesium depressed colonic ODC activity and depressed apparent absorption of organic matter, calcium, magnesium, and phosphorus. Ingestion of excess magnesium increased the mass of the cecal contents by twofold, caused hypertrophy of cecal walls, and increased the total amount of protein and total nitroreductase and beta-glucuronidase activity in the ceca of rats. Ingestion of supplemental calcium had less dramatic effects and increased the mass of cecal contents by only 28% and decreased the total amount of protein in the ceca. On the basis of their different effects on cecal microflora, magnesium appears to have less potential than does calcium as a protective agent against colon cancer.

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora.

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.