Design and Synthesis of an Azo Reductase Responsive Flavonol-Indomethacin Hybrid Used for the Diagnosis and Treatment of Colitis.

Human intestinal bacteria are the primary producers of azo reductase, and the content of azo reductase is closely associated with various intestinal diseases, including ulcerative colitis (UC). The rapid detection of changes in azo reductase levels is crucial for diagnosing and promptly intervening in UC. In this study, a therapeutic agent, FAI, specifically targeting UC, was designed and synthesized. This agent was developed by linking the anti-inflammatory drug indomethacin to flavonols with antioxidant activity via an azo bond (off-on). Breakage of the azo bond breaks results in the release of both fluorophores and drugs, achieving targeted tracing and integrated treatment effects. In vivo and in vitro fluorescence imaging experiments were used to demonstrate the potential of FAI in the diagnosis of UC, together with synergistic therapeutic effects through the release of both fluorophores and anti-inflammatory agents. Therefore, this diagnostic agent shows promise as a potential tool for diagnosing and treating UC.

Sustained release of 5-aminosalicylic acid from azoreductase-responsive polymeric prodrugs for prolonged colon-targeted colitis therapy.

Ulcerative colitis (UC) is a challenging inflammatory gastrointestinal disorder, whose therapies encounter limitations in overcoming insufficient colonic retention and rapid systemic clearance. In this study, we report an innovative polymeric prodrug nanoformulation for targeted UC treatment through sustained 5-aminosalicylic acid (5-ASA) delivery. Amphiphilic polymer-based 13.5 nm micelles were engineered to incorporate azo-linked 5-ASA prodrug motifs, enabling cleavage via colonic azoreductases. In vitro, micelles exhibited excellent stability under gastric/intestinal conditions while demonstrating controlled 5-ASA release over 24 h in colonic fluids. Orally administered micelles revealed prolonged 24-h retention and a high accumulation within inflamed murine colonic tissue. At an approximately 60% dose reduction from those most advanced recent studies, the platform halted DSS colitis progression and outperformed standard 5-ASA therapy through a 77-97% suppression of inflammatory markers. Histological analysis confirmed intact colon morphology and restored barrier protein expression. This integrated prodrug nanoformulation addresses limitations in colon-targeted UC therapy through localized bioactivation and tailored pharmacokinetics, suggesting the potential of nanotechnology-guided precision delivery to transform disease management.

A nitroreductase responsive probe for early diagnosis of pulmonary fibrosis disease.

Idiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease. However, the definitive diagnosis of IPF is impeded by the limited capabilities of current diagnostic methods, which may fail to capture the optimal timing for treatment. The main goal of this study is to determine the feasibility of a nitroreductase (NTR) responsive probe, 18F-NCRP, for early detection and deterioration monitoring of IPF. 18F-NCRP was obtained with high radiochemical purity (>95 %). BLM-injured mice were established by intratracheal instillation with bleomycin (BLM) and characterized through histological analysis. Longitudinal PET/CT imaging, biodistribution study and in vitro autoradiography were performed. The correlations between the uptake of 18F-NCRP and mean lung density (tested by CT), as well as histopathological characteristics were analyzed. In PET imaging study, 18F-NCRP exhibited promising efficacy in monitoring the progression of IPF, which was earlier than CT. The ratio of uptake in BLM-injured lung to control lung increased from 1.4-fold on D15 to 2.2-fold on D22. Biodistribution data showed a significant lung uptake of 18F-NCRP in BLM-injured mice. There was a strong positive correlation between the 18F-NCRP uptake in the BLM-injured lungs and the histopathological characteristics. Given that, 18F-NCRP PET imaging of NTR, a promising biomarker for investigating the underlying pathogenic mechanism of IPF, is attainable as well as desirable, which might lay the foundation for establishing an NTR-targeted imaging evaluation system of IPF.

Investigating FSGS-like injury in zebrafish larvae by nifurpirinol: efficacy and molecular insight.

Identifying effective drugs for focal segmental glomerulosclerosis (FSGS) treatment holds significant importance. Our high-content drug screening on zebrafish larvae relies on nitroreductase/metronidazole (NTR/MTZ)-induced podocyte ablation to generate FSGS-like injury. A crucial factor for successful drug screenings is minimizing variability in injury induction. For this, we introduce nifurpirinol (NFP) as a more reliable prodrug for targeted podocyte depletion. NFP showed a 2.3-fold increase in efficiency at concentrations 1,600-fold lower compared with MTZ-mediated injury induction. Integration into the screening workflow validated its suitability for the high-content drug screening. The presence of crucial FSGS hallmarks, such as podocyte foot process effacement, proteinuria, and activation of parietal epithelial cells, was observed. After the isolation of the glomeruli from the larvae, we identified essential pathways by proteomic analysis. This study shows that NFP serves as a highly effective prodrug to induce the FSGS-like disease in zebrafish larvae and is well-suited for a high-content drug screening to identify new candidates for the treatment of FSGS.NEW & NOTEWORTHY This research investigated the use of nifurpirinol in nanomolar amounts as a prodrug to reliably induce focal segmental glomerulosclerosis (FSGS)-like damage in transgenic zebrafish larvae. Through proteomic analysis of isolated zebrafish glomeruli, we were further able to identify proteins that are significantly regulated after the manifestation of FSGS. These results are expected to expand our knowledge of the pathomechanism of FSGS.

Enzyme-Triggered Degradation of Supramolecularly Cross-Linked Polymersomes of Azobenzene-Based Polyurethane: Cell-Selective Anticancer Drug Release.

Enzyme-responsive self-assembled nanostructures for drug delivery applications have gained a lot of attention, as enzymes exhibit dysregulation in many disease-associated microenvironments. Azoreductase enzyme levels are strongly elevated in many tumor tissues; hence, here, we exploited the altered enzyme activity of the azoreductase enzyme and designed a main-chain azobenzene-based amphiphilic polyurethane, which self-assembles into a vesicular nanostructure and is programmed to disassemble in response to a specific enzyme, azoreductase, with the help of the nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme in the hypoxic environment of solid tumors. The vesicular nanostructure sequesters, stabilizes the hydrophobic anticancer drug, and releases the drug in a controlled fashion in response to enzyme-triggered degradation of azo-bonds and disruption of vesicular assembly. The biological evaluation revealed tumor extracellular matrix pH-induced surface charge modulation, selective activated cellular uptake to azoreductase overexpressed lung cancer cells (A549), and the release of the anticancer drug followed by cell death. In contrast, the benign nature of the drug-loaded vesicular nanostructure toward normal cells (H9c2) suggested excellent cell specificity. We envision that the main-chain azobenzene-based polyurethane discussed in this manuscript could be considered as a possible selective chemotherapeutic cargo against the azoreductase overexpressed cancer cells while shielding the normal cells from off-target toxicity.

Crystal structures of NAD(P)H nitroreductases from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen-insensitive nitroreductases (Kp-NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp-NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)-binding mode is provided. The structures with PDB codes 7tmf (Kp-NR1a), 7tmg (Kp-NR1b) and 8dor (Kp-NR2) were determined at resolutions of 1.97, 1.90 and 1.35 Å, respectively. The Kp-NR1a and Kp-NR1b structures adopt an αß fold, in which four-stranded antiparallel ß-sheets are surrounded by five helices. With domain swapping, the ß-sheet was expanded with a ß-strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173-Ala185: in Kp-NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp-NR1b. The FMN interactions within Kp-NR1/NR2 involve hydrogen-bond and π-stacking interactions. Kp-NR2 contains four-stranded antiparallel ß-sheets surrounded by eight helices with two short helices and one ß-sheet. Structural and sequence alignments show that Kp-NR1a/b and Kp-NR2 are homologs of the Escherichia coli oxygen-insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp-NR1a/b and Kp-NR2 may detoxify polynitroaromatic compounds and Kp-NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping-pong bi-bi mechanism, respectively.

Nitroreductase DnrA, Utilizing Strategies Secreted in Bacillus sp. Za and SCK6, Enhances the Detoxification of Acifluorfen.

The residues of acifluorfen present a serious threat to the agricultural environment and sensitive crops. DnrA, a nitroreductase, is an intracellular enzyme that restricts the application of wild-type Bacillus sp. Za in environmental remediation. In this study, two strategies were employed to successfully secrete DnrA in strains SCK6 and Za, and the secretion expression conditions were optimized to achieve rapid degradation of acifluorfen. Under the optimal conditions, the relative activities of the DnrA supernatant from strains SCK6-D and Za-W were 3.06-fold and 3.53-fold higher than that of strain Za, respectively. While all three strains exhibited similar tolerance to different concentrations of acifluorfen, strains SCK6-D and Za-W demonstrated significantly faster degradation efficiency compared to strain Za. Furthermore, the DnrA supernatant from strains SCK6-D and Za-W could effectively reduce the toxicity of acifluorfen on maize and cucumber seedlings. This study provides an effective technical approach for the rapid degradation of acifluorfen.

Q48K mutation in the type IB nitroreductase NrmA is responsible for nitrofurantoin resistance in Enterococcus faecium.

OBJECTIVES: Nitrofurantoin is recommended as first-line therapy for the optimal treatment of uncomplicated urinary tract infections (UTIs) caused by enterococci and Escherichia coli. However, the mechanisms of nitrofurantoin resistance in enterococci have not been elucidated. This study aimed to investigate the mechanisms of nitrofurantoin resistance in E. faecium, focusing on the role of the nitroreductase NrmA. METHODS: Enterococcus strains isolated from the urinary tract samples were collected and were tested for nitrofurantoin susceptibility. Potential genes associated with nitrofurantoin resistance were screened in the NCBI nucleotide database and by polymerase chain reaction (PCR). Complementation assays and enzyme kinetic tests were performed to assess the impact of the Q48K mutation in NrmA on nitrofurantoin resistance. RESULTS: Of the 128 E. faecium isolates tested, 59 (46.1%) were resistant to nitrofurantoin. Analysis revealed the presence of a type IB nitroreductase, designated NrmA, in all E. faecium strains studied, shared 18.7% sequence identity with nitroreductase NfsB in E. coli. Different from NrmA in nitrofurantoin-susceptible E. faecium, nitrofurantoin-resistant strains had a single amino acid substitution, i.e., a lysine instead of a glutamine at position 48 (Q48K mutation). Complementation assays of nitrofurantoin-resistant E. faecium HS17-112 showed that the nitrofurantoin minimal inhibitory concentration of the complemented strain HS17-112: pIB166-nrmA (wild type [WT]) decreased from 128 mg/L to 4 mg/L. Compared with NrmA (WT), NrmA (Q48K) showed significantly reduced catalytic efficiency, with a kcat/Km value decreasing from 0.122 µM-1 s-1 to 0.000042 µM-1 s-1. CONCLUSION: The Q48K mutation in nitroreductase NrmA is responsible for nitrofurantoin resistance in E. faecium.

Designing a Hypoxia-Activated Sensing Platform Using an Azo Group-Triggered Reaction with the Formation of Silicon Nanoparticles.

Hypoxia is known as a specific signal of various diseases, such as liver fibrosis. We designed a hypoxia-sensitive fluorometric approach that cleaved the azo bond (N═N) in the presence of hypoxia-controlled agents (sodium dithionite and azoreductase). 4-(2-Pyridylazo) resorcinol (Py-N═N-RC) bears a desirable hypoxia-responsive linker (N═N), and its azo bond breakup can only occur in the presence of sodium dithionite and azoreductase and leads to the release of 2,4-dihydroxyaniline, which can react with 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane to generate yellow fluorescent silicon nanoparticles. This approach exhibited high selectivity and sensitivity toward both sodium dithionite and azoreductase over other potential interferences. The mouse liver microsome, which is known to contain azoreductase, was applied and confirmed the feasibility of the designed platform. Py-N═N-RC is expected to be a practical substrate for hypoxia-related biological analyses. Furthermore, silicon nanoparticles were successfully applied for Hela cell imaging owing to their negligible cytotoxicity and superb biocompatibility.

Dual Enzyme-Locked Activation Reporter for Accurate Liver Cancer Surveillance.

Activatable probes with a higher signal-to-background ratio and accuracy are essential for monitoring liver cancer as well as intraoperative fluorescence navigation. However, the presence of only one biomarker is usually not sufficient to meet the high requirement of a signal-to-background ratio in cancer surveillance, leading to the risk of misdiagnosis. In this work, a dual-locked activation response probe, Si-NTR-LAP, for nitroreductase and leucine aminopeptidase was reported. This dual-locked probe provides better tumor recognition and a higher signal-to-noise ratio than that of single-locked probes (Si-LAP and Si-NTR). In both the subcutaneous tumor model and the more complex orthotopic hepatocellular carcinoma model, the probe was able to identify tumor tissue with high specificity and accurately differentiate the boundaries between tumor tissue and normal tissue. Therefore, the dual-locked probe may provide a new and practical strategy for applying to real patient tumor tissue samples.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Hemicyanine-Based Highly Water-Soluble Probe for Extracellular Nitroreductase.

Nitroreductase (NTR) has long been a target of interest for its important role involved in the nitro compounds metabolism. Various probes have been reported for NTR analysis, but rarely able to distinguish the extracellular NTR from intracellular ones. Herein we reported a new NTR sensor, HCyS-NO2, which was a hemicyanine molecule with one nitro and two sulfo groups attached. The nitro group acted as the reporting group to respond NTR reduction. Direct linkage of nitro group into the hemicyanine π conjugate system facilitated the intramolecular electron transfer (IET) process and thus quenched the fluorescence of hemicyanine core. Upon reduction with NTR, the nitro group was rapidly converted into the hydroxylamino and then the amino group, eliminating IET process and thus restoring the fluorescence. The sulfo groups installed significantly increased the hydrophilicity of the molecule, and introduced negative charges at physiological pH, preventing the diffusion into bacteria. Both gram-negative and gram-positive bacteria were able to turn on the fluorescence of HCyS-NO2, without detectable diffusion into cells, providing a useful tool to probe the extracellular reduction process.

Selective pressure leads to an improved synthetic consortium fit for dye degradation.

Microorganisms have great potential for bioremediation as they have powerful enzymes and machineries that can transform xenobiotics. The use of a microbial consortium provides more advantages in application point of view than pure cultures due to cross-feeding, adaptations, functional redundancies, and positive interactions among the organisms. In this study, we screened about 107 isolates for their ability to degrade dyes in aerobic conditions and without additional carbon source. From our screening results, we finally limited our synthetic consortium to Gordonia and Rhodococcus isolates. The synthetic consortium was trained and optimized for azo dye degradation using sequential treatment of small aromatic compounds such as phenols that act as selective pressure agents. After four rounds of optimization with different aims for each round, the consortium was able to decolorize and degrade various dyes after 48 h (80%-100% for brilliant black bn, methyl orange, and chromotrop 2b; 50-70% for orange II and reactive orange 16; 15-30% for chlorazol black e, reactive red 120, and allura red ac). Through rational approaches, we can show that treatment with phenolic compounds at micromolar dosages can significantly improve the degradation of bulky dyes and increase its substrate scope. Moreover, our selective pressure approach led to the production of various dye-degrading enzymes as azoreductase, laccase-like, and peroxidase-like activities were detected from the phenol-treated consortium. Evidence of degradation was also shown as metabolites arising from the degradation of methyl red and brilliant black bn were detected using HPLC and LC-MS analysis. Therefore, this study establishes the importance of rational and systematic screening and optimization of a consortium. Not only can this approach be applied to dye degradation, but this study also offers insights into how we can fully maximize microbial consortium activity for other applications, especially in biodegradation and biotransformation.

DprE1 and Ddn as promising therapeutic targets in the development of novel anti-tuberculosis nitroaromatic drugs.

Tuberculosis remains the second deadliest infectious disease in humans and a public health threat due to the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains. Therefore, it is urgent to identify new anti-tuberculosis treatments and novel therapeutic targets to prevent the emergence of resistance. In recent years, the study of anti-tuberculosis properties of nitroaromatic compounds has led to the identification of two novel biological targets, the deazaflavin (F420)-dependent nitroreductase Ddn and the decaprenylphosphoryl-ß-d-ribose 2'-epimerase DprE1. This review aims to show why Ddn and DprE1 are promising therapeutic targets and highlight nitroaromatic compounds interest in developing new anti-tuberculosis treatments active against MDR-TB and XDR-TB. Despite renewed interest in the development of new anti-tuberculosis nitroaromatic compounds, pharmaceutical companies often exclude nitro-containing molecules from their drug discovery programs because of their toxic and mutagenic potential. This exclusion results in missed opportunities to identify new nitroaromatic compounds and promising therapeutic targets.

A Tumor-Targeting Dual-Modal imaging probe for nitroreductase in vivo.

Nitroreductase (NTR) overexpression often occurs in tumors, highlighting the significance of effective NTR detection. Despite the utilization of various optical methods for this purpose, the absence of an efficient tumor-targeting optical probe for NTR detection remains a challenge. In this research, a novel tumor-targeting probe (Cy-Bio-NO2) is developed to perform dual-modal NTR detection using near-infrared fluorescence and photoacoustic techniques. This probe exhibits exceptional sensitivity and selectivity to NTR. Upon the reaction with NTR, Cy-Bio-NO2 demonstrates a distinct fluorescence "off-on" response at 800 nm, with an impressive detection limit of 12 ng/mL. Furthermore, the probe shows on-off photoacoustic signal with NTR. Cy-Bio-NO2 has been successfully employed for dual-modal NTR detection in living cells, specifically targeting biotin receptor-positive cancer cells for imaging purposes. Notably, this probe effectively detects tumor hypoxia through dual-modal imaging in tumor-bearing mice. The strategy of biotin incorporation markedly enhances the probe's tumor-targeting capability, facilitating its engagement in dual-modal imaging at tumor sites. This imaging capacity holds substantial promise as an accurate tool for cancer diagnosis.

Harnessing the Potential of a Nitroreductase-Responsive Fluorescent Probe for the Diagnosis of Bacterial Keratitis.

Bacterial keratitis, an ocular emergency, is the predominant cause of infectious keratitis. However, diagnostic procedures for it are invasive, time-consuming, and expeditious, thereby limiting effective treatment for the disease in the clinic. It is imperative to develop a timely and convenient method for the noninvasive diagnosis of bacterial keratitis. Fluorescence imaging is a convenient and noninvasive diagnostic method with high sensitivity. In this study, a type of nitroreductase-responsive probe (NTRP), which responds to nitroreductase to generate fluorescence signals, was developed as an activatable fluorescent probe for the imaging diagnosis of bacterial keratitis. Imaging experiments both in vitro and in vivo demonstrated that the probe exhibited "turn-on" fluorescence signals in response to nitroreductase-secreting bacteria within 10 min. Furthermore, the fluorescence intensity reached its highest at 4 or 6 h in vitro and at 30 min in vivo when the excitation wavelength was set at 520 nm. Therefore, the NTRP has the potential to serve as a feasible agent for the rapid and noninvasive in situ fluorescence diagnosis of bacterial keratitis.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

A novel low-background nitroreductase fluorescent probe for real-time fluorescence imaging and surgical guidance of thyroid cancer resection.

Thyroid cancer always appears insidiously with few noticeable clinical symptoms. Due to its limitations, conventional ultrasound imaging can lead to missed or misdiagnosed cases. Surgery is still the primary treatment method of thyroid cancer, but removal of surrounding healthy tissues to minimize recurrence leads to overtreatment and added patient suffering. To address this challenge, herein, a nitroreductase (NTR) fluorescent probe, Ox-NTR, has been developed for detecting thyroid cancer and tracking the surgical removal of thyroid tumors by fluorescence imaging. The conjugated structure of oxazine 1 was disrupted, significantly reducing the issue of high background signals, thus effectively achieving low background fluorescence. Under hypoxic conditions, the nitro group of Ox-NTR can be reduced to an amine and subsequently decomposed into oxazine 1, emitting intense red fluorescence. Ox-NTR has a low detection limit of 0.09 µg/mL for NTR with excellent photostability and selectivity. Cellular studies show that Ox-NTR can effectively detect NTR levels in hypoxic thyroid cancer cells. Moreover, the ability of Ox-NTR of rapid response to thyroid cancer in vivo is confirmed by fluorescence imaging in mice, distinguishing tumors from normal tissues due to its superior low background fluorescence. Utilizing this fluorescence imaging method during surgical resection can guide the removal of tumors, preventing both missed tumor tissues and accidental removal of healthy tissue. In summary, the novel Ox-NTR offers precise detection capabilities that provide significant advantages over traditional imaging methods for thyroid cancer diagnosis and treatment, making it a valuable tool to guide tumor removal in surgical procedures.

Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Sequence similarity network analysis of drug- and dye-modifying azoreductase enzymes found in the human gut microbiome.

Drug metabolism by human gut microbes is often exemplified by azo bond reduction in the anticolitic prodrug sulfasalazine. Azoreductase activity is often found in incubations with cell cultures or ex vivo gut microbiome samples and contributes to the xenobiotic metabolism of drugs and food additives. Applying metagenomic studies to personalized medicine requires knowledge of the genes responsible for sulfasalazine and other drug metabolism, and candidate genes and proteins for drug modifications are understudied. A representative gut-abundant azoreductase from Anaerotignum lactatifermentan DSM 14214 efficiently reduces sulfasalazine and another drug, phenazopyridine, but could not reduce all azo-bonded drugs in this class. We used enzyme kinetics to characterize this enzyme for its NADH-dependent reduction of these drugs and food additives and performed computational docking to provide the groundwork for understanding substrate specificity in this family. We performed an analysis of the Flavodoxin-like fold InterPro family (IPR003680) by computing a sequence similarity network to classify distinct subgroups of the family and then performed chemically-guided functional profiling to identify proteins that are abundant in the NIH Human Microbiome Project dataset. This strategy aims to reduce the number of unique azoreductases needed to characterize one protein family in the diverse set of potential drug- and dye-modifying activities found in the human gut microbiome.