Nitroreductase Activity Research Articles

One step synthesis of red-emitting fluorescence turn-on probe for nitroreductase and its application to bacterial detection and oral cancer cell imaging.

Nitroreductase (NTR) belongs to a class of flavin mononucleotide-dependent and flavin adenine dinucleotide-dependent cytoplasmic enzymes; its contents in tumor cells increase during hypoxia. The development of fluorescent probes for detection of NTR activity is of great significance for the study of the state of hypoxia in living organisms. In this paper, a red-emitting fluorescence turn-on probe EBI-NO2 was synthesized using a one-step method. The fluorescence of the probe was enhanced by 60 folds in the presence of NTR. The probe also had high selectivity towards NTR, and its detection limit was as low as 1ng/mL. The reaction mechanism was verified using MS, molecular docking and theoretical calculations. In addition, it was successfully applied in real-time monitoring of NTR produced during growth of Escherichia coli (BL21) and in visualization of NTR in oral cancer cells (Cal-27) under hypoxia. This work provides a new imaging tool that can be applied to investigate the physiological and pathological changes in hypoxia oral cells.

Reduction of nitroarenes by magnetically recoverable nitroreductase immobilized on Fe3O4 nanoparticles

Enzymes as catalysts have attracted significant attention due to their excellent specificity and incomparable efficiency, but their practical application is limited because these catalysts are difficult to separate and recover. A magnetically recoverable biocatalyst has been effectively prepared through the immobilization of a nitroreductase (oxygen-insensitive, purified from Enterobacter cloacae) onto the Fe3O4 nanoparticles. The magnetic nanoparticles (MNPs) were synthesized by a coprecipitation method in an aqueous system. The surfaces of the MNPs were modified with sodium silicate and chloroacetic acid (CAA). Using 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) through a covalent binding, nitroreductase was loaded onto the modified magnetic carriers through covalent coupling, and thus, a magnetically recoverable biocatalyst was prepared. The free and immobilized nitroreductase activity was also investigated by the reduction of p-nitrobenzonitrile using nicotinamide adenine dinucleotide phosphate (NAPDH) as a cofactor. The activity of the immobilized enzyme was able to maintain 83.23% of that of the free enzyme. The prepared enzyme can easily reduce substituted nitrobenzene to substituted aniline at room temperature and atmospheric pressure, and the yield is up to 60.9%. Most importantly, the loaded nitroreductase carriers can be easily separated and recycled from the reaction system using an externally applied magnetic field. The magnetically recoverable biocatalyst can be recycled and reused 7 times while maintaining high activities and the activity of the magnetic catalyst can be maintained at more than 85.0% of that of the previous cycle. This research solves the recovery problem encountered in industrial applications of biocatalysts and presents a clean and green method of preparing substituted aniline.

Precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity with a targetable and activatable fluorescent probe.

An activatable and mitochondrial-targetable fluorescent probe was developed. This designed probe showed ratiometric fluorescence and light-up near-infrared emission responsiveness to nitroreductase, achieving precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity.

Rational design of fluorescent probes for targeted in vivo nitroreductase visualization.

Nitroreductase (NTR) has been recognized as a biomarker for identifying the hypoxic status of cancers. Therefore, it is of high scientific interest to design effective fluorescent probes for tracking NTR activity. However, studies on elucidation of the structure-performance relationship of fluorescent probes and those providing valuable insight into optimized probe design have rarely been reported. Three BODIPY based fluorescent probes were made by conjugation of para-, ortho-, and meta-nitrobenzene to the BODIPY core via a thiolether bond, respectively. Our study revealed that the linkage and nitro substituent position significantly influence the capability of nitroreductase detection.

An activatable NIR fluorescent rosol for selectively imaging nitroreductase activity

Hypoxia (i.e., pO2 ≤ ∼1.5 %) is an important characteristic of tumor microenvironments that directly correlates with resistance against first-line therapies and tumor proliferation/infiltration. The ability to accurately identify hypoxic tumor cells/tissue could afford tailored therapeutic regimens for personalized treatment, the development of more-effective therapies, and discerning the mechanisms underlying disease progression. Fluorogenic constructs currently developed for identifying such tumor cells/tissue operate by targeting the bioreductive activity of primarily the nitroreductase (NTR) class of enzymes, but collectively such constructs unfortunately present photophysical and/or physicochemical shortcomings that could limit their effectiveness upon implementation. To overcome these limitations, we present the rational design, development, and evaluation of the first activatable ultracompact xanthene core-based molecular probe (NO2-Rosol) for selectively imaging NTR activity that affords an “OFF-ON” near-infrared (NIR) fluorescence response (ca. > 700 nm) alongside a remarkable Stokes shift (ca. > 150 nm) via NTR activity-facilitated modulation to its energetics whereby the resultant interplay discontinues an intramolecular d-PET fluorescence-quenching mechanism transpiring between directly-linked electronically-uncoupled π-systems comprising its components. DFT calculations guided selection of a suitable fluorogenic scaffold and nitroaromatic moiety candidate that when adjoined could (i) afford such photophysical response upon bioreduction by upregulated NTR activity in hypoxic tumor cells/tissue and (ii) employ a retention mechanism strategy that capitalizes on an inherent physical property of the NIR fluorogenic scaffold for achieving signal amplification. NO2-Rosol demonstrated 705 nm NIR fluorescence emission and a 157 nm Stokes shift, selectivity for NTR over relevant bioanalytes, and a 28- and 12-fold fluorescence enhancement in solution and between cells cultured under different oxic conditions, respectively. In establishing feasibility for NO2-Rosol to provide favorable contrast levels in in solutioand in vitrostudies, we anticipate NO2-Rosol similarly doing so in future studies towards its translation.

Induction of the Antioxidant Response by the Transcription Factor NRF2 Increases Bioactivation of the Mutagenic Air Pollutant 3-Nitrobenzanthrone in Human Lung Cells.

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity. The genes encoding these enzymes are induced by the transcription factor NF-E2 p45-related factor 2 (NRF2), which raises the possibility that NRF2 activation exacerbates 3-NBA toxification. Since A549 cells possess constitutively active NRF2, we examined the effect of heterozygous (NRF2-Het) and homozygous NRF2 knockout (NRF2-KO) by CRISPR-Cas9 gene editing on the activation of 3-NBA. To evaluate whether NRF2-mediated gene induction increases 3-NBA activation, we examined the effects of NRF2 activators in immortalized human bronchial epithelial cells (HBEC3-KT). Changes in AKR1C1-1C3 and NQO1 expression by NRF2 knockout or use of NRF2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. We observed decreases in 3-NBA activation in the A549 NRF2 KO cell lines (53% reduction in A549 NRF2-Het cells and 82% reduction in A549 NRF2-KO cells) and 40-60% increases in 3-NBA bioactivation due to NRF2 activators in HBEC3-KT cells. Together, our data suggest that activation of the transcription factor NRF2 exacerbates carcinogen metabolism following exposure to diesel exhaust which may lead to an increase in 3-NBA-derived DNA adducts.

A genetically encoded fluorescent biosensor for detecting nitroreductase activity in living cancer cells

Genetically encoded fluorescent biosensors are particularly promising sensors to examine biochemical processes in a complex cellular context. RNA mimics of GFP are RNA-fluorophore complexes that emit green fluorescence comparable in brightness to fluorescent proteins. The fluorophore is non-fluorescent until it binds with specific RNA. We designed and developed a dual activated fluorescent probe based on RNA mimics of GFP for the detection of an intracellular nitroreductase. Our probe only fluoresces in the presence of both the specific RNA and nitroreductase. Since we used RNA to tag cells, our probe only fluoresces when nitroreductase exists in such cells. Our detection system should benefit the study of cell biomarkers in heterogeneous cell populations. More importantly, the strategy of quenching the RNA/DFHBI complex by alkylation of the hydroxyl group in DFHBI opens new possibilities for developing various genetically encoded fluorescent biosensors based on RNA/DFHBI complexes.

Soil Extracellular Enzyme Activities and Uptake of N by Oilseed Rape Depending on Fertilization and Seaweed Biostimulant Application

The present study has aimed at enhancing the insufficient knowledge of functional soil enzymes properties influenced by inorganic fertilization and biostimulant application to increase the uptake of nitrogen affecting the winter oilseed rape yield. Field experiments were conducted in Poland (53° N, 18° E) in Alfisol (USDA). In this experiment, the NPK rates applied were as follows: high 180 N, 70 P and 160 K 132 N (kg ha−1) or low 144 N, 35 P and 66 K (kg ha−1); fertilization with elemental S 36 or 0 (kg ha−1); and the seaweed biostimulant Kelpak was applied or there was no such treatment. Due to low NPK fertilization rates, the activity of dehydrogenases, peroxidases, and catalase increased in subsistent generative development stages from flowering to ripening. At the ripening stage, the activity of these enzymes, as well as nitroreductase activity, were inhibited by high NPK fertilizer rates. The seaweed biostimulant application and S fertilization increased N accumulation in plants of oilseed rape in generative development, by 16% and 13%, respectively, as compared with the lack of these treatments. The application of S increased the uptake of nitrogen in shoots and in whole oilseed rape plants only after application of higher rates of NPK.

Ratiometric Surface Enhanced Raman Scattering Immunosorbent Assay of Allergenic Proteins via Covalent Organic Framework Composite Material Based Nanozyme Tag Triggered Raman Signal "Turn-on" and Amplification.

The exploration of nanomaterials with mimic enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in an enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au-S bond and electrostatic force to further produce a Raman "hot spot". Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm-1 was weakened, and a new signal at 1591 cm-1 generated by 4-ATP was turned on, leading to the generation of a ratiometric SERS signal. Based on this performance, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I1591/I1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65-6.2 × 104 ng/mL, covering more than 3 orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery, and lower cross-reactivity, providing new insight into the application of SERS technology for trace target analysis.

A resorufin-based fluorescent turn-on probe responsive to nitroreductase activity and its application to bacterial detection

We present two resorufin-based fluorescent turn-on probes responsive to nitroreductase (NTR) activity (NTR probes 1 and 2) and their use toward the detection of bacteria. The probes exhibit a fluorescence turn-on at 586 nm in response to NTR activity, which corresponds to a vivid pink color. These optical changes are based on the NTR-mediated reduction of the nitro groups in the probes, which results in the production of highly fluorescent resorufin. The detection ability, mechanism, and enzyme kinetics of the probes for NTR activity have been thoroughly investigated. The enzyme efficiencies of the probes were superior to those previously reported. In addition, NTR probe 1 exhibited an excellent detection ability for NTR with high selectivity and no background signal when compared to 2. Moreover, NTR probe 1 was applicable for bacterial detection using a wash-free process because it gave rise to a fluorescence turn-on signal in response to bacterial NTR activity.

Evaluating the level of nitroreductase activity in clinical Klebsiella pneumoniae isolates to support strategies for nitro drug and prodrug development

To understand the potential utility of novel nitroreductase (NR)-activated prodrugs, NR enzyme activity was assessed in clinical Klebsiella pneumoniae isolates using a NR-activated fluorescent probe. NR activity was constant throughout the bacterial growth cycle, but individual K. pneumoniae isolates exhibited a wide range of NR activity levels. The genes of major NR enzymes (nfsA and nfnB) showed a number of sequence variants. Aside from a C-terminal extension of NfnB, which may be responsible for lower NR activity in specific isolates, the genetic differences did not explain the variation in activity. Analysis of important clinical strains (ST11, ST258, ST14 and ST101) showed significant variation in NR activity between isolates within the same sequence type despite conservation of nfsA/nfnB sequences. Addition of methyl viologen (MV), a known activator of soxRS, caused a significant increase in NR activity for all strains, with proportionally larger increases in activity seen for strains with low uninduced NR levels. Real-time PCR on selected strains following exposure to MV showed upregulation of soxS (15-32-fold) and nfsA (5-22-fold) in all strains tested. Expression of nfnB was upregulated 2-5-fold in 4/6 strains tested. High levels of NR activity in the absence of MV activation correlated with nitrofurantoin susceptibility. These data provide evidence that NR gene mutations and regulatory pathways influence NR activity in K. pneumoniae isolates and this is likely to impact treatment efficacy with novel nitro-containing drugs or prodrugs.

An efficient fluorescence sensor for nitroreductase selective imaging based on intramolecular photoinduced electron transfer

Timely and effective detection of bacterial pathogens is of great importance to reduce morbidity rates from bacterial infections. Recently, enzyme-activated fluorogenic probes, which invoke enzymatic catalysis to trigger fluorescence emission, have been superior sensors for bacterial infections needed for accurate diagnoses. Here, a fluorescent sensor for nitroreductase (NTR) detection is described. It is based on a cyanine fluorophore and utilizes photoinduced electron transfer to generate a rapid 10-fold fluorescence response after being catalytically reduced by NTR. It has enabled selective and sensitive visualization of NTR activity in vitro and in living bacterial pathogens. Thus, the probe has great potential to provide a rapid, noninvasive tool to diagnose infections and guide antimicrobial selection.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases Using a Photo-Crosslinking Fluorescent Sensor.

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines, and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, three-dimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.

Safety Evaluation of Oral Care Probiotics Weissella cibaria CMU and CMS1 by Phenotypic |and Genotypic Analysis.

Weissella cibaria CMU and CMS1 are known to exert beneficial effects on the oral cavity but have not yet been determined to be generally recognized as safe (GRAS), although they are used as commercial strains in Korea. We aimed to verify the safety of W. cibaria CMU and CMS1 strains through phenotypic and genotypic analyses. Their safety was evaluated by a minimum inhibitory concentration assay for 14 antibiotics, DNA analysis for 28 antibiotic resistance genes (ARGs) and one conjugative element, antibiotic resistance gene transferability, virulence gene analysis, hemolysis, mucin degradation, toxic metabolite production, and platelet aggregation reaction. W. cibaria CMU showed higher kanamycin resistance than the European Food Safety Authority (EFSA) cut-off, but this resistance was not transferred to the recipient strain. W. cibaria CMU and CMS1 lacked ARGs in chromosomes and plasmids, and genetic analysis confirmed that antibiotic resistance of kanamycin was an intrinsic characteristic of W. cibaria. Additionally, these strains did not harbor virulence genes associated with pathogenic bacteria and lacked toxic metabolite production, β-hemolysis, mucin degradation, bile salt deconjugation, β-glucuronidase, nitroreductase activity, gelatin liquefaction, phenylalanine degradation, and platelet aggregation. Our findings demonstrate that W. cibaria CMU and CMS1 can achieve the GRAS status in future.

A Highly Sensitive Chemiluminescent Probe for Detecting Nitroreductase and Imaging in Living Animals.

Hypoxia is a common characteristic of solid tumors, which is caused by the imbalance of oxygen supply and consumption. As the expression level of nitroreductase (NTR) increases in hypoxic solid tumors, NTR is one of the common biomarkers of hypoxia and widely used to evaluate the degree of tumor hypoxia. In this study, we designed and synthesized a highly water-soluble chemiluminescent probe, CL-NTR, for the detection of NTR activity in hypoxic tumors. It was found that the probe could be used to detect NTR with high sensitivity, and the total light photons increased tremendously with 6000-fold after the probe was treated with NTR. The chemiluminescence total light photons emission was directly proportional to the concentration of nitroreductase in the range of 3-55 ng/mL, with a detection limit of 0.947 ng/mL. Finally, the probe was successfully used to evaluate NTR activity in living mice by chemiluminescent imaging. In general, this probe has a remarkable response to NTR, which provides a promising method for the determination of NTR activity in vivo.

Role of Human Aldo-Keto Reductases in the Metabolic Activation of the Carcinogenic Air Pollutant 3-Nitrobenzanthrone.

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been previously implicated as the major nitroreductase responsible for 3-NBA activation, but it has recently been reported that human aldo-keto reductase 1C3 (AKR1C3) displays nitroreductase activity toward the chemotherapeutic agent PR-104A. We sought to determine whether AKR1C isoforms could display nitroreductase activity toward other nitrated compounds and bioactivate 3-NBA. Using discontinuous enzymatic assays monitored by UV-HPLC, we determined that AKR1C1-1C3 catalyze three successive two-electron nitroreductions toward 3-NBA to form the reduced product 3-aminobenzanthrone (3-ABA). Evidence of the nitroso- and hydroxylamino- intermediates were obtained by UPLC-HRMS. Km, kcat, and kcat/ Km values were determined for recombinant AKR1C and NQO1 and compared. We found that AKR1C1, AKR1C3, and NQO1 have very similar apparent catalytic efficiencies (8 vs 7 min-1 mM-1) despite the higher kcat of NQO1 (0.058 vs 0.012 min-1). AKR1C1-1C3 possess a Km much lower than that of NQO1, which suggests that they may be more important than NQO1 at the low concentrations of 3-NBA to which humans are exposed. Given that inhalation represents the primary source of 3-NBA exposure, we chose to evaluate the relative importance of AKR1C1-1C3 and NQO1 in human lung epithelial cell lines. Our data suggest that the combined activities of AKR1C1-1C3 and NQO1 contribute equally to the reduction of 3-NBA in A549 and HBEC3-KT cell lines and together represent approximately 50% of the intracellular nitroreductase activity toward 3-NBA. These findings have significant implications for the metabolism of nitrated polycyclic aromatic hydrocarbons and suggest that the hitherto unrecognized nitroreductase activity of AKR1C enzymes should be further investigated.

Degradation of trinitrotoluene by transgenic nitroreductase in Arabidopsis plants

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. TNT is toxic to many organisms, it is known to be a potential human carcinogen, and is persistent in the environment. This study presents a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of NAD(P)H-flavin nitroreductase (NFSB) from the Sulfurimonas denitrificans DSM1251. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerance and a strikingly higher capacity to remove TNT from their media. The highest specific rate constant of TNT disappearance rate was 1.219 and 2.297 mL/g fresh weight/h for wild type and transgenic plants, respectively. Meanwhile, the nitroreductase activity in transgenic plant was higher than wild type plant. All this indicates that transgenic plants show significantly enhanced tolerances to TNT; transgenic plants also exhibit strikingly higher capabilities of removing TNT from their media and high efficiencies of transformation.

Endoplasmic reticulum-targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues

Hypoxia conditions could increase the activity of intracellular nitroreductase (NTR) and lead to many malignant diseases. Therefore, monitoring the activity of NTR is of great significance to study the related diseases. Organelles play crucial roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum (ER) possesses single membrane structure, and it is the largest organelle in the cell. ER performs the synthesis, processing and modification of proteins and lipid, stabilizing the intracellular Ca2+ concentration and other physiological functions in living cells. Therefore, it is of great significance to develop ER-target probes in living system. Toward this goal, a new endoplasmic reticulum-targeted two-photon fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to NTR. Particularly, probe Na-NTR-ER has been successfully applied for the monitoring of NTR in ER with a high the Pearson's colocalization coefficient as 0.90 in HeLa cells and cancerous mouse tissues up to the depth of 100 μm with significant fluorescence signals.

Physiological aspects of nitro drug resistance in Giardia lamblia

For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4.Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases.

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).