RESUMO
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.
Assuntos
Leucil Aminopeptidase , Neoplasias Hepáticas , Nitrorredutases , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Humanos , Animais , Leucil Aminopeptidase/metabolismo , Leucil Aminopeptidase/análise , Nitrorredutases/metabolismo , Nitrorredutases/análise , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Camundongos , Corantes Fluorescentes/química , Imagem ÓpticaRESUMO
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.
Assuntos
Corantes Fluorescentes , Nitrorredutases , Imagem Óptica , Animais , Humanos , Camundongos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias/diagnóstico por imagem , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Nitrorredutases/metabolismo , Nitrorredutases/análise , Técnicas Fotoacústicas , Dióxido de Nitrogênio/síntese química , Dióxido de Nitrogênio/químicaRESUMO
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.
Assuntos
Corantes Fluorescentes , Ceratite , Nitrorredutases , Corantes Fluorescentes/química , Nitrorredutases/metabolismo , Nitrorredutases/análise , Ceratite/diagnóstico , Ceratite/microbiologia , Animais , Humanos , Imagem Óptica/métodos , CamundongosRESUMO
Hypoxia is commonly regarded as a typical feature of solid tumors, which originates from the insufficient supply of oxygen. Herein, the development of an efficient method for assessing hypoxia levels in tumors is strongly desirable. Nitroreductase (NTR) is an overexpressed reductase in the solid tumors, has been served as a potential biomarker to evaluate the degrees of hypoxia. In this work, we elaborately synthesized a new near-infrared (NIR) fluorescence probe (MR) to monitor NTR activity for assessment of hypoxia levels in living cells and in tumors. Upon exposure of NTR, the nitro-unit of MR could be selectively reduced to amino-moiety with the help of nicotinamide adenine dinucleotide. Moreover, the obtained fluorophore emitted a prominent NIR fluorescence, because it possessed a classical "push-pull" structure. The MR displayed several distinguished characters toward NTR, including intense NIR fluorescent signals, large Stokes shift, high selectivity and low limit of detection (46 ng/mL). Furthermore, cellular confocal fluorescence imaging results validated that the MR had potential of detecting NTR levels in hypoxic cells. Significantly, using the MR, the elevated of NTR levels were successfully visualized in the tumor-bearing mouse models. Therefore, this detecting platform based on this probe may be tactfully constructed for monitoring the variations of NTR and estimating the degrees of hypoxia in tumors.
Assuntos
Corantes Fluorescentes , Nitrorredutases , Nitrorredutases/metabolismo , Nitrorredutases/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Animais , Camundongos , Humanos , Imagem Óptica/métodos , Raios Infravermelhos , Camundongos Nus , Camundongos Endogâmicos BALB C , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismoRESUMO
This article reports the synthesis and characterization of a novel self-immolative linker, based on thiocarbonates, which releases a free thiol upon activation via enzymes. We demonstrate that thiocarbonate self-immolative linkers can be used to detect the enzymes penicillin G amidase (PGA) and nitroreductase (NTR) with high sensitivity using absorption spectroscopy. Paired with modern thiol amplification technology, the detection of PGA and NTR were achieved at concentrations of 160 nM and 52 nM respectively. In addition, the PGA probe was shown to be compatible with both biological thiols and enzymes present in cell lysates.
Assuntos
Nitrorredutases/análise , Penicilina Amidase/análise , Compostos de Sulfidrila/química , Estrutura Molecular , Nitrorredutases/metabolismo , Penicilina Amidase/metabolismo , Espectrometria de FluorescênciaRESUMO
A leishmaniose é uma zoonose de ampla distribuição mundial, causada pelos parasitas tripanossomatídeos do gênero Leishmania. Infelizmente, o arsenal terapêutico disponível é precário, mas vê-se crescente o interesse científico pela busca do potencial de derivados nitroheterocíclicos como alternativas terapêuticas. Nesse contexto, este trabalho analisou o potencial de derivados 5-nitro-2-furfurilidênicos contra diferentes cepas de Leishmania, assim como investigou um possível modo de ação para esta classe de nitrocompostos. Para tal, a quimioteca foi sintetizada de acordo com publicações prévias do grupo. O potencial de inibição de crescimento das culturas de promastigotas de L. (L.) infantum (Linf) e L. (L.) major (Lmaj) foi determinado, utilizando miltefosina (MILT) (Linf - IC50: 8,28±0,33 µM), anfotericina B (AMB) (Linf - IC50: 0,02±0,002 µM) e nifurtimox (NFX) (Lmaj - IC50: 3,5±0,09 µM) como referência. A maioria dos compostos apresentaram maior potencial que as referênias, destacando o composto 40 (Linf - IC50: 0,2±0,019 µM/ Lmaj - IC50: 0,087 ± 0,001 µM) como mais eficaz. Contra as formas amastigotas intracelulares, para Linf os compostos 40, 13 e 15 foram mais eficazes em reduzir a carga parasitária dos macrófagos infectados que fármacos de referência. Para Lmajor, o composto 40 (IC50: 0,006 ± 0,0003 µM) foi mais ativo que o NFX (IC50: 2,15 ± 0,01 µM). Também foi determinada a atividade da quimioteca frente a enzima nitrorredutase (NTR1), utilizando cepas de T. brucei superexpressantes de NTR1, e os compostos analisados foram até 18 vezes mais eficazes que à cepa wild-type. Ademais, a partir da análise exploratória de dados por análise de componentes principais (PCA) e de grupamentos hierárquicos (HCA), foi reconhecida a influência das propriedades relacionadas com o equilíbrio hidrófilo-lipófilo e da natureza estérica/geométrica das moléculas para atividade anti-Leishmania
Leishmaniasis is a worldwide zoonosis caused by trypanosomatid parasites of the genus Leishmania. Unfortunately, the available therapeutic arsenal is precarious, but there is growing scientific interest in searching the potential of nitroheterocyclic derivatives as therapeutic alternatives. In this context, this work analyzed the potential of 5-nitro-2-furfurylidene derivatives against different Leishmania strains, as well as investigated the potential mode of action for this nitro compounds class. To this end, the chemolibrary was synthesized according to our group's previous publications. The growth inhibitory potential potential for promastigote cultures of L. (L.) infantum (Linf) and L. (L.) major (Lmaj) was determined using miltefosine (MILT) (Linf - IC50: 8.28±0.33 µM), amphotericin B (AMB) (Linf - IC50: 0.02±0.002 µM) and nifurtimox (NFX) (Lmaj - IC50: 3.5±0.09 µM) as reference. Most of the compounds were more potent than the references, highlighting compound 40 (Linf - IC50: 0.2±0.019 µM/ Lmaj - IC50: 0.087 ± 0.001 µM) as the most effective. Against intracellular amastigote, for Linf, compounds 40, 13 and 15 were more effective in reducing the parasite load of infected macrophages than reference drugs. For Lmajor, compound 40 (IC50: 0.006 ± 0.0003 µM) was more active than NFX (IC50: 2.15 ± 0.01 µM). The activity against nitroreductase (NTR1) enzyme was determined using overexpressing NTR1 mutant T. brucei strains, and the analyzed compounds were up to 18 times more effective than wild-type. Furthermore, exploratory data analysis using principal component analysis (PCA) and hierarchical clustering (HCA) methods were used. The influence of properties related to the hydrophiliclipophilic balance and the steric/geometric nature of the molecules was associated with the anti-Leishmanial activity
Assuntos
Terapias Complementares/instrumentação , Leishmaniose/patologia , Análise de Componente Principal/classificação , Leishmania/metabolismo , Nitrorredutases/análise , Preparações Farmacêuticas/análise , Análise de Dados , Nitrocompostos/agonistasRESUMO
Hypoxia is a pathological hallmark of solid tumors. Detection of hypoxia is therefore of great interest for tumor diagnosis and treatment. As a well-established biomarker of hypoxia, nitroreductase (NTR) has been widely exploited in the development of hypoxia-responsive fluorescent probes on the basis of its enzymatic activity to reduce nitroaryl groups. However, studies on the relationship between the nitroaryl structure and the probe performance for optimal probe design are still rare. Here we report a comparative investigation of nitroaryl groups and identification of the optimal nitroaryl structure for developing new fluorescent probes with extremely high efficiency in the detection of NTR and the imaging of hypoxic tumor cells. Specifically, we synthesized a series of resorufin-based fluorescent probes containing different nitroaryl groups, compared their fluorescence responses to NTR, and identified 2-nitro-N-methyl-imidazolyl as the optimal nitroaryl group that is much more efficient than the most widely used 4-nitrophenyl for NTR detection. The structure-performance relationship was then studied by theoretical molecular docking, revealing the unique features of 2-nitro-N-methyl-imidazolyl in binding and reaction with NTR. We further incorporated the 2-nitro-N-methyl-imidazolyl group into a near-infrared (NIR) hemicyanine fluorophore and developed a NIR fluorescent probe NFP-7 for the detection of NTR and hypoxic tumor cells. NFP-7 exhibits a strong fluorescence increase toward NTR in vitro with an ultrafast (within 40 seconds to fluorescence maximum) and ultrasensitive (0.2 ng mL-1 detection limit) response. NFP-7 has also been demonstrated for imaging the degree of hypoxia in live tumor cells and, more importantly, in a murine tumor model. Our study provides important insights into hypoxia probe development and new tools for hypoxia imaging.
Assuntos
Descoberta de Drogas , Corantes Fluorescentes/química , Nitrorredutases/análise , Imagem Óptica , Oxazinas/química , Animais , Corantes Fluorescentes/síntese química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Confocal , Simulação de Acoplamento Molecular , Estrutura Molecular , Nitrorredutases/metabolismo , Oxazinas/síntese química , Células Tumorais CultivadasRESUMO
Aromatic nitro compounds are reduced to their corresponding amino derivatives by nitroreductases (NTR), while identification and characterization of the corresponding enzymes in mammalian systems are yet unrevealed. However, mammalian NTR activity has been considered as a favorable target in development of theranostic agents for cancer and hypoxia of solid tumors. Currently, small molecule-based fluorescent probes have emerged as a valuable assay tool for NTR activity. However, there has been a limit to comparing NTR activity between different cells, since most probes have relied on fluorescence changes that are affected by not only enzymatic activity but also nonenzymatic factors. Here, we developed a self-calibrating bipartite fluorescent probe, consisting of NTR-sensitive nitronaphthalimide and nonsensitive coumarin moieties. Thereby, it was possible to compare the relative NTR activity by monitoring fluorescence ratios in noncancerous and some cancerous cells and to demonstrate for certain that the elevated NTR activity is associated with cancer cells and hypoxia states.
Assuntos
Materiais Biocompatíveis/química , Hipóxia Celular , Corantes Fluorescentes/química , Neoplasias/metabolismo , Nitrorredutases/metabolismo , Materiais Biocompatíveis/síntese química , Calibragem , Corantes Fluorescentes/síntese química , Teste de Materiais , Estrutura Molecular , Nitrorredutases/análise , Imagem Óptica , Tamanho da PartículaRESUMO
The study aimed to evaluate the total content and bioavailable forms of Zn, Cu, Pb and Ni and enzymatic activity (nitro reductase and peroxidases) in the mineral levels of surface soils within the zone of influence of various tree species. The conducted variance analysis confirmed the significant impact of the studied tree habitats on the total content and bioavailable forms of metals and on enzymatic activity. The total content of analysed metals were low and in no case exceeded the possible concentrations. The high bioavailability (AF %) values calculated for habitats of different species compositions (of 53.78% for Zn, 76.82% for Cu, 60.81% for Pb and 44.72% for Ni) may pose a risk of accumulation of these metals in plants. A significant correlation was found between nitrate reduction activity and Pb content (r = 0.510) and Cu (r = 0.678). Principal component analysis allowed two principal components to be distinguished (PC1 and PC2) that accounted for 60.95% of the total change in variance.
Assuntos
Metais Pesados/análise , Nitrorredutases/análise , Peroxidases/análise , Poluentes do Solo/análise , Solo/química , Árvores , Disponibilidade Biológica , Ecossistema , Concentração de Íons de Hidrogênio , Polônia , Análise de Componente Principal , Especificidade da EspécieRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Compostos de Boro/química , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Nitrorredutases/análise , Biomarcadores Tumorais/metabolismo , Compostos de Boro/síntese química , Corantes Fluorescentes/síntese química , Humanos , Modelos Moleculares , Estrutura Molecular , Nitrobenzenos/química , Nitrorredutases/metabolismoRESUMO
Rhodamine dyes bearing aromatic nitro group has been synthesized for nitroreductase enzyme chemosensing applications. The probe is showing very selective turn-on fluorescent response towards nitroreductase enzymes and in hypoxic conditions. The sensor displays a remarkable fluorescent enhancement at 557 nm (λex = 500 nm) without the interference of other biologically relevant species under hypoxic conditions in a physiological medium. The nitro group in the sensor is reduced by the nitroreductase enzyme to the amino group, resulting in the hydrolysis of the probe and subsequent release of highly fluorescent rhodamine 6G dye is observed. This rhodamine based fluorescent probe has been utilized for the imaging of nitroreductase enzymes as well as hypoxia in live cells.
Assuntos
Hipóxia Celular , Colorimetria/métodos , Microscopia de Fluorescência , Nitrorredutases/análise , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Microscopia de Fluorescência/métodos , Rodaminas/químicaRESUMO
Hypoxia, as a condition in which a region of body has low oxygen tension, is closely related to a variety of pathological conditions, and in many diseases local hypoxia occurs that would further increase the severity of diseases. Hence the extent of hypoxia could reflect the related pathological conditions and diseases, and the detection of hypoxia is of great significance. In hypoxia, the elevated level of nitroreductase (NTR) usually occurs, which could serve as a biomarker for hypoxia and thus the related diseases. Herein, an activatable fluorescent probe TPAQS-NO2 based on aggregation-induced emission (AIE) was designed for hypoxia detection via responding to NTR. The probe consists of an electron acceptor quinolinium and an electron donor triphenylamine group. The activated probe shows a large Stokes shift (186 nm). The probe TPAQS-NO2 was successfully used for detecting the early-stage and the advanced-stage tumors via NTR detection in 4T1 tumor-bearing mouse model. Furthermore, the probe TPAQS-NO2 was applied for detecting NTR in the cerebral ischemia (CIS) mouse model. The probe could offer an effective approach for detecting hypoxia-related pathological conditions.
Assuntos
Isquemia Encefálica/diagnóstico por imagem , Corantes Fluorescentes/química , Hipóxia/diagnóstico por imagem , Neoplasias/diagnóstico por imagem , Nitrorredutases/análise , Compostos de Anilina/síntese química , Compostos de Anilina/química , Animais , Isquemia Encefálica/fisiopatologia , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Neoplasias/fisiopatologia , Imagem Óptica , Compostos de Quinolínio/síntese química , Compostos de Quinolínio/químicaRESUMO
Nitroreductase (NTR) with a high expression level in tumors has been considered as a biomarker of highly aggressive hypoxia tumors. Thus, it is important to develop powerful tools for tumor hypoxia detection. Here, we developed a two-photon fluorescent probe hTP-NNO2 for NTR detection. The probe with one-step synthesis exhibited high yield. hTP-NNO2 showed high selectivity and sensitivity for NTR and the detection limit was as low as 43 ng mL-1. hTP-NNO2 also showed low cytotoxicity and high stability, indicating that hTP-NNO2 is suitable for NTR detection in real-time and in situ under physiological conditions. hTP-NNO2 was used for NTR imaging in hypoxia cells and the fluorescence intensity of hTP-NNO2 increased with decreasing oxygen concentration. Benefiting from the advantages of two-photon fluorescent probes, we performed NTR detection in deep brain tissue with an imaging depth of up to 100 µm. hTP-NNO2 was further successfully applied for NTR detection in zebrafish and tumors. These results indicated that we developed a promising fluorescence imaging tool for NTR detection in vitro and in vivo.
Assuntos
Encéfalo/enzimologia , Corantes Fluorescentes/química , Nitrorredutases/análise , Imagem Óptica/métodos , Hipóxia Tumoral , Células A549 , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Camundongos , Camundongos Nus , Microscopia Confocal , Nitrorredutases/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Development of a mitochondria-targeting fluorescent probe with large Stokes shift and long-wavelength emission was benefit for accurate detection of hypoxic status, which was known as a major factor of the tumor physiology and influence important pathological processes. However, an efficient optical approach for simultaneously achieving such merits was still lacking. In this work, a turn-on fluorescence probe (HBT-NP) was designed to assess the hypoxic condition of tumor cells by detecting nitroreductase (NTR). Probe HBT-NP was constructed by conjugating 4-nitrobenzyl moiety as reaction site for NTR to 2-(benzo[d]thiazol-2-yl)-4-methylphenol derived fluorescent dye HBT-Py which demonstrated large Stokes shift (Δλ = 243 nm) and long wavelength emission (λem = 640 nm) due to intrinsic mechanism of ESIPT together with ICT process. Upon incubated with NTR, HBT-NP could successively undergo nitro reduction reaction and then release HBT-Py. The reaction mechanism was further confirmed by mass spectra and HPLC analysis, and the docking calculation also indicated that the binding mode and docking affinity of probe HBT-NP with NTR play an important role in catalytic reduction reaction process. As a result, HBT-NP displayed a wide linear range (0.1-1.5 µg/mL) and low detection limit (2.8 ng/mL) response to NTR, and could be used to evaluate hypoxic condition of cancer cells with precise mitochondria-targeting.
Assuntos
Benzotiazóis/química , Hipóxia Celular/fisiologia , Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Nitrorredutases/análise , Benzotiazóis/metabolismo , Benzotiazóis/toxicidade , Linhagem Celular Tumoral , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Nitrorredutases/metabolismo , Ligação Proteica , Compostos de Piridínio/química , Compostos de Piridínio/metabolismo , Compostos de Piridínio/toxicidade , Compostos de Quinolínio/química , Compostos de Quinolínio/metabolismo , Compostos de Quinolínio/toxicidade , Espectrometria de FluorescênciaRESUMO
A nitroreductase (NTR) responsive fluorescent probe, Na-NO2, comprising p-nitrobenzyl as the unique recognition group and 1,8-naphthalimide as fluorophore, was synthesized. Na-NO2 showed remarkable fluorescence "turn-on" signal in the presence of NTR under DMSO/H2O (1:19, v/v) buffered with PBS (pH = 7) solution in the presence of NADH (300 µM). Furthermore, the probe has a low detection limit down to 3.4 ng/mL and it is very sensitive towards the NTR in Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), normal and tumor cells such as HL-7702, HepG-2 and MCF-7.
Assuntos
Escherichia coli/enzimologia , Corantes Fluorescentes/química , Naftalimidas/química , Nitrorredutases/análise , Imagem Óptica , Staphylococcus aureus/enzimologia , Linhagem Celular , Corantes Fluorescentes/síntese química , Células Hep G2 , Humanos , Células MCF-7 , Naftalimidas/síntese química , Nitrorredutases/metabolismoRESUMO
All cellular processes are the results of synchronized actions of several intracellular biochemical pathways. Recent emphasis is to visualize such pathways using appropriate small molecular reagents, dye-labeled proteins, and genetically encoded fluorescent biosensors that produce a luminescence ON response either on selective binding or on reacting with an analyte that is produced through a specific biochemical/enzymatic transformation. Studying such enzymatic processes by probing the fluorescence response as the read-out signal is expected to provide important insights into crucial biochemical transformations induced by an enzyme in its native form. Many of such studies are extended for monitoring enzymatic transformations under in vitro or in vivo condition. A few of the recent reports reveal that such molecular probes are even capable of quantifying abnormal levels of enzymes in real-time and is linked to the key area of clinical diagnostics and chemical biology. A synchronized analysis of all such reports helps in developing a rationale for designing purpose-built molecular probes or chemodosimeters as well as newer reagents for studying crucial enzymatic process or quantification of the respective enzyme. In this review, an attempt will be there to highlight several recent bioimaging reagents and studies that have provided insights into crucial biochemical or enzymatic transformations.
Assuntos
Enzimas/metabolismo , Corantes Fluorescentes/química , Bibliotecas de Moléculas Pequenas/química , Aminopeptidases/análise , Aminopeptidases/metabolismo , Animais , Enzimas/análise , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/metabolismo , Humanos , Monofenol Mono-Oxigenase/análise , Monofenol Mono-Oxigenase/metabolismo , Nitrorredutases/análise , Nitrorredutases/metabolismo , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
OBJECTIVES: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. RESULTS: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. CONCLUSIONS: Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.
Assuntos
Bacillus subtilis/enzimologia , Biodegradação Ambiental , Dinitrobenzenos/metabolismo , Poluentes Ambientais/metabolismo , Nitrorredutases/análise , Vibrio vulnificus/enzimologia , Cinética , Nitrorredutases/isolamento & purificação , OxirreduçãoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Nitrorredutases/análise , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Regulação Bacteriana da Expressão Gênica/genética , Variação Genética/genética , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Simulação de Acoplamento Molecular , Nitrorredutases/genética , Ligação ProteicaRESUMO
Development of a highly selective and sensitive imaging probe for accurate detection of myocardial hypoxia will be helpful to estimate the degree of ischemia and subsequently guide personalized treatment. However, an efficient optical approach for hypoxia monitoring in myocardial ischemia is still lacking. In this work, a cardiomyocyte-specific and nitroreductase-activatable near-infrared nanoprobe has been developed for selective and sensitive imaging of myocardial hypoxia. The nanoprobe is a liposome-based nanoarchitecture which is functionalized with a peptide (GGGGDRVYIHPF) for targeting heart cells and encapsulating a nitrobenzene-substituted BODIPY for nitroreductase imaging. The nanoprobe can specifically recognize and bind to angiotensin II type 1 receptor that is overexpressed on the ischemic heart cells by the peptide and is subsequently uptaken into heart cells, in which the probe is released and activated by hypoxia-related nitroreductase to produce fluorescence emission at 713 nm. The in vitro response of the nanoprobe toward nitroreductase resulted in 55-fold fluorescence enhancement with the limit of detection as low as 7.08 ng/mL. Confocal fluorescence imaging confirmed the successful uptake of nanoprobe by hypoxic heart cells and intracellular detection of nitroreductase. More significantly, in vivo imaging of hypoxia in a murine model of myocardial ischemia was achieved by the nanoprobe with high sensitivity and good biocompatibility. Therefore, this work presents a new tool for targeted detection of myocardial hypoxia and will promote the investigation of the hypoxia-related physiological and pathological process of ischemic heart disease.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Hipóxia/diagnóstico por imagem , Isquemia Miocárdica/diagnóstico por imagem , Nitrorredutases/análise , Animais , Compostos de Boro/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Corantes Fluorescentes/toxicidade , Limite de Detecção , Lipossomos/química , Lipossomos/toxicidade , Masculino , Camundongos Endogâmicos ICR , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/toxicidade , Ratos , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
The overexpression of nitroreductase (NTR) in hypoxia has been recognized as a biomarker of highly aggressive disease, and the development of a hypoxia-sensitive two-photon (TP) bioimaging probe with both excitation and emission wavelengths in the red-light region provides favorable deep-tissue imaging with a low background fluorescence signal. Although quite a few TP hypoxia-sensitive fluorescent probes have been reported for NTR detection, their short emission wavelength (<550 nm) limits their application. Herein, we report a red light emissive TP hypoxia-sensitive turn-on probe (NRP) by employing Nile Red as a red-emitting fluorophore and p-nitrobenzene as an NTR recognition group with improved sensitivity. The NRP probe showed obvious strong red-fluorescence enhancement in the presence of NTR and high selectivity toward NTR in aqueous solution. Our in vitro experimental results illustrated that the NRP loaded tumor cells treated under hypoxia display remarkably strong fluorescence in both OP and TP microscopy at 655 nm with 45-fold enhancement, which affords deep-tissue penetration ability. The NRP probe was also successfully applied for imaging NTR in liver tissue slices and a 4T1-bearing mice model, which is important for bioimaging applications.