[Developments of Profluorescent Nitroxide Probes for Highly Sensitive and Selective Detection of Biological Redox Molecules].

Disruption of the redox balance in vivo is closely involved in the development of various diseases associated with oxidative stress. Therefore, methods for the in vivo analysis of antioxidants and free radicals are essential to elucidate the pathogenic mechanisms of such diseases. Although profluorescent nitroxide probes can be used to evaluate redox molecules with high sensitivity, these probes have low selectivity. Recently, we developed two profluorescent nitroxide probes, 15-((9-(ethylimino)-10-methyl-9Hbenzo[a]phenoxazin-5-yl)amino)-3,11-dioxa-7-azadispiro-hexadecan-7-yloxyl (Nile-DiPy) and 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), which had high sensitivity and selectivity toward ascorbic acid and lipid-derived radicals, respectively. These probes can react sensitively and selectively to each target molecule and can be used in animal experiments. In this paper, we review the design strategies and application of these profluorescent nitroxide probes.

Anti-influenza A virus activity and structure-activity relationship of a series of nitrobenzoxadiazole derivatives.

Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed. In this work, we assayed a library of nitrobenzoxadiazole derivatives against the influenza virus A/Puerto Rico/8/34 H1N1 (PR8) strain. We identified three promising 4-thioether substituted nitrobenzoxadiazoles (12, 17, and 25) that were able to inhibit viral replication at low micromolar concentrations in two different infected cell lines using a haemagglutination assay. We further assessed these molecules using an In-Cell Western assay, which confirmed their potency in the low micromolar range. Among the three molecules, 12 and 25 displayed the most favourable profile of activity and selectivity and were selected as hit compounds for future optimisation studies.

A FRET-based ratiometric fluorescent probe for sulfide detection in actual samples and imaging in Daphnia magna.

Sulfide plays an important role in many important life processes, and abnormal levels of sulfide that may cause diseases. Sulfide is also essential in industrial production and food processing, and it is well concerned for environmental issues and food safety. In order to study the physiological and pathological effects of sulfide and the impact on the environment, it is still urgent to develop a convenient and effective sulfide detection technology. Here, we developed a ratiometric fluorescent probe 7-Nitro-1,2,3-benzoxoxadiazole-Acridoneacetylpiperazine (NBD-AAP) which is based on the fluorescence resonance energy transfer (FRET) between acridone and NBD fluorophores. The NBD-AAP probe could produce a ratiometric response to micromolar sulfide in buffer (pH = 7.4), exhibiting a significant enhancement in fluorescent emission ratio (F427/F552) and obvious visual phenomenon (orange to pink under visible light and yellow to blue under UV light). At the same time, this NBD-AAP probe also has excellent properties including high selectivity and low detection limit (0.19 µM). In addition, this probe has been successfully used for detecting the sulfide in actual samples (monosodium glutamate, beer, environmental water) and imaging in Daphnia magna. These results indicate that NBD-AAP has broad application prospects in sulfide detection and in vivo imaging studies.

A novel highly sensitive fluorescent probe for bioimaging biothiols and its applications in distinguishing cancer cells from normal cells.

In recent years, targeting drugs made by physical loading or chemical bonding of drugs on small molecular carriers have shown a very wide application prospect in the field of tumor and cancer treatment. How to achieve the release of drugs in cancer cells has become the core of this research. One of the most important bases for drug localization is to use the difference of small molecular biothiol concentration between cancer cells and normal cells. Details of the changes of biothiol levels in the growth and reproduction of cancer cells are still poorly understood, and the main reason is the lack of sensitive real-time imaging tools for biothiols in cancer cells. In this work, we reasonably designed and synthesized the combination of 4-hydroxy-1,8-naphthalimide and NBD-Cl as a concise fluorescent probe HN-NBD for imaging biothiols in live cells and zebrafish. In addition, due to the advantages of HN-NBD design, it is sufficiently sensitive to biothiols, and further imaging can distinguish cancer cells from normal cells. Probe HN-NBD would be of great significance to biomedical researchers for the study of biothiol-related diseases, the screening of new anticancer drugs, and the early diagnosis and treatment of cancers.

Nitrobenzoxadiazole Ether-Based Near-Infrared Fluorescent Probe with Unexpected High Selectivity for H2S Imaging in Living Cells and Mice.

H2S is an important endogenous gasotransmitter, and its detection in living systems is of great significance. Especially, selective and sensitive near-infrared (NIR) fluorescent H2S probes with rapid response and large Stokes shift are highly desirable because of their superiority for in vivo detection. Probes with nitrobenzoxadiazole (NBD) ether as reaction sites have been well-explored recently to detect biothiols or H2S/biothiols simultaneously, rather than to detect H2S selectively. In this work, a new NBD ether-based NIR fluorescent probe was developed, which was unexpectedly found to show high selectivity for H2S over various other analytes including biothiols, making it practical for specific detection of H2S both in vitro and in vivo. Upon response to H2S, this probe showed rapid and significant turn-on NIR emission changes centered at 744 nm within 3 min, together with a remarkable large Stokes shift (166 nm) and high sensitivity (LOD: 26 nM). Moreover, imaging exogenous and endogenous H2S in living cells and rapid imaging of H2S in living mice with this probe was successfully applied with excellent performance.

Molecular engineering of a colorimetric two-photon fluorescent probe for visualizing H2S level in lysosome and tumor.

As a multifunctional signaling molecule, hydrogen sulfide (H2S) plays an essential role in diverse physiological and pathological processes. The two-photon fluorescence probes detecting H2S selectively in vivo could be useful tools to better study the mechanism of diseases. Then, an efficient two-photon lysosome-specific probe 1 has been developed to detect endogenous H2S in living cells and mice. Probe 1 displays excellent properties with 28-fold fluorescence enhancement, marked color changes in naked-eye and fluorescence, high selectivity and sensitivity, and low detection limit (0.22 µM) to H2S. These remarkable properties of probe 1 enable its practical applications in detecting H2S in environment (wastewater) and food (beer). Moreover, as a two-photon probe under near infrared excitation at 790 nm, probe 1 can monitor the level changes of endogenous H2S of lysosome and tumor in living system with good membrane permeability and high imaging resolution. Specially, the probe detecting H2S distribution in lysosome could provide more evidences to explain the association of target-organelle and H2S.

Synthesis and characterisation of a new benzamide-containing nitrobenzoxadiazole as a GSTP1-1 inhibitor endowed with high stability to metabolic hydrolysis.

The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.

Fluorescent probe for sensitive discrimination of Hcy and Cys/GSH in living cells via dual-emission.

Abnormal levels of Cys, Hcy and GSH are associated with various diseases, thus monitoring biothiols is of great significance. In this work, a dual-emission responsive near-infrared fluorescent probe NIR-NBD for detecting Hcy and Cys/GSH was developed based on the conjugation of a dicyanoisophorone based fluorophore (NIR-OH) and 7-nitrobenzofurazan (NBD). To our surprise, the addition of Hcy induced significant fluorescence enhancement at both 549 and 697 nm; while Cys/GSH resulted in major fluorescence emission at 697 nm. The detection limit was determined to be 33.2 nM for Cys, 33.5 nM for Hcy, and 34.4 nM for GSH. Therefore, the probe can be used for discriminative detection of Hcy and Cys/GSH. Moreover, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing Hcy and Cys/GSH in living cells.

A near-infrared Nile red fluorescent probe for the discrimination of biothiols by dual-channel response and its bioimaging applications in living cells and animals.

Biothiols, including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and H2S, play important roles in human physiological processes. However, it is a great difficulty to distinguish biothiols from each other because of their similar chemical properties. Based on Nile red, we have designed and synthesized a near-infrared fluorescent probe for discriminating Cys/Hcy from GSH/H2S by a dual-channel detection method. Using an ether bond, near-infrared Nile red was attached to 7-nitrobenzofurazan to construct the probe. Due to the photo-induced electron transfer, the probe showed almost no fluorescence from the green to red emission band. But upon the addition of Cys (0-150 µM) or Hcy (0-200 µM), the probe exhibited a noteworthy fluorescence "turn-on" signal in two unique emission bands (Green and Red) with a fast response (within 30 min). In contrast, the probe displayed an increase in fluorescence only in the red channel when encountering GSH (0-70 µM) or H2S (0-50 µM), and GSH/H2S could be tested respectively by different response time. The limit of detection was calculated to be 0.09 µM (Cys), 0.30 µM (Hcy), 0.24 µM (GSH), and 0.04 µM (H2S), respectively (based on S/N = 3). The desirable dual-channel detection could be achieved in serum samples and living cells. Moreover, the probe could be applied for bioimaging in mice, which indicated its potential application in the clinic.

Highly sensitive and selective light-up fluorescent probe for monitoring gallium and chromium ions in vitro and in vivo.

Over the past decades, gallium (Ga) compounds have gained importance in the field of cancer treatment. Gallium acts as an iron mimic and disturbs iron-dependent propagation and other processes in tumor cells. However, the toxicity of gallium was also well documented in vitro and in vivo in animals. Though the oral administration of gallium in humans is less toxic, it has also been shown that a long period of administration could induce tumor fibrosis. Chromium (Cr), a naturally occurring heavy metal, is commonly used in industrial processes and can cause severe health problems in humans. It has been found to be closely involved in the metabolism of nucleic acids, proteins and fats in humans. Cr(iii) salts can be used as micronutrients and dietary supplements. However, similar to gallium (Ga3+), chromium (Cr3+) can build up to an excessive degree that is harmful to the human body. Therefore, it would be of great interest to develop chemosensing for the selective and sensitive detection of gallium and chromium ions in vitro and in vivo. Herein, we reported that an NBD-based (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) fluorescent probe (NBDT) was fabricated with demonstrated extraordinary specificity and sensitivity. A swift response toward Ga3+ and Cr3+ ions was discovered using fluorescence enhancement over a wide pH range and with cycle stability. Furthermore, lighted up by Ga3+ and Cr3+ ions in vitro, this NBDT sensor was successfully applied to detect exogenous Ga3+ and Cr3+ ions in MDA-MB-231 and HepG2 cells. Additionally, using zebrafish as the in vivo model, we demonstrated the capability of this NBDT for detecting and imaging Ga3+ and Cr3+ ions in zebrafish. Taken together, this NBDT has indicated great potential for detecting and monitoring Ga3+ and Cr3+ ions in vitro and in vivo.

Molecular Glue that Spatiotemporally Turns on Protein-Protein Interactions.

We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.

Chemical Proteomics and Super-resolution Imaging Reveal That Chloroquine Interacts with Plasmodium falciparum Multidrug Resistance-Associated Protein and Lipids.

It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.

Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full peptidyl moiety of (pseudo)glutathione.

CONTEXT: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice. OBJECTIVE: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers. MATERIALS AND METHODS: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported. RESULTS: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX). DISCUSSION AND CONCLUSIONS: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.

Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines.

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochemistry, as a derivatization agent in analytical chemistry, and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines containing two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compound formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated aromatic systems for amine derivatization in analytical and biochemistry applications. The reaction with polyamines opens the way to libraries of NBD-tagged compounds.

Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG.

Sodium-coupled glucose co-transporters SGLT1 and SGLT2 play important roles in intestinal absorption and renal reabsorption of glucose, respectively. Blocking SGLT2 is a novel mechanism for lowering the blood glucose level by inhibiting renal glucose reabsorption and selective SGLT2 inhibitors are under development for treatment of type 2 diabetes. Furthermore, it has been reported that perturbation of SGLT1 is associated with cardiomyopathy and cancer. Therefore, both SGLT1 and SGLT2 are potential therapeutic targets. Here we report the development of a non-radioactive cell-based method for the screening of SGLT inhibitors using COS-7 cells transiently expressing human SGLT1 (hSGLT1), CHO-K1 cells stably expressing human SGLT2 (hSGLT2), and a novel fluorescent d-glucose analogue 1-NBDG as a substrate. Our data indicate that 1-NBDG can be a good replacement for the currently used isotope-labeled SGLT substrate, (14)C-AMG. The Michaelis constant of 1-NBDG transport (0.55 mM) is similar to that of d-glucose (0.51 mM) and AMG (0.40 mM) transport through hSGLT1. The IC50 values of a SGLT inhibitor phlorizin for hSGLT1 obtained using 1-NBDG and (14)C-AMG were identical (0.11 µM) in our cell-based system. The IC50 values of dapagliflozin, a well-known selective SGLT2 inhibitor, for hSGLT2 and hSGLT1 determined using 1-NBDG were 1.86 nM and 880 nM, respectively, which are comparable to the published results obtained using (14)C-AMG. Compared to (14)C-AMG, the use of 1-NBDG is cost-effective, convenient and potentially more sensitive. Taken together, a non-radioactive system using 1-NBDG has been validated as a rapid and reliable method for the screening of SGLT1 and SGLT2 inhibitors.

Syntheses of 2-NBDG analogues for monitoring stereoselective uptake of D-glucose.

2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.

Novel irreversible fluorescent probes targeting the 18 kDa translocator protein: synthesis and biological characterization.

The 18 kDa translocator protein (TSPO) is a mitochondrial protein whose basal density is altered in several diseases, with the result that the evaluation of its expression levels by means of molecular imaging techniques represents a promising diagnostic approach. Experimental procedures using a labeled ligand often cause loss of the bound probe, and consequently high affinity ligands covalently binding the receptor protein are needed to overcome this problem. We have previously described a series of N,N-dialkyl-(2-phenylindol-3-yl)glyoxylamides as potent and selective TSPO ligands. Starting from these derivatives, we designed novel TSPO irreversible ligands bearing an electrophilic isothiocyanato group (7, 8), together with an irreversible NBD-fluorescent probe (18). The TSPO affinity of the new irreversible ligands was measured on rat tissue homogenates by [(3)H]Ro 5-4864 radiobinding kinetic assays, all compounds showing high affinities for the target protein. Further biological characterization of the fluorescent irreversible TSPO probe 18 was carried out by using fluorescent spectroscopy in human glioma cells.

Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin.

Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.

Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.

Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.