A photo-responsive chemical modulation of m6A RNA demethylase FTO.

The adenine N6-methylation m6A is a crucial modification that is associated with several biological functions. One of the two m6A demethylases FTO has arisen as an attractive target for the development of novel cancer therapies. Here, we describe a new design, synthesis and evaluation of a photo-responsive and selective inhibitor of FTO.

Nanomolar detection of adenosine triphosphate (ATP) using a nanostructured fluorescent chemosensing ensemble.

We report a novel nanostructured chemosensing ensemble PyNp-C13/UD, obtained by self-assembling uranine dye (UD) and an amphiphilic pyridinium salt PyNp-C13. The ensemble was developed for the fluorescence turn-on sensing of ATP in aqueous solutions and inside living cells. The assembly operates via an indicator displacement assay (IDA) method with an ultra-low detection limit of 6.8 nM.

A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Novel Fluorescein-Based Fluorescent Probe for Detecting H2S and Its Real Applications in Blood Plasma and Biological Imaging.

A broad-spectrum fluorescent probe, which can be applied to monitoring H2S in various biological systems, has been rationally designed and synthesized. This specific probe was applied to localize the endogenous H2S in living Raw264.7 macrophage cells, HepG2 cells, and H9C2 cells. At the same time, the probe has successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS and CSE activity in H9C2 cells. This probe could serve as a powerful molecular imaging tool to further explore the physiological function and the molecular mechanisms of endogenous H2S in living animal systems.

Fluorescein dye derivatives and their nanohybrids: Synthesis, characterization and antimicrobial activity.

Fluorescein (resorcinolphthalein) is a synthetic organic photoactive dye compound soluble in water, alcohol and polar solvents. It is widely used as a fluorescent tracer in medicinal and biological applications and tumor infected tissues tracer. In this study, fluorescein (F) was condensed by five coupling agents namely: p,p-phenylene diamine, p-hydroxy aniline, o-hydroxy aniline, p-methoxy aniline and p-methyl aniline in a molar ratio of 2(F):1 (coupling agent). The chemical structures of the synthesized fluorescein derivatives were confirmed using: microelemental analysis, FTIR spectroscopy, 1H-NMR spectroscopy, and mass spectroscopy. The synthesized compounds were loaded on chemically prepared silver nanoparticles via reduction reaction of silver nitrate. The structures and properties of the formed fluorescein derivatives silver nanohybrids were determined using: UV/Vis spectroscopy, TEM images and dynamic light scattering (DLS). The synthesized compounds and their nanohybrids were evaluated for their antimicrobial activities against different bacterial strains and fungi. The results showed that the formed fluorescein derivatives silver nanohybrids are in moderate diameter range, and the loading of the synthesized compounds protect the silver nanoparticles against coagulation. The antimicrobial activity against the studied microorganisms was comparable to the standard used. Moreover, the antimicrobial activity was increased considerably in case of using fluorescein derivatives silver nanohybrids. The antimicrobial activities were correlated to the chemical structures of the compounds, diameter of the formed nanohybrids and to the nature of the tested bacterial strains. The mechanism of the antimicrobial action of the synthesized compounds and their nanohybrids was proposed.

Fluorescein-labeled Bacitracin and Daptomycin Conjugates: Synthesis, Fluorescence Imaging and Evaluation.

BACKGROUND: Previously, glycopeptides antibiotics such as vancomycin, ramoplanin and an antifungal antibiotic nystatin have been studied for their diagnostic and therapeutic potential. OBJECTIVE: To further explore the diagnostic and chemotherapeutic potential of other antibiotics we have now employed daptomycin, a lipopetide antibiotic and bacitracin, a polypeptide antibiotic in uptake and vitality tests on human cell lines. METHOD: Fluorescent conjugates of bacitracin and daptomycin were synthesized using fluorescein isothiocynate (FITC) for confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). The cellular uptake of the synthesized daptomycin and bacitracin conjugates was studied on seven human cell lines, two healthy and five malignant using CLSM and FACS. To examine the cell membrane damage caused by the conjugates FACS experiments were carried out using propidium iodide. RESULTS: The uptake pattern was different for both antibiotics for all the cell lines. The cytoplasmic uptake of daptomycin conjugate was lower than the bacitracin conjugate, resulting in decreased cell membrane damage. CONCLUSION: No preferential uptake into malignant or healthy cells was found for the two different antibiotic conjugates and the uptake patterns were also different between the two antibiotics. However, the lower cytotoxicity and different uptake mechanism makes daptomycin conjugate a prospective candidate for further study as a diagnostic agent for various intracellular infections.

Synthesis and characterization of Pt(IV) fluorescein conjugates to investigate Pt(IV) intracellular transformations.

Pt(IV) anticancer compounds typically operate as prodrugs that are reduced in the hypoxic environment of cancer cells, losing two axial ligands in the process to generate active Pt(II) species. Here we report the synthesis of two fluorescent Pt(IV) prodrugs of cisplatin in order to image and evaluate the Pt(IV) reduction process in simulated and real biological environments. Treatment of the complexes dissolved in PBS buffer with reducing agents typically encountered in cells, glutathione or ascorbate, afforded a 3- to 5-fold fluorescence turn-on owing to reduction and loss of their fluorescein-based axial ligands, which are quenched when bound to platinum. Both Pt(IV) conjugates displayed moderate cytotoxicity against human cancer cell lines, with IC50 values higher than that of cisplatin. Immunoblotting and DNA flow cytometry analyses of one of the complexes, Pt(IV)FL2, revealed that it damages DNA, causes cell cycle arrest in S or G2/M depending on exposure time, and ultimately triggers apoptotic cell death. Fluorescence microscopic studies prove that Pt(IV)FL2 enters cells intact and undergoes reduction intracellularly. The results are best interpreted in terms of a model in which the axial fluorescein ligands are expelled through lysosomes, with the platinum(II) moiety generated in the process binding to genomic DNA, which results in cell death.

Synthesis, biological evaluation, and in vivo imaging of the first camptothecin-fluorescein conjugate.

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. In vivo and ex vivo imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution in vivo.

A fluorescein-based probe with high selectivity to cysteine over homocysteine and glutathione.

A fluorescent probe based on fluorescein displays excellent selectivity and sensitivity for cysteine and its application for bio-imaging is described.

Photochemical control of FlAsH labeling of proteins.

Spatiotemporal control of protein fluorescence is a powerful tool in tracking protein movements within cells. Here we report an approach to using genetically encoded photo-caged amino acids to control labeling protein tetracysteine tags with biarsenical fluorescein dyes (FlAsH).

Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells.

Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and beta-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.

Rhodamine-based fluorogenic probe for imaging biological thiol.

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modification of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells.

A phosphinate-based red fluorescent probe for imaging the superoxide radical anion generated by RAW264.7 macrophages.

4',9'-Bis(diphenylphosphinyl)naphthofluorescein (PNF-1) has been designed and synthesized as a highly selective, sensitive, cell-permeable, red fluorescent probe for detecting O(2) (.-) in biological systems. The design strategy for the probe is based on the nucleophilic mechanism of O(2) (.-) to mediate deprotection of the probe to naphthofluorescein, the emission spectrum of which is just in the spectral region of low background fluorescence interference in biological systems. Upon treatment with O(2) (.-), the probe exhibits a strong fluorescence response and high selectivity for O(2) (.-), rather than other reactive oxygen species or biological compounds. A linear calibration curve for PNF-1 showed a detection limit of 0.1 nM O(2) (.-). This new type of fluorescent probe allows nanomolar changes in O(2) (.-) concentrations in living cells to be detected by confocal microscopy.

A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies.

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.

Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7-positive tumour by approx. 60%. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.