- Shining new light on ancient drugs: preparation and subcellular localisation of novel fluorescent analogues of Cinchona alkaloids in intraerythrocytic Plasmodium falciparum
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Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.
- Woodland, John G.,Hunter, Roger,Smith, Peter J.,Egan, Timothy J.
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- Fluorescent Penetration Enhancers Reveal Complex Interactions among the Enhancer, Drug, Solvent, and Skin
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Skin penetration/permeation enhancers facilitate drug delivery through the skin barrier. However, the specific mechanisms that govern the enhancer interactions with the skin, drug, and donor solvent are not fully understood. We designed and synthesized fluorescent-labeled enhancers by attaching 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD) groups to 6-aminohexanoic acid esters. These NBD esters (applied at a 1% concentration) enhanced the permeation of the model drugs theophylline and hydrocortisone through human skin in vitro up to 6.6- and 3.9-times, respectively. The enhancement effects were strongly affected by the ester chain length (C8-C12) and the polarity of the donor solvent. Using high-performance liquid chromatography with fluorescence detection, no NBD esters were detected in the acceptor buffer, but their hydrolysis product, NBD acid, was detected, whereas both acid and esters were found in the skin. The enhancer hydrolysis occurred in the lower stratum corneum and epidermis; more hydrophilic NBD acid, which is an inactive enhancer, penetrated deeper. This illustrates the principle of biodegradable enhancers. The enhancer concentrations in the skin depended not only on the enhancer chain length and the donor solvent, but also on the drug used. Thus, the drug, when coapplied with the enhancer, modulates the enhancer penetration into the skin and, consequently, its effect. Finally, active (NBD-C8 ester) and inactive (NBD acid) enhancers were visualized in human skin by confocal laser scanning microscopy. Both compounds were found mostly in the stratum corneum intercellular spaces, suggesting that although both are located within the skin barrier lipids, only the active ester is able to effectively interact with the lipids, which was proved by infrared spectroscopy of enhancer-treated stratum corneum. This proof-of-concept study illustrates the use of fluorescent enhancers to obtain insight into the skin penetration/permeation process; interactions among the enhancer, drug, solvent, and skin; and enhancer metabolism.
- Kope?ná, Monika,Ková?ik, Andrej,Ku?era, Ond?ej,Machá?ek, Miloslav,Sochorová, Michaela,Audrlická, Pavla,Vávrová, Kate?ina
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- A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs
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Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.
- Jida, Mouhamad,Sanchez, Cecilia P.,Urgin, Karène,Ehrhardt, Katharina,Mounien, Saravanan,Geyer, Aurelia,Elhabiri, Mourad,Lanzer, Michael,Davioud-Charvet, Elisabeth
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- Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases**
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Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.
- Arenz, Christoph,Basu, Shibom,Bechara, Cherine,Bossis, Guillaume,Cong, Xiaojing,Del Nero, Elise,Drapeau, Marion,Fontanel, Simon,Gabellier, Ludovic,Golebiowski, Jér?me,Granier, Sebastien,Healey, Robert D.,Hornemann, Thorsten,Jeannot, Sylvain,Karsai, Gergely,Leyrat, Cedric,Maurel, Damien,Saied, Essa M.,Saint-Paul, Julie
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- Identification and Characterization of a Single High-Affinity Fatty Acid Binding Site in Human Serum Albumin
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A single high-affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12 fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site-specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA-binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow-sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X-ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug-protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.
- Wenskowsky, Lea,Schreuder, Herman,Derdau, Volker,Matter, Hans,Volkmar, Julia,Nazaré, Marc,Opatz, Till,Petry, Stefan
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supporting information
p. 1044 - 1048
(2018/01/01)
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- A specific marker of the capsaicin fluorescent probe and its synthetic method and application
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The invention relates to a specifically marked capsaicin fluorescent probe and a synthetic method and application thereof, which belong to the technical field of biology. Firstly, the invention discloses the specifically marked capsaicin fluorescent probe
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Paragraph 0053; 0054
(2018/01/11)
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- Glycerophosphoinositols: Total synthesis of the first fluorescent probe derivative
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The first fluorescent glycerophosphoinositol probe was synthesized in moderate good yield (37%). The total synthesis applied a convergent synthetic strategy involving two successive coupling reactions between the three key moieties: myo-inositol, glycerol
- Greco, Graziella,D'Antona, Nicola,Gambera, Giovanni,Nicolosi, Giovanni
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supporting information
p. 2111 - 2114
(2014/11/08)
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- From in vitro to in cellulo: Structure-activity relationship of (2-nitrophenyl)methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa
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Recent studies have shown that compounds based on a (2-nitrophenyl)methanol scaffold are promising inhibitors of PqsD, a key enzyme of signal molecule biosynthesis in the cell-to-cell communication of Pseudomonas aeruginosa. The most promising molecule displayed anti-biofilm activity and a tight-binding mode of action. Herein, we report on the convenient synthesis and biochemical evaluation of a comprehensive series of (2-nitrophenyl)methanol derivatives. The in vitro potency of these inhibitors against recombinant PqsD as well as the effect of selected compounds on the production of the signal molecules HHQ and PQS in P. aeruginosa were examined. The gathered data allowed the establishment of a structure-activity relationship, which was used to design fluorescent inhibitors, and finally, led to the discovery of (2-nitrophenyl)methanol derivatives with improved in cellulo efficacy providing new perspectives towards the application of PqsD inhibitors as anti-infectives. This journal is the Partner Organisations 2014.
- Storz, Michael P.,Allegretta, Giuseppe,Kirsch, Benjamin,Empting, Martin,Hartmann, Rolf W.
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supporting information
p. 6094 - 6104
(2014/08/05)
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- Versatile probes for the selective detection of vicinal-dithiol-containing proteins: Design, synthesis, and application in living cells
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Endogenous vicinal-dithiol-containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p-aminophenylarsenoxide (PAO). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease-related VDPs and for the discovery of new drug targets. Copyright
- Huang, Chusen,Yin, Qin,Meng, Jiangjiang,Zhu, Weiping,Yang, Yi,Qian, Xuhong,Xu, Yufang
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supporting information
p. 7739 - 7747
(2013/07/19)
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- One-pot fluorescent labeling protocol for complex hydroxylated bioactive natural products
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Tagging of small bioactive molecules with a fluorophore is a highly sensitive method to trace their cellular activities through real-time visual information. Here we disclose a 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-based, high-yielding, one-pot labeling protocol for hydroxylated molecules using Yamaguchi coupling as the key reaction. This methodology was successfully applied on several sensitive and complex hydroxylated bioactive compounds including 7-deacetylazadiradione, simvastatin, camptothecin, andrographolide, cinchonine, β-dihydroartemisinin, and azadirachtin A. Further, utility of this protocol was illustrated on the cytotoxic activity of azadiradione derivatives against several cancer cell lines through cell imaging of two qualified fluorescent probes.
- Haldar, Saikat,Kumar, Santosh,Kolet, Swati P.,Patil, Harshal S.,Kumar, Dhiraj,Kundu, Gopal C.,Thulasiram, Hirekodathakallu V.
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p. 10192 - 10202
(2013/11/06)
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- Synthesis of fluorescent C24-ceramide: Evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin
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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.
- Novotny, Jakub,Pospěchová, Kate?ina,Hrabálek, Alexandr,?áp, Robert,Vávrová, Kate?ina
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supporting information; experimental part
p. 6975 - 6977
(2010/06/16)
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- Identification of a tunable site in bryostatin analogs: C20 bryologs through late stage diversification
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The C20 region of our bryostatin analogs was identified as a nonpharmacophoric site that could be varied to tune analogs for function and physical properties without significantly affecting their binding affinity for PKC. The use of this site in a late-stage diversification strategy has enabled the facile synthesis of a variety of new C20 analogs, all of which retain nanomolar affinity for PKC, in agreement with our pharmacophore hypothesis.
- Wender, Paul A.,Baryza, Jeremy L.
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p. 1177 - 1180
(2007/10/03)
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- The synthesis of acid- and base-labile lipopeptides on solid support
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Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68% yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.
- Ludolph, Bjoern,Waldmann, Herbert
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p. 3683 - 3691
(2007/10/03)
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