activity-based probes
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Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.
The function of enzymatic proteins is given by their ability to bind specific small molecules into their active sites. These sites can often be found in pockets on a hypothetical boundary between the protein and its environment. Detection, analysis, and visualization of pockets find its use in protein engineering and drug discovery. Many definitions of pockets and algorithms for their computation have been proposed. Kawabata and Go defined them as the regions of empty space into which a small spherical probe can enter but a large probe cannot and developed programs that can compute their approximate shape. In this article, this definition was slightly modified in order to capture the existence of large internal holes, and a Voronoi-based method for the computation of the exact shape of these modified regions is introduced. The method first puts a finite number of large probes on the protein exterior surface and then, considering both large probes and atomic balls as obstacles for the small probe, the method computes the exact shape of the regions for the small probe. This is all achieved with Voronoi diagrams, which help with the safe navigation of spherical probes among spherical obstacles. Detected regions are internally represented as graphs of vertices and edges describing possible movements of the center of the small probe on Voronoi edges. The surface bounding each region is obtained from this representation and used for visualization, volume estimation, and comparison with other approaches. © 2019 Wiley Periodicals, Inc.
Bacterial β sliding clamp (β-clamp) is an emerging drug target currently lacking small-molecule inhibitors with good in vivo activity. Thus, there is a need for fast and simple screening methods for identifying inhibitor candidates. Here we demonstrate the use of nuclear magnetic resonance spectroscopy (NMR) for evaluating compound binding to the E. coli β-clamp. To identify suitable molecular probes, a series of tetrahydrocarbazoles were synthesized, some of which contain fluorine. Key challenges in the synthesis were formation of regioisomers during the Fischer indole reaction and reducing racemization at the stereogenic center. The tetrahydrocarbazoles were assayed against the E. coli β-clamp by saturation-transfer difference (STD) NMR, waterLOGSY and T1ρ. Analysis by isothermal titration calorimetry gave KD-values of 1.7-14 μM for three fluorinated probe candidates, and NMR chemical shift perturbation experiments confirmed these molecules to directly interact with the β-clamp binding pocket. Binding of the fluorinated molecules to β-clamp was easily observed with 19F-observed T2-based binding experiments, and proof of concept for a fluorine-based binding assay for E. coli β-clamp binders is provided.
- MeSH
- Escherichia coli * účinky léků MeSH
- halogenace MeSH
- karbazoly * chemie chemická syntéza farmakologie metabolismus MeSH
- magnetická rezonanční spektroskopie * MeSH
- molekulární sondy chemie chemická syntéza metabolismus MeSH
- molekulární struktura MeSH
- proteiny z Escherichia coli metabolismus antagonisté a inhibitory chemie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M-1s-1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.
- MeSH
- fluorescenční barviva chemie farmakologie MeSH
- fotochemické procesy MeSH
- histondeacetylasy chemie genetika metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- pozitronová emisní tomografie * MeSH
- sirtuiny antagonisté a inhibitory chemie metabolismus MeSH
- thioamidy chemie farmakologie MeSH
- transport elektronů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.
- MeSH
- biosenzitivní techniky * MeSH
- delece genu MeSH
- DNA katalytická chemie MeSH
- DNA chemie MeSH
- G-kvadruplexy * MeSH
- ionty analýza chemie MeSH
- kovy analýza chemie MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie genetika MeSH
- nanočástice chemie MeSH
- nukleové kyseliny analýza chemie MeSH
- organické látky analýza chemie MeSH
- proteiny analýza chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.
- MeSH
- akrylamidy chemická syntéza chemie metabolismus MeSH
- fluorescenční barviva chemická syntéza chemie metabolismus MeSH
- fluorescenční mikroskopie MeSH
- inhibitory cysteinových proteinas chemická syntéza chemie metabolismus MeSH
- katalytická doména MeSH
- kathepsin K antagonisté a inhibitory chemie metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- racionální návrh léčiv MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 μM-1 s-1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 μM-1 s-1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.
- Publikační typ
- časopisecké články MeSH
Nanoencapsulation is a promising approach to enhance the therapeutic potential of a drug. Herein, three selected naphthoquinone (NTQ) derivatives, based on the IC50 value against Trypanosoma evansi, were encapsulated using gum damar as biocompatible and biodegradable natural gum via nanoprecipitation method. Nanoformulation of NTQs (NNTQs) was less than 150 nm in size, was found to be stable and released the drug in a sustained manner. All the three NNTQs exhibited significant antitrypanosomal effect and morphological changes at approximately two to three times lesser drug concentrations. The nanoformulations exhibited enhanced production of reactive oxygen species (ROS) in the axenic culture of T. evansi and less cytotoxic effect on horse peripheral blood mononuclear cells relative to pure NTQs. As evidenced by flow cytometry, the NNTQs showed dose-dependent and time-dependent increased transition of live cells (AV-PI-) to early apoptotic cells (AV+PI-), late apoptotic cells (AV-PI+), and necrotic cells (AV+PI+) using annexin V/propidium iodide probe analysis. The results concluded that NNTQs induced more ROS, apoptosis and necrotic effects that exhibited more inhibitory effect on the growth of T. evansi with respect to respective NTQ by themselves.
- MeSH
- koně MeSH
- leukocyty mononukleární MeSH
- naftochinony * farmakologie MeSH
- nanokapsle * MeSH
- reaktivní formy kyslíku MeSH
- Trypanosoma * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A complex OsO(4), 2,2'-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion.
Pro správné pochopení fyziologických procesů v buňce a případně jejich odchylek jsou molekulárně‑biologické analýzy nezbytným nástrojem využívaným v biomedicínském výzkumu a také v klinické diagnostice. Existuje množství technik, které umožňují určit lokalizaci studovaných proteinů a jejich interakční aktivitu. Tyto přístupy využívají především interakce specificky se vážících molekul s cílovými proteiny (protilátky) nebo synteticky připravené rekombinantní proteiny (GFP fúzní protein; metody fluorescenčního/bioluminiscenčního rezonančního přenosu energie). „Proximity ligation assay“ (PLA) in situ představuje novou techniku zobrazující proteiny na úrovni jednotlivých buněk a tkání s využitím reportérové molekuly DNA a DNA modifikujících procesů. Tato metoda umožňuje přímou vizualizaci proteinů, jejich hladiny, modifikace a interakce v jednotlivých fixovaných buňkách a tkáních. Sondy jsou tvořeny specifickými protilátkami s navázaným oligonukleotidem, který slouží jako reportérová molekula. Pokud dojde k navázání sond v těsné blízkosti, následuje vznik kružnicové DNA, jež slouží jako templát pro amplifikaci otáčivou kružnicí. Amplifikační reakce umožňuje vizualizaci sledované interakce. Ve srovnání s dostupnými molekulárně‑biologickými metodami vycházejícími z genového inženýrství, PLA in situ umožňuje studovat endogenní proteiny v jejich přirozených podmínkách, a může být tudíž použita pro studium klinického materiálu. PLA in situ je využitelná v jakékoliv výzkumné oblasti zaměřené na studium proteinových interakcí, jako je studium buněčných signálních drah, identifikace cílů farmakologicky účinných látek či v onkologické diagnostice.
To understand cellular processes and events responsible for their perturbations, proteomic analyses are needed in biomedical research and clinical diagnostics. Several techniques based on specifically binding reagents (antibodies) or recombinant proteins (GFP fusion protein, methods of fluorescence/bioluminescence resonance energy transfer) are generally used to study protein location and activity resulting from secondary modifications and interactions. The in situ proximity ligation assay represents a novel technique of in situ protein imaging using DNA as a reporter molecule and DNA amplification processes. This method enables direct visualization of single molecules, their levels, modifications and pattern of interactions in individual fixed cells and tissues. Proximity probes consist of specific antibody with attached oligonucleotides that are used as reporter molecules for identification of such events. Proximity probes guide the formation of a circular DNA strand when bound in close proximity. The DNA circle after that serves as a template for rolling‑circle amplification allowing the interaction to be visualized. Compared to available proteomic techniques benefiting from genetic engineering, in situ PLA enables study of endogenous proteins in their natural environment and thus can be used for clinical specimens. The areas of applicability where proximity ligation procedure can be used include any research field where protein interaction measurements are important, such as signaling pathway studies, monitoring of pharmacological treatment targets and oncological diagnostics. Key words: in situ PLA – protein interaction – protein detection methods – proximity ligation This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805) and BBMRI_CZ (LM2010004). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 31. 1. 2014 Accepted: 25. 3. 2014
- Klíčová slova
- in situ PLA, metody detekce proteinů, proteinové interakce,
- MeSH
- chemické techniky analytické * MeSH
- fyziologie buňky MeSH
- mapování interakce mezi proteiny * metody MeSH
- molekulární sondy - techniky * MeSH
- molekulární sondy MeSH
- oligonukleotidové sondy MeSH
- oligonukleotidy metabolismus MeSH
- proteiny MeSH
- protilátky MeSH
- techniky amplifikace nukleových kyselin MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
