A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Spangler, Corinna M.; Spangler, Christian; Göttle, Martin; Shen, Yuequan; Tang, Wei-Jen; Seifert, Roland; Schäferling, Michael

doi:10.1016/j.ab.2008.06.014

Cited by 27 publications

(17 citation statements)

References 31 publications

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“…28 The displacement of water ligands by chelating polyphosphate anions is reversible, and the response in luminescence intensity (or lifetime) can be used to monitor the conversion of ATP in enzymatic reactions in real‐time 23. 29 However, in these cases, the sensitizing antenna chromophores such as tetracycline or norfloxacin only form weakly bound complexes with the lanthanide ion. Thus, an excess amount of sensitizer usually has to be added, which leads to compounds with undefined structure and a luminescence response that is prone to many interferences, because oxyanions generally have a high affinity toward europium ions.…”

Section: Introductionmentioning

confidence: 99%

Multidentate Europium Chelates as Luminoionophores for Anion Recognition: Impact of Ligand Design on Sensitivity and Selectivity, and Applicability to Enzymatic Assays

Schäferling

Ääritalo

Soukka

2014

Chemistry A European J

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The design of photoluminescent molecular probes for the selective recognition of anions is a major challenge for the development of optical chemical sensors. The reversible binding of anions to lanthanide centers is one promising option for the realization of anion sensors, because it leads in some cases to a strong luminescence increase by the replacement of quenching water molecules. Yet, it is an open problem to gain control of the sensitivity and selectivity of the luminescence response. Primarily, the selective detection of (poly)phosphate species such as nucleotides has emerged as a demanding task, because they are involved in many biological processes and enzymatic reactions. We designed a series of pyridyl-based multidentate europium complexes (seven-, six-, and five-dentate) including sensitizing chromophores and studied their luminescence intensity and lifetime responses to different (poly)phosphates (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), pyrophosphate, and phosphate anions), and carboxyanions (citrate, malate, oxalacetate, succinate, α-ketoglutarate, pyruvate, oxalate, carbonate). The results reveal that the number of free coordination sites has a significant impact on the sensitivity and selectivity of the response. Because of its reversibility, the lanthanide probes can be applied to monitor the activity of ATP-consuming enzymes such ATPases and apyrases, which is demonstrated by means of the five-dentate complex.

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Section: Introductionmentioning

confidence: 99%

Multidentate Europium Chelates as Luminoionophores for Anion Recognition: Impact of Ligand Design on Sensitivity and Selectivity, and Applicability to Enzymatic Assays

Schäferling

Ääritalo

Soukka

2014

Chemistry A European J

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“…The lower basal fluorescence of bis-MANT-analog and the higher CaM-dependent fluorescence signals at the excitation wavelength of 350 nm as compared to mono-MANT nucleotides could be used as non-radioactive highthroughput screening assay for EF inhibitors, including compounds binding to the catalytic site as well as compounds impeding with the interaction of EF and CaM (Lee et al 2004). Such FRET assay constitutes a valuable complementation of the sensitive radiometric EF activity assay used in this study and the recently described fluorometric EF activity assay (Spangler et al 2008). …”

Section: Discussionmentioning

confidence: 98%

Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5′-triphosphates

Taha

Dove

Geduhn

et al. 2011

Naunyn-Schmiedeberg's Arch Pharmacol

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Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl (ANT) groups at the 2'(3')-O-ribosyl position, with the unique preference for the base cytosine (Taha et al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the most potent EF inhibitor (K (i), 100 nM) among 16 compounds studied. Here, we examined the interaction of EF with a series of 18 2',3'-O-mono- and bis-(M)ANT-substituted nucleotides, recently shown to be very potent inhibitors of the AC toxin from Bordetella pertussis, CyaA (Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We analysed purified EF and EF mutants in radiometric AC assays and in fluorescence spectroscopy studies and conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the most potent EF inhibitor (K (i), 80 nM). In contrast to the observations made for CyaA, introduction of a second (M)ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to bis-MANT-ATP, but FRET to bis-MANT-CTP was only small. Mutations N583Q, K353A and K353R differentially altered the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP. The nucleotide binding site of EF accommodates bulky bis-(M)ANT-substituted purine and pyrimidine nucleotides, but the fit is suboptimal compared to CyaA. These data provide a basis for future studies aiming at the development of potent EF inhibitors with high selectivity relative to mammalian ACs.

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“…The facts that the respective nucleotides are now commercially available and that EF can be readily purified in large amounts facilitate the establishment of a high-throughput screening assay. Such FRET assay constitutes a valuable complementation of the sensitive radiometric AC assay using [α- 32 P]ATP as substrate and our recently developed non-radioactive terbium-norfloxacin assay, detecting PP i [22]. …”

Section: Discussionmentioning

confidence: 99%

Distinct interactions of 2′- and 3′-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the adenylyl cyclase toxin of Bacillus anthracis, edema factor

Suryanarayana

Wang

Richter

et al. 2009

Biochemical Pharmacology

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Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. Bacillus anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (K i values in the 0.1-5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′-and 3′-MANTisomers can bind to EF. The aim of the present study was to examine the effects of defined 2′-and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT-fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxyisomers and the 2′(3′)-racemates. K i values of MANT-nucleotides for inhibition of catalysis correlated with K d values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′-MANT-and 3′-MANTisomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions.

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A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Cited by 27 publications

References 31 publications

Multidentate Europium Chelates as Luminoionophores for Anion Recognition: Impact of Ligand Design on Sensitivity and Selectivity, and Applicability to Enzymatic Assays

Multidentate Europium Chelates as Luminoionophores for Anion Recognition: Impact of Ligand Design on Sensitivity and Selectivity, and Applicability to Enzymatic Assays

Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5′-triphosphates

Distinct interactions of 2′- and 3′-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the adenylyl cyclase toxin of Bacillus anthracis, edema factor

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