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

Abstract

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.

Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors ofBordetella pertussisAdenylyl Cyclase Toxin

“…Nonetheless, the inhibitory profiles of edema factor and CyaA are different. Most notably, MANT-ATP, defined 2Ј-MANT-and 3Ј-MANT-ATP isomers, and MANT-CTP inhibit catalytic activity of CyaA with approximately 10-fold lower potency than catalysis by edema factor, respectively (Göttle et al, 2007;Suryanarayana et al, 2009;Taha et al, 2009). Based on these differences it is also reasonable to assume that the enzymological analysis of edema factor with our new compound library will give different results than the analysis of CyaA.…”

An overview of investigational toxin-directed therapies for the adjunctive management ofBacillus anthracisinfection and sepsis

“…Adefovir inhibited EF-stimulated cAMP production and its cardiovascular effects in perfused heart and aortic ring models [22,25]. Other agents shown to have the potential to inhibit EF's enzymatic activity include 4-[4-(4-nitrophenyl)-thiazolylamino]-benzene-sulfonamide [140,141], 3-{(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid [142,143], and (M)Ant nucleotides [144-149] and allosteric inhibitors [150]. …”

Designing inhibitors of anthrax toxin

“…The most potent EF inhibitor, 3′-(N-methyl)anthraniloyl-2′-deoxyadenosine-5′-triphosphate (K i = 10 nM) (157, 158), was one of the investigated (M)ANT-nucleotides, which carries a fluorescent (N-methyl)anthraniloyl-group to bind different nucleotide-binding proteins (147) (Figure 5B, left) . However, an additional problem that the developers of the ET inhibitors has faced was related to the high hydrophilicity and, therefore, low membrane permeability of the inhibitors determined by the nucleotide-based structure of the compounds.…”

“…(B) Small-molecule EF inhibitors. Left : Structure of compound 22, one of the (M)ANT nucleotides inhibiting with IC 50 = 10 nM (157). Right : Structure for an optimized EF inhibitor, compound 22 (159) inhibiting EF-induced secretion of cAMP by cultured cells with IC 50 = 2.71 μM.…”