title: 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.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Regensburg, 90430, Regensburg, Germany.
authors.forename: Hesham
authors.initials: H
authors.lastname: Taha
authors.forename: Stefan
authors.initials: S
authors.lastname: Dove
authors.forename: Jens
authors.initials: J
authors.lastname: Geduhn
authors.forename: Burkhard
authors.initials: B
authors.lastname: König
authors.forename: Yuequan
authors.initials: Y
authors.lastname: Shen
authors.forename: Wei-Jen
authors.initials: WJ
authors.lastname: Tang
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001408:Bacillus anthracis; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D009154:Mutation
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D003597:Cytosine Nucleotides; C030325:anthrax toxin; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases