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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. B. anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study [Taha HM, Schmidt J, Göttle M, Suryanarayana S, Shen Y, Tang WJ, et al. Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides. Mol Pharmacol 2009;75:693-703] 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 microM 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'-MANT-isomers 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'-deoxy-isomers 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'- and 3'-MANT-isomers 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.
AuthorsSrividya Suryanarayana, Jenna L Wang, Mark Richter, Yuequan Shen, Wei-Jen Tang, Gerald H Lushington, Roland Seifert
JournalBiochemical pharmacology (Biochem Pharmacol) Vol. 78 Issue 3 Pg. 224-30 (Aug 01 2009) ISSN: 1873-2968 [Electronic] England
PMID19492438 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References
  • Antigens, Bacterial
  • Bacterial Toxins
  • anthrax toxin
  • ortho-Aminobenzoates
  • 3'-O-(N-methylanthraniloyl) ATP
  • 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Adenylyl Cyclases
Topics
  • Adenosine Triphosphate (analogs & derivatives, chemistry)
  • Adenylyl Cyclases (chemistry)
  • Antigens, Bacterial (chemistry)
  • Bacillus anthracis (enzymology)
  • Bacterial Toxins (chemistry)
  • Fluorescence Resonance Energy Transfer
  • Guanosine Triphosphate (chemistry)
  • Isomerism
  • Models, Molecular
  • ortho-Aminobenzoates (chemistry)

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