Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides.

Abstract

Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides ( Mol Pharmacol 70: 878-886, 2006 ). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2'(3')-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (K(i), 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. 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 MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 is crucial for FRET to MANT-ATP and MANT-CTP and that the mutations N583Q, K353A, and K353R differentially alter the inhibitory potencies of MANT-ATP and MANT-CTP. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors.

• Authors: Hesham M Taha, Jennifer Schmidt, Martin Göttle, Srividya Suryanarayana, Yuequan Shen, Wei-Jen Tang, Andreas Gille, Jens Geduhn, Burkhard König, Stefan Dove, Roland Seifert
• Journal: Molecular pharmacology (Mol Pharmacol) Vol. 75 Issue 3 Pg. 693-703 (Mar 2009) ISSN: 1521-0111 [Electronic] United States
• PMID: 19056899 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 2'(3')-O-methylanthraniloyl-gamma-amido-adenosine triphosphate
• Anthrax Vaccines
• Antigens, Bacterial
• Bacterial Toxins
• Purine Nucleotides
• Pyrimidine Nucleotides
• anthrax toxin
• ortho-Aminobenzoates
• Adenylyl Imidodiphosphate
• Adenosine Diphosphate
• 3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate
• Adenylyl Cyclases

Topics

• Adenosine Diphosphate (analogs & derivatives, chemistry, metabolism)
• Adenylyl Cyclases (chemistry, metabolism)
• Adenylyl Imidodiphosphate (analogs & derivatives, chemistry, metabolism)
• Animals
• Anthrax Vaccines (chemistry, genetics, metabolism)
• Antigens, Bacterial (chemistry, genetics, metabolism)
• Bacterial Toxins (chemistry, genetics, metabolism)
• Catalysis
• Cattle
• Crystallography, X-Ray
• Fluorescence Resonance Energy Transfer
• Mutagenesis, Site-Directed
• Protein Binding (genetics)
• Purine Nucleotides (chemistry, metabolism)
• Pyrimidine Nucleotides (chemistry, metabolism)
• ortho-Aminobenzoates (chemistry, metabolism)