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Molecular Analysis of the Interaction of Anthrax Adenylyl Cyclase Toxin, Edema Factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-Substituted Purine and Pyrimidine Nucleotides

Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany (H.M.T., J.S., M.G.); Department of Molecular Biosciences, the University of Kansas, Lawrence, Kansas (S.S.); Ben May Department for Cancer Research, the University of Chicago, Chicago, Illinois (W.-J.T.); the College of Life Sciences, Nankai University, People's Republic of China (Y.S.); Institute of Organic Chemistry, University of Regensburg, Germany (J.G., B.K.); Department of Pharmacology, University of Heidelberg, Heidelberg, Germany (A.G.); Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg, Regensburg, Germany (S.D.), Department of Pharmacology, Medical School of Hannover, Hannover, Germany (R.S.)

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[Abstract/Free Full Text] ). 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.

Received for publication September 29, 2008.

Accepted for publication December 4, 2008.

Address correspondence to: Dr. Roland Seifert, Department of Pharmacology, Medical School of Hannover, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany. E-mail: seifert.roland{at}mh-hannover.de

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