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 calmodulindependent 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 2Ј(3Ј)-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.

Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

“…MANT-ITP was synthesized according to Hiratsuka (1983) with the modifications described by Taha et al (2009). MANT-GTP and MANT-XTP were obtained from Jena Bioscience (Jena, Germany).…”

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

“…MANT-ATP (1) was less potent than MANT-ITP (2), but at all other combinations of adenosine and inosine derivatives the corresponding ANT-ATPs exhibited higher inhibition potency (4 3 5, 6 3 7, 8 3 9, 10 3 11). MANT-CTP (3) is the most potent MANT nucleotide inhibitor of edema factor AC toxin from Bacillus anthracis with a K i CyaA Inhibition by Bis-Halogen-ANT Nucleotides value of 100 nM (Taha et al, 2009). This preference for cytosine was not observed for CyaA (Göttle et al, 2007).…”

“…ANT-ADP were synthesized according to Hiratsuka (1983) with modifications described in Taha et al (2009). Methylisatoic anhydride, isatoic anhydride, chloroisatoic anhydride, bromoisatoic anhydride, aminoisatoic anhydride, ATP, ITP, CTP, IMP, and bovine serum albumin, fraction V, highest quality, were purchased from Sigma-Aldrich (Seelze, Germany).…”

“…However, the K i value of bis-MANT-IMP is still very low, i.e., in the range of 20 to 40 M, depending on the experimental conditions. Because the length of the phosphate chain is important for high potency of AC inhibitors (Mou et al, 2005(Mou et al, , 2006Göttle et al, 2007;Taha et al, 2009), we focused our synthesis effort on NTPs. The ANT group was also modified by halogens and an acetylated amino group at the 5-position of the phenyl ring system.…”

“…For all experiments double-distilled water was used. Sources of all other biochemical reagents have been described previously (Gille et al, 2004;Göttle et al, 2007;Taha et al, 2009).…”