ABSTRACT
Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.
Subject(s)
Anthrax/drug therapy , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/antagonists & inhibitors , Drug Design , Small Molecule Libraries/pharmacology , Adenosine Triphosphate/metabolism , Adenylyl Cyclase Inhibitors , Animals , Anthrax/microbiology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Antigens, Bacterial/chemistry , Bacillus anthracis/drug effects , Bacillus anthracis/metabolism , Bacillus anthracis/pathogenicity , Bacterial Toxins/chemistry , Binding Sites , Cyclic AMP/metabolism , Disease Models, Animal , Ligands , Models, Molecular , Molecular Docking Simulation , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic useABSTRACT
Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15µg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.
Subject(s)
Bacillus anthracis/metabolism , Bacterial Toxins/antagonists & inhibitors , Drug Design , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/metabolism , Administration, Oral , Animals , Antigens, Bacterial/metabolism , Bacterial Toxins/metabolism , Benzoates/chemical synthesis , Benzoates/chemistry , Binding Sites , Cell Line , Computer Simulation , Cyclic AMP/metabolism , Fluorenes/chemistry , Mice , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
The synthesis and development of a novel class of molecules that inhibit anthrax edema factor, an adenylyl cyclase, is reported. These molecules are derived from the initial discovery that histidine and imidazole adducts of the prostaglandin PGE(2) reduce the net secretory response of cholera toxin-challenged mice and act directly on the action of anthrax edema factor, a calmodulin-dependent adenylyl cyclase. The simple enones examined in this letter were prepared by palladium-catalyzed Suzuki reaction.
