title: Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design.
abstract: Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, beta-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-beta-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin (= PA + LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.
authors.affiliation: Innovative Biologics, Inc., 10900 University Boulevard, Manassas, VA 20110, USA.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Karginov
authors.forename: Ekaterina M
authors.initials: EM
authors.lastname: Nestorovich
authors.forename: Mahtab
authors.initials: M
authors.lastname: Moayeri
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Bezrukov
mesh_terms: D000818:Animals; D000881:Anthrax / Q000188:drug therapy; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004594:Electrophysiology; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D008297:Male; D051379:Mice; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D051381:Rats; D011916:Rats, Inbred F344; D047392:beta-Cyclodextrins / Q000737:chemistry* / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D047392:beta-Cyclodextrins; C505831:per-6-(3-aminopropylthio)-beta-cyclodextrin