title: Impact of Dendrimer Terminal Group Chemistry on Blockage of the Anthrax Toxin Channel: A Single Molecule Study.
abstract: Nearly all the cationic molecules tested so far have been shown to reversibly block K⁺ current through the cation-selective PA63channels of anthrax toxin in a wide nM-mM range of effective concentrations. A significant increase in channel-blocking activity of the cationic compounds was achieved when multiple copies of positively charged ligands were covalently linked to multivalent scaffolds, such as cyclodextrins and dendrimers. Even though multivalent binding can be strong when the individual bonds are relatively weak, for drug discovery purposes we often strive to design multivalent compounds with high individual functional group affinity toward the respective binding site on a multivalent target. Keeping this requirement in mind, here we perform a single-channel/single-molecule study to investigate kinetic parameters of anthrax toxin PA63channel blockage by second-generation (G2) poly(amido amine) (PAMAM) dendrimers functionalized with different surface ligands, including G2-NH₂, G2-OH, G2-succinamate, and G2-COONa. We found that the previously reported difference inIC50values of the G2-OH/PA63and G2-NH₂/PA63binding was determined by both on- and off-rates of the reversible dendrimer/channel binding reaction. In 1 M KCl, we observed a decrease of about three folds in k o n and a decrease of only about ten times in t r e s with G2-OH compared to G2-NH₂. At the same time for both blockers, k o n and t r e s increased dramatically with transmembrane voltage increase. PAMAM dendrimers functionalized with negatively charged succinamate, but not carboxyl surface groups, still had some residual activity in inhibiting the anthrax toxin channels. At 100 mV, the on-rate of the G2-succinamate binding was comparable with that of G2-OH but showed weaker voltage dependence when compared to G2-OH and G2-NH₂. The residence time of G2-succinamate in the channel exhibited opposite voltage dependence compared to G2-OH and G2-NH₂, increasing with thecis-negative voltage increase. We also describe kinetics of the PA63ion current modulation by two different types of the "imperfect" PAMAM dendrimers, the mixed-surface G2 75% OH 25% NH₂ dendrimer and G3-NH₂ dendron. At low voltages, both "imperfect" dendrimers show similar rate constants but significantly weaker voltage sensitivity when compared with the intact G2-NH₂ PAMAM dendrimer.
authors.affiliation: Department of Biology, The Catholic University of America, Washington DC, WA 20064, USA. 00yamini@cua.edu.
authors.forename: Goli
authors.initials: G
authors.lastname: Yamini
authors.affiliation: Department of Biology, The Catholic University of America, Washington DC, WA 20064, USA. 56kalu@cua.edu.
authors.forename: Nnanya
authors.initials: N
authors.lastname: Kalu
authors.affiliation: Department of Biology, The Catholic University of America, Washington DC, WA 20064, USA. nestorovich@cua.edu.
authors.forename: Ekaterina M
authors.initials: EM
authors.lastname: Nestorovich
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D050091:Dendrimers / Q000737:chemistry*; D008024:Ligands
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D050091:Dendrimers; D008024:Ligands; C104700:PAMAM Starburst; C030325:anthrax toxin
keywords: Bacillus anthracis; multivalency; planar lipid bilayer technique; pore blockage; protective antigen