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Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Förstner P, Bayer F, Kalu N, Felsen S, Förtsch C, Aloufi A, Ng DY, Weil T, Nestorovich EM, Barth H - Biomacromolecules (2014)

Bottom Line: Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development.These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells.We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center , D-89081 Ulm, Germany.

ABSTRACT
Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

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PAMAM Dendrimer-Induced PA63 and C2IIa PoreInhibitionat Multichannel Level. (A) Multichannel PA63 conductancechanged by G0 (top) and G1 (bottom) dendrimer addition to the cis-compartment of bilayer chamber. (B) Multichannel C2IIaconductance changed by G3 (top) and G8 (bottom) dendrimer additionto the cis-compartment of bilayer chamber. The currentrecordings were additionally filtered over a 100 ms time interval.A 0.1 M KCl solutions at pH 6 were buffered by 5 mM MES. Recordingswere taken at 20 mV applied voltage. The dashed lines represent zerocurrent levels. The lowest and greatest dendrimer concentrations thatare not marked in the figures are given in FigureS4 (titration curves). (C) IC50 values of the PA63/G1-NH2 binding reaction increase with KCl bulkconcentration (filled circles). The salt dependence effect is strongerthan the one reported earlier for the AmPrβCD blocker (stars).14 The AmPrβCD data are reprinted with permissionfrom ref (14). Copyright2010 Elsevier.
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fig1: PAMAM Dendrimer-Induced PA63 and C2IIa PoreInhibitionat Multichannel Level. (A) Multichannel PA63 conductancechanged by G0 (top) and G1 (bottom) dendrimer addition to the cis-compartment of bilayer chamber. (B) Multichannel C2IIaconductance changed by G3 (top) and G8 (bottom) dendrimer additionto the cis-compartment of bilayer chamber. The currentrecordings were additionally filtered over a 100 ms time interval.A 0.1 M KCl solutions at pH 6 were buffered by 5 mM MES. Recordingswere taken at 20 mV applied voltage. The dashed lines represent zerocurrent levels. The lowest and greatest dendrimer concentrations thatare not marked in the figures are given in FigureS4 (titration curves). (C) IC50 values of the PA63/G1-NH2 binding reaction increase with KCl bulkconcentration (filled circles). The salt dependence effect is strongerthan the one reported earlier for the AmPrβCD blocker (stars).14 The AmPrβCD data are reprinted with permissionfrom ref (14). Copyright2010 Elsevier.

Mentions: We first compared PA63 and C2IIa channel blockage by the cationic PAMAM dendrimers overa range of generations using the planar lipid bilayer technique (Figure 1). All tested dendrimers inhibited PA63 and C2IIa channel conductance in a concentration-dependent mannerwhen added to the cis-compartment of a bilayer chamber.Figure 1A,B show four representative recordingsof the raw data titration curves in multichannel PA63 (Figure 1A) and C2IIa (Figure 1B)membranes modified by an increasing concentration of G0 (Figure 1A, top), G1 (Figure 1A, bottom),G3 (Figure 1B, top), and G8 (Figure 1B, bottom) PAMAM dendrimers.


Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Förstner P, Bayer F, Kalu N, Felsen S, Förtsch C, Aloufi A, Ng DY, Weil T, Nestorovich EM, Barth H - Biomacromolecules (2014)

PAMAM Dendrimer-Induced PA63 and C2IIa PoreInhibitionat Multichannel Level. (A) Multichannel PA63 conductancechanged by G0 (top) and G1 (bottom) dendrimer addition to the cis-compartment of bilayer chamber. (B) Multichannel C2IIaconductance changed by G3 (top) and G8 (bottom) dendrimer additionto the cis-compartment of bilayer chamber. The currentrecordings were additionally filtered over a 100 ms time interval.A 0.1 M KCl solutions at pH 6 were buffered by 5 mM MES. Recordingswere taken at 20 mV applied voltage. The dashed lines represent zerocurrent levels. The lowest and greatest dendrimer concentrations thatare not marked in the figures are given in FigureS4 (titration curves). (C) IC50 values of the PA63/G1-NH2 binding reaction increase with KCl bulkconcentration (filled circles). The salt dependence effect is strongerthan the one reported earlier for the AmPrβCD blocker (stars).14 The AmPrβCD data are reprinted with permissionfrom ref (14). Copyright2010 Elsevier.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215879&req=5

fig1: PAMAM Dendrimer-Induced PA63 and C2IIa PoreInhibitionat Multichannel Level. (A) Multichannel PA63 conductancechanged by G0 (top) and G1 (bottom) dendrimer addition to the cis-compartment of bilayer chamber. (B) Multichannel C2IIaconductance changed by G3 (top) and G8 (bottom) dendrimer additionto the cis-compartment of bilayer chamber. The currentrecordings were additionally filtered over a 100 ms time interval.A 0.1 M KCl solutions at pH 6 were buffered by 5 mM MES. Recordingswere taken at 20 mV applied voltage. The dashed lines represent zerocurrent levels. The lowest and greatest dendrimer concentrations thatare not marked in the figures are given in FigureS4 (titration curves). (C) IC50 values of the PA63/G1-NH2 binding reaction increase with KCl bulkconcentration (filled circles). The salt dependence effect is strongerthan the one reported earlier for the AmPrβCD blocker (stars).14 The AmPrβCD data are reprinted with permissionfrom ref (14). Copyright2010 Elsevier.
Mentions: We first compared PA63 and C2IIa channel blockage by the cationic PAMAM dendrimers overa range of generations using the planar lipid bilayer technique (Figure 1). All tested dendrimers inhibited PA63 and C2IIa channel conductance in a concentration-dependent mannerwhen added to the cis-compartment of a bilayer chamber.Figure 1A,B show four representative recordingsof the raw data titration curves in multichannel PA63 (Figure 1A) and C2IIa (Figure 1B)membranes modified by an increasing concentration of G0 (Figure 1A, top), G1 (Figure 1A, bottom),G3 (Figure 1B, top), and G8 (Figure 1B, bottom) PAMAM dendrimers.

Bottom Line: Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development.These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells.We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center , D-89081 Ulm, Germany.

ABSTRACT
Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

Show MeSH
Related in: MedlinePlus