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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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Single channelanalysis of the PA63/G1-NH2 binding reaction.(A) Conductance of single PA63 (top)and C2IIa (bottom) channels in the absence (left) and presence (right)of PAMAM dendrimers G1 in the cis-side of the bilayerchamber shows the bimodal character of the dendrimer-induced action.Recordings are shown at 50 ms time resolution. 1 M KCl solutions atpH 6 were buffered by 5 mM MES. Recordings were taken at 50 mV appliedvoltage. Higher concentrations of G1 compared to the ones reportedin Table 1 were needed because of the increasedsupporting electrolyte concentrations (1 M vs 0.1 M) used for thesingle-channel measurements. “*” and “+”indicate two different modes of the dendrimer binding. (B) Power spectraldensities of PAMAM dendrimer G1 induced PA63 current fluctuations.PA63 single channel current fluctuations in presence ofG1 (black spectrum) can be fitted by the single Lorentzian in contrastto 1/f noise in the blocker-free (con) solutions(shaded spectrum). (C, D) Kinetic parameters of PAMAM dendrimers G1binding as functions of transmembrane voltage compared with the dataearlier reported for AmPrβCD.14 (C)The on-rate constant, kon, of G1 bindingto PA63 shows strong voltage dependence (filled circles)in contrast to the AmPrβCD blocker (stars). (D) The G1 bindingtime (filled circles) shows strong nonexponential voltage dependencein contrast to the AmPrβCD binding time (stars), which is nearlyexponential (linear when plotted in a semi logarithmic scale). TheAmPrβCD data are reprinted with permission from ref (14). Copyright 2010 Elsevier.
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fig3: Single channelanalysis of the PA63/G1-NH2 binding reaction.(A) Conductance of single PA63 (top)and C2IIa (bottom) channels in the absence (left) and presence (right)of PAMAM dendrimers G1 in the cis-side of the bilayerchamber shows the bimodal character of the dendrimer-induced action.Recordings are shown at 50 ms time resolution. 1 M KCl solutions atpH 6 were buffered by 5 mM MES. Recordings were taken at 50 mV appliedvoltage. Higher concentrations of G1 compared to the ones reportedin Table 1 were needed because of the increasedsupporting electrolyte concentrations (1 M vs 0.1 M) used for thesingle-channel measurements. “*” and “+”indicate two different modes of the dendrimer binding. (B) Power spectraldensities of PAMAM dendrimer G1 induced PA63 current fluctuations.PA63 single channel current fluctuations in presence ofG1 (black spectrum) can be fitted by the single Lorentzian in contrastto 1/f noise in the blocker-free (con) solutions(shaded spectrum). (C, D) Kinetic parameters of PAMAM dendrimers G1binding as functions of transmembrane voltage compared with the dataearlier reported for AmPrβCD.14 (C)The on-rate constant, kon, of G1 bindingto PA63 shows strong voltage dependence (filled circles)in contrast to the AmPrβCD blocker (stars). (D) The G1 bindingtime (filled circles) shows strong nonexponential voltage dependencein contrast to the AmPrβCD binding time (stars), which is nearlyexponential (linear when plotted in a semi logarithmic scale). TheAmPrβCD data are reprinted with permission from ref (14). Copyright 2010 Elsevier.

Mentions: Quantitative analysis of the single-channel blockageproved difficult at the physiological salt concentrations becausethe residence time of the compound in the channel was very long. Toobtain reliable statistics on the kinetic parameters of the bindingreaction, we switched to 1 M KCl. This switch allowed us to more fullycharacterize and quantify this process on a single-channel level (Figure 3). Typical recordings of ion current through thesingle PA63 and C2IIa pores modified by PAMAM-NH2 dendrimer G1 are shown in Figure 3A. Usingsingle-channel analyses, we found that the inhibitive action of dendrimersis bimodal. First, cationic PAMAM dendrimers when added to the cis-side of the membrane (side of toxin addition) generateintense fluctuations in the current through a single channel (markedby “*”) similar to those observed previously with thesmall-molecule88 and cyclodextrin-basedblockers.6,15 These fluctuations are the fast transientsbetween a fully open and blocked channel resulting from reversiblebinding of the cationic dendrimers to the residues inside the pores.Second, significantly longer voltage-dependent channel blockage eventswere also observed (Figure 3A, marked by “+”).The second mode of channel inhibition possessed the characteristicproperties of a typical voltage-induced closed state of β-barrelchannels. Earlier, similar two modes of the PA63 and C2IIapore blockages were reported for the β-cyclodextrin-based cationicblockers.


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)

Single channelanalysis of the PA63/G1-NH2 binding reaction.(A) Conductance of single PA63 (top)and C2IIa (bottom) channels in the absence (left) and presence (right)of PAMAM dendrimers G1 in the cis-side of the bilayerchamber shows the bimodal character of the dendrimer-induced action.Recordings are shown at 50 ms time resolution. 1 M KCl solutions atpH 6 were buffered by 5 mM MES. Recordings were taken at 50 mV appliedvoltage. Higher concentrations of G1 compared to the ones reportedin Table 1 were needed because of the increasedsupporting electrolyte concentrations (1 M vs 0.1 M) used for thesingle-channel measurements. “*” and “+”indicate two different modes of the dendrimer binding. (B) Power spectraldensities of PAMAM dendrimer G1 induced PA63 current fluctuations.PA63 single channel current fluctuations in presence ofG1 (black spectrum) can be fitted by the single Lorentzian in contrastto 1/f noise in the blocker-free (con) solutions(shaded spectrum). (C, D) Kinetic parameters of PAMAM dendrimers G1binding as functions of transmembrane voltage compared with the dataearlier reported for AmPrβCD.14 (C)The on-rate constant, kon, of G1 bindingto PA63 shows strong voltage dependence (filled circles)in contrast to the AmPrβCD blocker (stars). (D) The G1 bindingtime (filled circles) shows strong nonexponential voltage dependencein contrast to the AmPrβCD binding time (stars), which is nearlyexponential (linear when plotted in a semi logarithmic scale). TheAmPrβCD data are reprinted with permission from ref (14). Copyright 2010 Elsevier.
© Copyright Policy
Related In: Results  -  Collection

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fig3: Single channelanalysis of the PA63/G1-NH2 binding reaction.(A) Conductance of single PA63 (top)and C2IIa (bottom) channels in the absence (left) and presence (right)of PAMAM dendrimers G1 in the cis-side of the bilayerchamber shows the bimodal character of the dendrimer-induced action.Recordings are shown at 50 ms time resolution. 1 M KCl solutions atpH 6 were buffered by 5 mM MES. Recordings were taken at 50 mV appliedvoltage. Higher concentrations of G1 compared to the ones reportedin Table 1 were needed because of the increasedsupporting electrolyte concentrations (1 M vs 0.1 M) used for thesingle-channel measurements. “*” and “+”indicate two different modes of the dendrimer binding. (B) Power spectraldensities of PAMAM dendrimer G1 induced PA63 current fluctuations.PA63 single channel current fluctuations in presence ofG1 (black spectrum) can be fitted by the single Lorentzian in contrastto 1/f noise in the blocker-free (con) solutions(shaded spectrum). (C, D) Kinetic parameters of PAMAM dendrimers G1binding as functions of transmembrane voltage compared with the dataearlier reported for AmPrβCD.14 (C)The on-rate constant, kon, of G1 bindingto PA63 shows strong voltage dependence (filled circles)in contrast to the AmPrβCD blocker (stars). (D) The G1 bindingtime (filled circles) shows strong nonexponential voltage dependencein contrast to the AmPrβCD binding time (stars), which is nearlyexponential (linear when plotted in a semi logarithmic scale). TheAmPrβCD data are reprinted with permission from ref (14). Copyright 2010 Elsevier.
Mentions: Quantitative analysis of the single-channel blockageproved difficult at the physiological salt concentrations becausethe residence time of the compound in the channel was very long. Toobtain reliable statistics on the kinetic parameters of the bindingreaction, we switched to 1 M KCl. This switch allowed us to more fullycharacterize and quantify this process on a single-channel level (Figure 3). Typical recordings of ion current through thesingle PA63 and C2IIa pores modified by PAMAM-NH2 dendrimer G1 are shown in Figure 3A. Usingsingle-channel analyses, we found that the inhibitive action of dendrimersis bimodal. First, cationic PAMAM dendrimers when added to the cis-side of the membrane (side of toxin addition) generateintense fluctuations in the current through a single channel (markedby “*”) similar to those observed previously with thesmall-molecule88 and cyclodextrin-basedblockers.6,15 These fluctuations are the fast transientsbetween a fully open and blocked channel resulting from reversiblebinding of the cationic dendrimers to the residues inside the pores.Second, significantly longer voltage-dependent channel blockage eventswere also observed (Figure 3A, marked by “+”).The second mode of channel inhibition possessed the characteristicproperties of a typical voltage-induced closed state of β-barrelchannels. Earlier, similar two modes of the PA63 and C2IIapore blockages were reported for the β-cyclodextrin-based cationicblockers.

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