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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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Influence of the PAMAMdendrimer surface groups on the PA63 ion current inhibition.(A) The PAMAM G2-OH dendrimer-induced PA63 inhibition atmultichannel level. The current recordingswere additionally filtered over 100 ms time interval. 0.1 M KCl solutionsat pH 6 were buffered by 5 mM MES. Recordings were taken at 20 mVapplied voltage. The dashed line represents zero current level. (B)Typical multichannel titration curves of the PAMAM–OH dendrimer-inducedPA63 pore inhibition measured in 0.01 M (open squares),0.1 M (filled circles), and 1 M KCl (open triangles) at 20 mV appliedvoltage. (C) Typical multichannel titration curves of the PA63 current inhibition by G2-NH2 (open squares), G2-OH (filledcircles), and G2-SA (filled triangles) PAMAM dendrimers. Recordingwere taken at 20 mV in 0.1 M KCl solutions at pH 6.
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fig2: Influence of the PAMAMdendrimer surface groups on the PA63 ion current inhibition.(A) The PAMAM G2-OH dendrimer-induced PA63 inhibition atmultichannel level. The current recordingswere additionally filtered over 100 ms time interval. 0.1 M KCl solutionsat pH 6 were buffered by 5 mM MES. Recordings were taken at 20 mVapplied voltage. The dashed line represents zero current level. (B)Typical multichannel titration curves of the PAMAM–OH dendrimer-inducedPA63 pore inhibition measured in 0.01 M (open squares),0.1 M (filled circles), and 1 M KCl (open triangles) at 20 mV appliedvoltage. (C) Typical multichannel titration curves of the PA63 current inhibition by G2-NH2 (open squares), G2-OH (filledcircles), and G2-SA (filled triangles) PAMAM dendrimers. Recordingwere taken at 20 mV in 0.1 M KCl solutions at pH 6.

Mentions: Interestingly,PAMAM dendrimers G2 and G3 functionalized with surface hydroxyl groups(PAMAM–OH) also inhibited PA63 channels in a concentration-dependentmanner. However, the channel blocking activity of these compoundswas significantly decreased compared with the PAMAM dendrimers carryingthe positively charged surface amino groups (Table 1). Thus, we detected almost 20 times reduction in IC50 values between the PAMAM dendrimers G2 functionalized with the surfaceamino sites and those with the surface hydroxyl sites (Figure 2A). Note that we compared PAMAM G2-NH2 and G2-OH dendrimers because G0-OH and G1-OH are not available commercially.We also tested G3-OH and observed 9 times decrease in avidity (higherIC50) compared with G3-NH2. This “residual”activity of the PAMAM–OH dendrimers could be determined bytheir net positive charge. Even though the surface charge of PAMAM–OHis equal to zero, the poly(amido amine) interior structure of PAMAMdendrimers holds a significant number of the tertiary amino groupsthat could be positively charged at subacidic pH.


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)

Influence of the PAMAMdendrimer surface groups on the PA63 ion current inhibition.(A) The PAMAM G2-OH dendrimer-induced PA63 inhibition atmultichannel level. The current recordingswere additionally filtered over 100 ms time interval. 0.1 M KCl solutionsat pH 6 were buffered by 5 mM MES. Recordings were taken at 20 mVapplied voltage. The dashed line represents zero current level. (B)Typical multichannel titration curves of the PAMAM–OH dendrimer-inducedPA63 pore inhibition measured in 0.01 M (open squares),0.1 M (filled circles), and 1 M KCl (open triangles) at 20 mV appliedvoltage. (C) Typical multichannel titration curves of the PA63 current inhibition by G2-NH2 (open squares), G2-OH (filledcircles), and G2-SA (filled triangles) PAMAM dendrimers. Recordingwere taken at 20 mV in 0.1 M KCl solutions at pH 6.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Influence of the PAMAMdendrimer surface groups on the PA63 ion current inhibition.(A) The PAMAM G2-OH dendrimer-induced PA63 inhibition atmultichannel level. The current recordingswere additionally filtered over 100 ms time interval. 0.1 M KCl solutionsat pH 6 were buffered by 5 mM MES. Recordings were taken at 20 mVapplied voltage. The dashed line represents zero current level. (B)Typical multichannel titration curves of the PAMAM–OH dendrimer-inducedPA63 pore inhibition measured in 0.01 M (open squares),0.1 M (filled circles), and 1 M KCl (open triangles) at 20 mV appliedvoltage. (C) Typical multichannel titration curves of the PA63 current inhibition by G2-NH2 (open squares), G2-OH (filledcircles), and G2-SA (filled triangles) PAMAM dendrimers. Recordingwere taken at 20 mV in 0.1 M KCl solutions at pH 6.
Mentions: Interestingly,PAMAM dendrimers G2 and G3 functionalized with surface hydroxyl groups(PAMAM–OH) also inhibited PA63 channels in a concentration-dependentmanner. However, the channel blocking activity of these compoundswas significantly decreased compared with the PAMAM dendrimers carryingthe positively charged surface amino groups (Table 1). Thus, we detected almost 20 times reduction in IC50 values between the PAMAM dendrimers G2 functionalized with the surfaceamino sites and those with the surface hydroxyl sites (Figure 2A). Note that we compared PAMAM G2-NH2 and G2-OH dendrimers because G0-OH and G1-OH are not available commercially.We also tested G3-OH and observed 9 times decrease in avidity (higherIC50) compared with G3-NH2. This “residual”activity of the PAMAM–OH dendrimers could be determined bytheir net positive charge. Even though the surface charge of PAMAM–OHis equal to zero, the poly(amido amine) interior structure of PAMAMdendrimers holds a significant number of the tertiary amino groupsthat could be positively charged at subacidic pH.

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