Natl

Natl. azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a -cyclodextrin core via a polyethylene glycol linker of an appropriate length. The producing heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors display considerable promise as anthrax anti-toxins. by incubating Natural264.7 cells with a mixture of PA and LF in the presence of several concentrations of the inhibitor. The heptavalent molecule could inhibit cytotoxicity having a half-maximal inhibitory concentration (IC50) of ca. 10 nM on a per-peptide basis (Fig. 6A). Heptavalent molecules presenting only thioglycerol showed no inhibitory activity (Fig. 6A), and the monovalent peptide did not inhibit cytotoxicity at concentrations as high as 2 mM. The heptavalent inhibitor consequently offered a more than 100,000-fold enhancement in the activity of this peptide. To test whether the well-defined heptavalent inhibitor based on the PEG11 linker was resistant to proteolytic degradation, we also incubated the inhibitor with 80% serum at 37 C. Samples were withdrawn at numerous time intervals and their inhibitory activity was identified using the cytotoxicity assay. As seen in Number 6B, the heptavalent inhibitor did not display any significant loss in activity over a three day time period. Open in a separate window Number 6 Characterization of a well-defined heptavalent anthrax toxin inhibitor. and and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent anti-toxins might serve as useful adjuncts to antibiotics for the treatment of anthrax. The approach layed out in this work might also become broadly relevant to developing well-defined oligovalent molecules that inhibit pathogens or additional microbial toxins heat-labile enterotoxin by modular structure-based design. J. Am. Chem. Soc. 2000;122:2663C2664. [Google Scholar] 22. Kitov PI, Sadowska JM, Mulvey G, Armstrong GD, Ling H, Pannu NS, Go through RJ, Package DR. Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature. 2000;403:669C672. [PubMed] [Google Scholar] 23. Mulvey GL, Marcato P, Kitov PI, Sadowska J, Package DR, Armstrong GD. Assessment in mice of the restorative potential of tailored, multivalent Shiga toxin carbohydrate ligands. J. Infect. Dis. 2003;187:640C649. [PubMed] [Google Scholar] 24. Polizzotti BD, Maheshwari R, Vinkenborg J, Kiick KL. Effects of Saccharide Spacing and Chain Extension on Toxin Inhibition by Glycopolypeptides of Well-Defined Architecture. Macromolecules. 2007;40:7103C7110. [PMC free article] [PubMed] [Google Scholar] 25. Gu LQ, Braha O, Conlan S, Cheley S, Bayley H. Stochastic sensing of organic analytes by a pore-forming protein comprising a molecular adapter. Nature. 1999;398:686C690. [PubMed] [Google Scholar] 26. Liao KC, Mogridge J. Manifestation of Nlrp1b inflammasome parts in human being fibroblasts confers susceptibility to anthrax lethal toxin. Infect. Immun. 2009:4455C4462. [PMC free article] [PubMed] [Google Scholar] 27. Gujraty K, Sadacharan S, Frost M, Poon V, Kane RS, Mogridge J. Functional characterization of peptide-based anthrax toxin inhibitors. Mol. Pharm. 2005;2:367C372. [PubMed] [Google Scholar] 28. Cunningham K, Lacy DB, Mogridge J, Collier RJ. Mapping the lethal element and edema element binding sites on oligomeric anthrax protecting antigen. Proc. Natl. Acad. Sci. USA. 2002;99:7049C7053. [PMC free article] [PubMed] [Google Scholar] 29. Garcia AE, Sanbonmatsu KY. Exploring the energy scenery of a beta hairpin in explicit solvent. Proteins. 2001;42:345C354. [PubMed] [Google Scholar] 30. Sugita Y, Okamoto Y. Replica-exchange molecular dynamics methods for protein folding. Chem. Phys. Lett. 1999;314:141C151. [Google Scholar] 31. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J. Mol. Graph. 1996;14:33C8. 27C8. [PubMed] [Google F2rl3 Scholar] 32. Lacy DB, Wigelsworth DJ, Melnyk RA, Harrison SC, Collier RJ. Structure of heptameric protecting antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent pore formation. Proc. Natl. Acad. Sci. USA. 2004;101:13147C13151. [PMC free article] [PubMed] [Google Scholar] 33. Gray JJ, Moughon S, Wang C, Schueler-Furman O, Kuhlman B, Rohl CA, Baker D. Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. J Mol Biol. 2003;331:281C299. [PubMed] [Google Scholar] 34. Kolb HC, Finn MG, Sharpless KB. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. Engl. 2001;40:2004C2021. [PubMed].Angew. for an inhibitory peptide within the heptameric subunit of anthrax toxin. We developed an approach based on copper-catalyzed azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a -cyclodextrin core via a polyethylene glycol linker of an appropriate length. The resulting heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors show considerable promise as anthrax anti-toxins. by incubating RAW264.7 cells with a mixture of PA and LF in the presence of several concentrations of the inhibitor. The heptavalent molecule could inhibit cytotoxicity with a half-maximal inhibitory concentration (IC50) of ca. 10 nM on a per-peptide basis (Fig. 6A). Heptavalent molecules presenting only thioglycerol showed no inhibitory activity (Fig. 6A), and the monovalent peptide did not inhibit cytotoxicity at concentrations as high as 2 mM. The heptavalent inhibitor therefore provided a more than 100,000-fold enhancement in the activity of this peptide. To test whether the well-defined heptavalent inhibitor based on the PEG11 linker was resistant to proteolytic degradation, we also incubated the inhibitor with 80% serum at 37 C. Samples were withdrawn at various time intervals and their inhibitory activity was decided using the cytotoxicity assay. As seen in Physique 6B, the heptavalent inhibitor did not show any significant loss in activity over a three day period. Open in a separate window Physique 6 Characterization of a well-defined heptavalent anthrax toxin inhibitor. and and showed GV-58 appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent anti-toxins might serve as valuable adjuncts to antibiotics for the treatment of anthrax. The approach outlined in this work might also be broadly applicable to designing well-defined oligovalent molecules that inhibit pathogens or other microbial toxins heat-labile enterotoxin by modular structure-based design. J. Am. Chem. Soc. 2000;122:2663C2664. [Google Scholar] 22. Kitov PI, Sadowska JM, Mulvey G, Armstrong GD, Ling H, Pannu NS, Read RJ, Bundle DR. Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature. 2000;403:669C672. [PubMed] [Google Scholar] 23. Mulvey GL, Marcato P, Kitov PI, Sadowska J, Bundle DR, Armstrong GD. Assessment in mice of the therapeutic potential of tailored, multivalent Shiga toxin carbohydrate ligands. J. Infect. Dis. 2003;187:640C649. [PubMed] [Google Scholar] 24. Polizzotti BD, GV-58 Maheshwari R, Vinkenborg J, Kiick KL. Effects of Saccharide Spacing and Chain Extension on Toxin Inhibition by Glycopolypeptides of Well-Defined Architecture. Macromolecules. 2007;40:7103C7110. [PMC free article] [PubMed] [Google Scholar] 25. Gu LQ, Braha O, Conlan S, Cheley S, Bayley H. Stochastic sensing of organic analytes by GV-58 a pore-forming protein made up of a molecular adapter. Nature. 1999;398:686C690. [PubMed] [Google Scholar] 26. Liao KC, Mogridge J. Expression of Nlrp1b inflammasome components in human fibroblasts confers susceptibility to anthrax lethal toxin. Infect. Immun. 2009:4455C4462. [PMC free article] [PubMed] [Google Scholar] 27. Gujraty K, Sadacharan S, Frost M, Poon V, Kane RS, Mogridge J. Functional characterization of peptide-based anthrax toxin inhibitors. Mol. Pharm. 2005;2:367C372. [PubMed] [Google Scholar] 28. Cunningham K, Lacy DB, Mogridge J, Collier RJ. Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen. Proc. Natl. Acad. Sci. USA. 2002;99:7049C7053. [PMC free article] [PubMed] [Google Scholar] 29. Garcia AE, Sanbonmatsu KY. Exploring the energy landscape of a beta hairpin in explicit solvent. Proteins. 2001;42:345C354. [PubMed] [Google Scholar] 30. Sugita Y, Okamoto Y. Replica-exchange molecular dynamics methods for protein folding. Chem. Phys. Lett. 1999;314:141C151. [Google Scholar] 31. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J. Mol. Graph. 1996;14:33C8. 27C8. [PubMed] [Google Scholar] 32. Lacy DB, Wigelsworth DJ, Melnyk RA, Harrison SC, Collier RJ. Structure of heptameric protective antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent pore formation. Proc. Natl. Acad. Sci. USA. 2004;101:13147C13151. [PMC free article] [PubMed] [Google Scholar] 33. Gray JJ, Moughon S, Wang C, Schueler-Furman O, Kuhlman B, Rohl CA, Baker D. Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. J Mol Biol. 2003;331:281C299. [PubMed] [Google Scholar] 34. Kolb HC, Finn MG, Sharpless KB. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. Engl. 2001;40:2004C2021. [PubMed] [Google Scholar] 35. Lutz J-F. 1,3-Dipolar Cycloadditions of Azides and Alkynes: a Universal Ligation Tool in Polymer and Materials Science. Angew. Chem. Int. Ed. Engl. 2007;46:1018C1025. [PubMed] [Google Scholar] 36. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise huisgen.