Glycopolymer–Lec,n Interac,ons and Inhibi,on of Pathogens using Mul,valent Scaffolds Sarah-Jane Richards, Mathew W. Jones, Yanzi Gou, David M. Haddleton and Matthew I. Gibson S-J.Richards@warwick.ac.uk
www2.warwick.ac.uk/go/gibsongroup
Background •  Polymers with pendent carbohydrate moie4es (glycopolymers) interact with lec4ns and have demonstrated binding affini4es several orders of magnitude greater than a single carbohydrate.3 •  Interference at this early stage is known as an4-­‐adhesion therapy and does not kill the pathogen. Importantly, it prevents binding and hence internalisa4on which markedly reduces the chance of becoming resistant.4 Protein-­‐carbohydrate interac4ons mediate a mul4tude of cri4cal biological recogni4on processes.1 The proteins responsible for deciphering this informa4on are termed lec4ns.2 Comparison of Surface Binding with Inhibitory Ac,vity Glycopolymer–Lectin Interactions
The nature of the interac4ons between glycopolymers and lec4ns, and the structural features necessary to obtain high-­‐affinity materials are not fully understood.2 In this study, we probed mul4valent interac4ons in the α-­‐
mannose -­‐ Concanavalin-­‐A (ConA) pairing: Binding Modes
P2 and P3 inhibited ConA 10x more effec4vely than P1. However, this is in contrast to the QCM studies: P1 P2 P3 •  QCM-­‐d also probes the viscoelas4city of films formed on a surface. •  Post-­‐polymerisa4on Modifica4on: α-­‐D-­‐mannose •  Large changes in dissipa4on (ΔD) indicate a flexible film, whilst small ΔD values suggest a rigid, non-­‐flexible coa4ng: The longer chain polymer (P3) spans the binding site of ConA was ‘clicked-­‐on’ to a poly-­‐(propargyl methacrylate) whereas the shorter polymers (P1/P2) can only bind one site. backbone. •  Polymer Chain Lengths: 2 (P1), 6 (P2) and 11 nm (P3). •  Binding inhibi4on was assessed using Quartz-­‐crystal microbalance with dissipa4on monitoring (QCM-­‐d) and Fluorescence-­‐linked sorbent assay (FLSA). •  Higher binding affinity = increased mass of glycoside binding to the lec4n surface. This property is used to screen new inhibitors. •  A combina4on of techniques is required to assess the efficacy of an inhibitor. Gou, Y.; Richards, S-­‐J.; Haddleton D. M.; Gibson, M. I. Polymer. Chem. 2012 Inhibi,on of Bacterial Toxins Role of Linker Length and Carbohydrate Density
Bacterial Toxin Binding
•  Polymers synthesised with ~ 6 Å (short) and 16 Å (long) linkers. The cholera toxin (CTx) secreted by Vibrio cholerae binds glycosides expressed on the cell surface.5 Materials with high-­‐affinity and selec4vity for these lec4ns could find applica4ons as an4-­‐adhesive agents.4 PNA binding site CTx binding site •  Probe influence of chain length, carbohydrate density and linker length on binding inhibi4on. •  Glycopolymer library produced by tandem post -­‐ polymerisa4on modifica4ons. •  Structural biology indicates CTx has deeper binding site than other galactose binding lec4ns such as Peanut agglu4nin (PNA). Tandem Post-Polymerisation Modification
‘Clickable’ units are not compa4ble with controlled radical polymerisa4on. Instead, tandem-­‐post polymerisa4on modifica4on are performed. •  Linker length has no effect on PNA inhibi4on (A). •  Longer linker has 2 – 3 fold lower MIC50 compared to shorter linker on inhibi4ng CTx (B). •  100 X more ac4ve than free galactose. •  Polymers synthesised with 10, 25 and 50 % galactose. •  By mass (A), low galactose densi4es lead to a rela4ve decrease in binding affinity/inhibitory ac4vity. •  By mole of galactose (B), 10 % and 100 % func4onalised polymers are the most ac4ve sugges4ng several features (e.g. sterics and site spanning) contribute to inhibitory ac4vity. Summary
•  Tandem post-­‐polymerisa4on modifica4on allows synthesis of polymers from same chain length distribu4on. •  Poly(pentafluorophenyl methacrylate) for easy modifica4on. •  β-­‐D-­‐galactose was ‘clicked-­‐on’ to pendent alkyne moie4es. •  Results in biocompa4ble methacrylamide based (co)polymers. •  Longer linker has beker binding site accessibility. •  Carbohydrate density has an effect. •  Inhibitors have to be developed for the binding site. Richards, S-­‐J.; Jones, M. W.; Hunaban, M. I.; Haddleton, D. M.; Gibson M. I. Angew. Chem Int. Ed., 2012 References
1.  C. R. Bertozzi, L. L. Kiessling, Science, 2001, 291, 2357. 2.  M. Ambrosi, N. R. Cameron, B. G. Davis, Org. Biomol. Chem., 2005, 3, 1593. 3.  S. G. Spain, M. I. Gibson, N. R. Cameron, J. Polym. Sci., Part A: Polym. Chem., 2007, 45, 5031. 4.  R. J. Pieters, Med. Res. Rev. 2007, 27, 796. 5.  B. D. Polizos, K. L. Kiick, Biomacromolecules, 2006, 7, 483. Acknowledgements
Mak Gibson Haddleton Group Gibson Group MOAC Yanzi Gou EPSRC Mat Jones