A Glycopeptide Dendrimer Inhibitor of the Galactose-Specific Lectin LecA and of Pseudomonas aeruginosa Biofilms

The spread of antibiotic resistant bacteria is one of the most pressing problems in human health today. [1] In the case of the opportunistic pathogen Pseudomonas aeruginosa , which causes lethal airway infections in cystic fibrosis and immuno-compromised patients, the formation of biofilms plays an important role in antibiotic resistance and disease progres-sion. [2] Biofilm formation is mediated in part by the galactose-specific lectin LecA (PA-IL) [3] and the fucose-specific lectin LecB (PA-IIL), [4] as evidenced by studies with deletion mutants [5] and the partial inhibitory effect of simple fucose and galactose derivatives in vitro and in vivo. [5a,6] Under-standing the glycoconjugate–lectin interaction is a key feature in developing potent biofilm inhibitors. Capitalizing on the well-known cluster effect observed on binding of multivalent carbohydrates to lectins, [7–11] we recently reported the first case of P. aeruginosa biofilm inhibition with a multivalent lectin inhibitor, the fucosylated glycopeptide dendrimer FD2 (cFuc-Lys-Pro-Leu) 4 ( Lys- Phe-Lys-Ile) 2 Lys- His-IleNH 2 , which targets LecB. [12,13] Herein we report the first case of P. aeruginosa biofilm inhibition with a multivalent ligand targeting the galactose-specific lectin LecA, biofilms by glycopeptide dendrimers. Biofilms were grown on steel coupons inoculated with PAO1 for 48 h at 37 8 C in the presence of ligands (20 m m galactosyl endgroup) followed by staining with acridine orange prior to analysis of surface coverage. Metal coupons incubated with bacteria only (control), growth media with no bacteria (media control), and with FD2 dendrimer, were used as positive and negative controls. [20] The strongest binding was observed with the second-generation glycopeptide dendrimer GalAG2. This dendrimer contains an aromatic aglycone linker that engages in an unprecedented T-stack interaction with His50 at the LecA galactose binding site, as evidenced by X-ray crystallography. This interaction enables additional contacts between the outer tripeptide branch of the dendrimer and the lectin; the contacts do not occur in the case of the thiopropyl linker in the GalB-type ligands, for which the tripeptide is disordered in the X-ray structure. Interestingly, both GalAG2 and GalBG2 dendrimers displayed potent biofilm inhibition, whereas the G1 analogues were much less active and the G0 analogs were inactive. Thus multivalency played a much more important role for biofilm inhibition than the nature of the linker. Future experiments will address activity improvement by dendrimer sequence optimization and the synthesis of analogues with higher multivalency.

To a solution of galactose pentaacetate (2.0 g, 5.12 mmol) in dry CH 2 Cl 2 (10 mL) at 0 °C, 33% HBr/AcOH (4.5 mL) was slowly added. The resulting orange solution was stirred at r.t. for 30 min then diluted with CH 2 Cl 2 (50 mL), washed with saturated aqueous NaHCO 3 (3 x 30 mL) and dried over Na 2 SO 4 . The solvent was removed under reduced pressure to give the crude α-bromoderivative a (2.0 g, 5.02 mmol) which was used without purification for the glycosidation. 1
The solvent was removed under reduced pressure to give a crude which was purified by flash column chromatography (eluent: petroleum ether/ethyl acetate 2:1 and 1:1) to afford b (1.83 g, 3.27 mmol, 64% in two steps) as a white foam slightly contaminated by benzyl-4-hydroxy-benzoate, which can be easily removed after hydrogenation.

Staining
Two hundred microlitres of acridine orange (0.1% w/v) were added to the steel coupon in order to stain the remaining attached cells. This was left for 2 minutes 30 seconds. Excess acridine orange was removed by the addition of 20ml sterile PBS and the dish agitated for 5 minutes and the waste removed. This step was repeated once further with sterile PBS and once with sterile distilled water and left to air dry. The coupons were air dried and mounted onto a microscope slide.

Imaging and analysis
The coupons were placed in the universal slide holder of an inverted Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss, Germany).
Surface attachment. Coupons were imaged for bacterial attachment using the x10 objective lens, 405nm laser with pinhole of 94.2nm, gain set at 708 and digital offset at -1. Six images were taken at random from each coupon. The images were uploaded to ImageJ (NIH, USA) and thresholded between 40 and 164. The area fraction was calculated and the average surface area and standard error was calculated.
Biomass. Coupons were imaged for biomass using the x40 objective lens, 405nm laser, pinhole 73.3, gain set at 722, digital offset -43. Z-stacks were captured with averaging of 4 for each slice.
Images were subject to thresholding at 126 and biomass calculated with connected volume filtering active, without smacking. The results were exported to excel and standardised to the activity of the wild-type PAO1 strain. The results were exported to GraphPad Prism 5 for analysis. S15

P. aeruginosa lectin LecA expression and purification
LecA was expressed and purified by affinity chromatography along an optimized protocol and in accordance to a previous report. 3 The plasmid pET25paIL was transformed into Escherichia coli BL21(DE3) cells. E. coli cells were grown in 6L of LB medium (10gm tryptone; 5gm yeast extract and 5gm Nacl in 1L of deionised water) at 30 °C. When the culture had reached an optical density           LecA-galactoside crystals belong to space groups P4 1 2 1 2, P2 1 2 1 2 1 and P2 1 2 1 2 with the corresponding asymmetric units containing four, eight and two monomers for NPG, GalAG0 and GalBG0 respectively. Further details on data collection statistics are given in (Table S1). Crystals were cryo-cooled at 100 K after soaking them for as short a time as possible in glycerol 25% v/v in precipitant solution. All data were collected at the SLS synchrotron (Villigen, Switzerland) at beamline PX-II/III ( Figure S28).

S24
Data were integrated and scaled with the X-ray detector software for processing single-crystal monochromatic diffraction data (XDS). 4 The structures of the co-crystallized ligands were solved by the molecular replacement technique with the Phaser program 5 , using the monomeric structure (PDB code 1OKO) 6

Ligand induced conformational changes in LecA
The T-shaped interaction mode between NPG and His50 within the LecA-NPG binding site    Molecular dynamic simulations. The prepared systems were used for molecular dynamics simulation. MD simulations for a period of 10 ns were performed using following three steps :

1) minimization 2) equilibration and 3) production run;
Minimization. The setup systems were minimized to remove close contacts between solute-solvent molecules using the LBFGS method with maximum iterations of 200 cycles and convergence threshold of 1.0 Kcal mol -1 Å -1 . The step size was 0.005 ps and switch criteria was 25.0 Kcal mol -1 Å -1 . The short range interactions used cutoff radius of 9.0 Å, whereas the long range coulombic interactions were taken into account using smooth Particle Mesh Ewald (PME) with Ewald tolerance of 1e-09 Å.
Equilibration protocol. During equilibration 2000 steps of steepest descent minimization of water molecules and ions were performed to allow water molecules to assume a lower energetic geometry, while the solute was restrained with force constant of 50 kcal/mol Å 2 . The resulting S29 systems were then subjected to 2000 steps of minimization with no restraints, reaching a root mean square gradient of 0.1 to assure the relaxation of the structures, followed by 12 ps of heating from 10 to 300 K in a constant volume ensemble with restraints on the solute heavy atoms (50 kcal/mol Å 2 ). This was followed by 12 ps of constant pressure unrestrained simulation at 300 K, where convergences of energies, temperature, pressure and density of the systems was monitored.
Production runs protocol. The output co-ordinates and velocities from the equilibrated run were subsequently used in productions runs over a period of 10 ns at 300 K using NPT ensemble. The bond lengths to hydrogens were constrained with a variant of the M-SHAKE algorithm 14