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. In the case of the opportunistic pathogen Pseudomonas aeruginosa, which causes lethal airway infections in cystic fibrosis and immunocompromised patients, the formation of biofilms plays an important role in antibiotic resistance and disease progression. Biofilm formation is mediated in part by the galactosespecific lectin LecA (PA-IL) and the fucose-specific lectin LecB (PA-IIL), as evidenced by studies with deletion mutants and the partial inhibitory effect of simple fucose and galactose derivatives in vitro and in vivo. Understanding 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, 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)2Lys-His-IleNH2, which targets LecB. Herein we report the first case of P. aeruginosa biofilm inhibition with a multivalent ligand targeting the galactose-specific lectin LecA, using the related b-phenylgalactosyl peptide dendrimer GalAG2. Considering the favorable properties of FD2 as lectin inhibitor, we set out to investigate if its peptide dendrimer portion might also be suitable for inhibitors of the galactosespecific P. aeruginosa lectin LecA. Because hydrophobic groups in the sugar anomeric position have been shown to enhance the affinity of galactosides to LecA, acetylprotected 4-carboxyphenyl b-galactoside (GalA) was attached to the peptide dendrimer. To probe the effect of the sugar-dendrimer linker on binding, carboxypropyl b-thiogalactoside (GalB) was also introduced as the last building block in solid-phase peptide synthesis to provide dendrimers GalA/ BG1 and GalA/BG2, and the linear peptides GalA/BG0. The dendrimers were obtained pure as trifluoroacetate salts after deacetylation on a solid support, acid-mediated cleavage from the support, and purification by preparative HPLC (Scheme 1). The binding affinity to LecA was evaluated in a hemagglutination assay that measured the inhibition of LecAinduced agglutination of rabbit erythrocytes in comparison to d-galactose as the reference. Thermodynamic parameters were obtained by isothermal titration calorimetry (ITC). A strong multivalency effect on binding was observed in both the GalA and the GalB dendrimer series. The strongest effect occurred with dendrimer GalAG2, which showed a 4000-fold increase in hemagglutination inhibition activity and a 875-fold increase in binding (Kd) to LecA compared to d-galactose (Table 1). Considering that the number of sugars and the peptidic scaffold are the same in both series, the presence of the phenyl group in GalA dendrimers led to a remarkable enhancement in affinity compared with the thiogalactoside GalB dendrimers. The dendrimers inhibited P. aeruginosa biofilms, as evidenced by using the steel coupon assay (Figure 1). A generation-dependent effect was observed in both the GalA and the GalB series, with essentially complete inhibition of biofilm formation with the second-generation (G2) dendrimers, as observed with the LecB inhibitor FD2, whereas the acetylated dendrimer AcG2, which lacks galactosyl groups, showed only a small inhibition of biofilm formation. Bacterial growth was unaffected by the ligands, thus ruling out toxicity effects. These data were consistent with a LecA-mediated inhibition of P. aeruginosa biofilms by the dendrimers. [*] Dr. R. U. Kadam, M. Bergmann, Dr. M. A. Swiderska, Priv.-Doz. Dr. A. Stocker, Priv.-Doz. Dr. T. Darbre, Prof. Dr. J.-L. Reymond Department of Chemistry and Biochemistry, University of Berne Freiestrasse 3, 3012 Berne (Switzerland) E-mail: [email protected]