Structure‐Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

Abstract

The galactopeptide dendrimer GalAG2 ((β-Gal-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA-KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide-lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X-ray crystallography. Two of these tripeptides, GalA-KPY (dissociation constant (K(D))=2.7 μM) and GalA-KRL (K(D)=2.7 μM), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2, particularly G2KPY ((β-Gal-OC6H4CO-Lys-Pro-Tyr)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine-tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.

Development of Pseudomonas aeruginosa Lectin LecA Inhibitor by using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds

“…However, it is interesting that an indole moiety connected to p ‐phenyl on peptide 9 k was tolerated by LecA with a comparable K D to 9 g . To understand how the ligand may interact with LecA, we performed molecular modeling analysis of truncated 6 k with the LecA complex structure from a protein data base (3ZYB) . The docking results (Figure ) indicated that the way of truncated ligand 6 k binding to LecA was similar to the interaction reported in the β‐phenylgalactopyranoside–LecA complex structure, where the hydroxyl groups (3′‐ and 4′‐OH) directly interact with calcium ions and the p ‐phenyl ring initiates T‐shape CH–π interactions with the His50 residue (Figure ).…”

Multivalency effects on Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA

“…17 The crystals of the GalAxPSG3·LecA complex were obtained by soaking. 17 The crystals of the GalAxPSG3·LecA complex were obtained by soaking.…”

“…15 Tight lectin binding and biofilm inhibition by these tetravalent G2 dendrimers depended on a multivalency effect since the lower generation analogs (G0 and G1) were essentially inactive. 17 Considering that many of the reported high affinity multivalent glycosidic ligands for lectins feature an 8-fold or higher multivalency, we asked the question whether the binding affinity and biological activity of dendrimers GalAG2 and GalBG2 might be increased in higher valency analogs as observed in other series of tight binding LecA ligands. 17 Considering that many of the reported high affinity multivalent glycosidic ligands for lectins feature an 8-fold or higher multivalency, we asked the question whether the binding affinity and biological activity of dendrimers GalAG2 and GalBG2 might be increased in higher valency analogs as observed in other series of tight binding LecA ligands.…”

Highly Ordered Self‐Assembly of Native Proteins into 1D, 2D, and 3D Structures Modulated by the Tether Length of Assembly‐Inducing Ligands

“…LecA (PA-IL) from Pseudomonas aeruginosa,w hich is ah omotetrameric protein with ac uboid-shaped structure (length % 7.2 nm, width % 3.5 nm, and height % 1.9 nm) is selected as the building block for assembly fabrication (Figure 1a). Based on the protein packing in crystal structure of the LecAsugar complex, [21] in our design, three kinds of packing patterns formed by LecA with different side lengths were possible,n amely,s hort-short, long-long,a nd diagonal-diagonal ( Figure 1d). [20] According to our previous reports, [10c, 13b] proteins are connected through the molecular recognition between lectin and sugar and the p-p stacking of rhodamine B( RhB) between the neighboring ligands ( Figure 1c), five assemblyinducing ligands RnG (n = 1t o5 )c ontaining galactopyranoside (Gal) and RhB are prepared with different tether lengths, that is,t he number of ethylene oxide repeating units varies from one to five (Figure 1b).…”