Glycopeptide Dendrimer Inhibitors Targeting LecA
FULL PAPER
100 mm CaCl2, pH 7.5). The purified protein was extensively dialyzed
against distilled water containing 2 mm CaCl2 for 7 days and characterized
by using SDS-PAGE and mass spectroscopy. Purified fractions of protein
were lyophilized and kept at À208C.
Materials and Methods
Synthetic procedures and characterization of the various galactosylated
tripeptides and peptide dendrimers are described in the Supporting Infor-
mation.
Hemagglutination assay:
Erythrocyte preparation: Rabbit red cells (erythrocytes 50%; Biomer-
ieux), separated from preservative by centrifugation (1500 rpm; 10 min),
were washed three times with 0.9% NaCl solution (saline) and suspend-
ed to a concentration of 5% v/v in phosphate-buffered saline (PBS;
0.01m; pH 7.4). The suspension was given papain treatment, which in-
volves incubation of 9 volumes of the 5% cell suspension with 1 volume
of the 1% w/v papain (crude preparation; Sigma) in 0.1% w/v l-cysteine
solution at 378 for 30 min. The enzyme-treated cells were washed three
times in PBS and then resuspended in PBS to a concentration of 5%.
In silico mutagenesis of glycopeptide ligands: Single-point mutant (SPM)
and combined mutant (CM) ligands were generated in silico based on
the native glycopeptide ligand sequence GalAG0 (GalA-Lys-Pro-Leu-
NH2) by using the mutagenesis utility tool in the PyMol v1.3 software.
Single-point mutations involved the mutation of each amino acid in the
sequence with the 20 natural amino acids, with the b-phenyl-galactosyl
part kept unaltered. This leads to 57 possible mutants in the SPM series
(Figure S67 and Table S2 in the Supporting Information). Subsequently,
the CMs were designed based on the best hits obtained from in silico
docking and scoring of the SPMs (Table S3 in the Supporting Informa-
tion).
LecA titration: In order to determine the lectin concentration needed to
agglutinate the cells, decreasing amounts of LecA were incubated with
the red blood cells. Serial twofold dilutions were made in the wells of
a microtiter plate (96-well microtiter nontreated V-bottom plates; Nunc,
Denmark). The twofold dilutions were made by adding buffer solution
(50 mL) to all 24 wells and LecA solution (50 mL; 0.34 mgmLÀ1) to the
first well. A 50 mL volume was then transferred from the first well to the
second. The second well was mixed and a 50 mL volume was transferred
to the third well. This procedure was repeated until the 24th well. To
each well, the red blood cell solution (50 mL; 5% in PBS) was added,
and the mixture was incubated for 30 min at 48C. After this time, the
plates were centrifuged for 30 s (1000 g), the wells were examined, and
the minimum amount of LecA required to agglutinate the cell suspension
was determined. This was then considered to be one HA unit. For the in-
hibition assay, an 8 HA unit LecA solution was made up.
Molecular docking and scoring:
Glycopeptide ligand geometry optimization: Glycopeptides with a core b-
galactose phenyl group (GalA) with the amino acids P1, P2, and P3 in
the corresponding positions were used for the molecular docking study.
Subsequently, the built ligands were geometry optimized with the Macro-
model v9.1 program (Schrodinger, LLC) by using the Optimized Poten-
tials for Liquid Simulations–All Atom (OPLS-AA) force field[24]with the
truncated Newton conjugate gradient protocol. Partial atomic charges
were assigned according to the OPLS-AA force field.
Protein structure preparation and refinement: The X-ray crystal structure
of lectin LecA from P. aeruginosa in complex with GalAG0 (PDB ID:
3ZYB) obtained from the RCSB Protein Data Bank (PDB; http://
study. Water molecules of crystallization were kept 5 ꢃ around the co-
crystallised ligand, and the protein was optimized for docking by using
the protein preparation and refinement utility provided by Schrçdinger
LLC. Partial atomic charges were assigned according to the OPLS-AA
force field.
Minimum inhibitory concentration determination: A 50 mL sample of
each inhibitor examined was serially diluted with PBS (50 mL) in the mi-
crotiter plate to produce twofold dilutions (as described above). The in-
hibitor solutions were incubated with the 8 HA unit LecA solution
(50 mL; conc. of LecA=5.31 mgmLÀ1) for 30 min at 48C. After this time,
the erythrocytes in PBS suspension (50 mL; conc.=5%) were added, and
the wells were mixed and incubated for 1 h at room temperature. The
plates were then centrifuged for 30 s (1000 g). Each test was performed
in triplicate. The activity of the tested compounds was recorded as the
minimum inhibitory concentration (MIC), which corresponded to the
highest dilution that caused complete inhibition of hemagglutination
(Figure S68–70 in the Supporting Information).
Docking methodology and protocol: All docking calculations were per-
formed by using the “Extra Precision” (XP) mode of the Glide pro-
gram.[25] The accuracy of a docking procedure can be evaluated by deter-
mining how closely the lowest energy pose (binding conformation) pre-
dicted by the object scoring function resembles an experimental binding
mode as determined by X-ray crystallography. In the present study, the
Extra Precision Glide docking procedure was validated by removing the
ligand from GalAG0 from the co-crystallised LecA complex and re-dock-
ing into the binding site of LecA. A good agreement was observed be-
tween the localization of the inhibitor upon docking and from the crystal
structure, that is, there were similar hydrogen-bonding interactions with
N107, D100, Q53, and H50 and a similar Ca2+ coordination. The pairwise
RMS value between the predicted conformation and the observed X-ray
crystallographic conformation of GalAG0 equalled 1.27 ꢃ, a value that
suggests the reliability of the docking program and the Glide parameter
set in reproducing the experimentally observed binding mode for LecA
from P. aeruginosa. The validated docking protocol was then used for
docking the SPM and CM ligands into the crystal structure of the protein
(Table S2 and S3 in the Supporting Information).
Biofilm formation on polystyrene microtiter plates: A modified version
of the method described by Diggle et al. was employed.[5c] The 96-well,
sterile, U-bottomed polystyrene microtiter plates (TPP, Switzerland)
were prepared by adding sterile deionized water (200 mL) to the periph-
eral wells to decrease evaporation from the test wells. Aliquots of 180 mL
of culture medium (10% w/v nutrient broth no. 2, Oxoid) containing the
appropriate concentration of the test compound were added to the inter-
nal wells. For better solubility, G2KPW and G2KPY were dissolved in
10% w/v nutrient broth containing 5% v/v DMSO. An inoculum of
P. aeruginosa strain PAO1 was prepared from a 5 mL overnight culture
grown in LB broth. Aliquots (20 mL) of overnight cultures, prewashed in
10% w/v nutrient broth and normalized to an optical density at 600 nm
(OD600) of 1, were inoculated into the test wells. Plates were incubated in
a humid environment for 25 h at 378C. Wells were washed with sterile
deionized water (200 mL) before staining with 10% w/v nutrient broth
(200 mL) containing 0.5 mm WST-8 and 20 mm phenazine ethosulfate for
3 h at 378C. Afterwards, the well supernatants were transferred to a poly-
styrene flat-bottomed 96-well plate (TPP, Switzerland), and the absorb-
ance was measured at 450 nm with a plate reader (SpectraMax250 from
Molecular Devices).
P. aeruginosa LecA expression and purification: LecA was expressed and
purified by affinity chromatography along an optimized protocol and in
accordance with a previous report.[26] The plasmid pET25paIL was trans-
formed into Escherichia coli BL21ACTHNUTRGNE(UNG DE3) cells. E. coli cells were grown in
Luria–Bertani (LB) medium (6 L; tryptone (10 g), yeast extract (5 g),
NaCl (5 g) in deionized water (1 L)) at 308C. When the culture had
reached an optical density of 0.5–0.6 at 600 nm, isopropyl-b-d-thiogalac-
topyranoside (IPTG) was added to a final concentration of 0.1 mm. Cells
were harvested after being left overnight with shaking at 220 rpm at
208C, washed, and resuspended in loading buffer (100 mL; 20 mm Tris–
HCl, 100 mm CaCl2, pH 7.5). The cells were broken by sonication. After
centrifugation at 5000 rpm for 45 min, the supernatant was loaded onto
an affinity chromatography column containing Sepharose 4B (250 mL).
LecA was eluted with 0.2m d-galactose in buffer (20 mm Tris–HCl,
Isothermal titration calorimetry: Lyophilized LecA was dissolved in
buffer (0.1m Tris–base, pH 7.5, 25 mm CaCl2). The protein concentration
was checked by measurement of the absorbance at 280 nm by using a the-
oretical molarity extinction coefficient of 27600mÀ1 cmÀ1. Ligands were
dissolved directly into the same buffer. ITC was performed with a iTC200
calorimeter (MicroCal Inc.). Titration was performed on 40 mm LecA in
a 200 mL sample cell by using 1–2 mL injections of 1–1.5 mm ligand every
Chem. Eur. J. 2013, 19, 17054 – 17063
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
17061
Kadam, Rameshwar U.
