[47]
carbamate), 250ꢅ20 mm, 10 mm; Chiral Technologies Europe,
field and partial atomic charges, the molecular geometries of (R)-
1 and (S)-1 were each separately energy minimized using the
adopted-based Newton–Raphson algorithm. Structures were con-
sidered fully optimized when the energy changes between itera-
Cedex, France) as the chiral stationary phase and MeOH/MeCN
(
98:2) containing dimethylethanolamine (DMEA; 0.2% v/v) as the
[18]
mobile phase. Enantiomeric excess (ee) of the isolated fractions
was determined using a CHIRALPAK AD (amylose tris(3,5-dimethyl-
phenyl carbamate),150ꢅ4.6 mm, 5 mm; Chiral Technologies Europe,
Cedex, France) analytical column and MeOH/MeCN (90:10) contain-
ing DMEA (0.2% v/v) as the mobile phase. The less polar enantio-
mer (R)-1 showed a retention factor (k’) of 0.604, while the more
polar enantiomer (S)-1 had a k’ value of 1.764 (see Supporting In-
formation in Reference [18])
À1 [48]
tions were less than 0.01 Kcalmol . Tacrine was retrieved from
[49]
the TcAChE–tacrine complex (PDB ID: 1ACJ). The coordinates of
[50]
hAChE (PDB ID: 1B41 ), and hBuChE complexed with choline
[51]
[51]
(PDB ID: 1P0M ) and butyrate (PDB ID: 1P0I ) were obtained
from the Protein Data Bank (PDB). For docking studies, initial pro-
teins were prepared by removing all water molecules, heteroa-
toms, and any co-crystallized solvent and ligands (choline and bu-
tyrate). To correct for poor or missing assignments of explicit hy-
drogen atoms in the PDB structures, and to accommodate bond
order information not possible in the PDB file format, proper
bonds, bond orders, hybridization and charges were assigned
using the protein model tool in Discovery Studio. A CHARMm
forcefield was applied using the receptor–ligand interactions tool
in Discovery Studio. Docking calculations were performed using
Determination of the inhibitory potency on Ab
ated by recombinant hAChE: Aliquots of Ab1–40 (2 mL; Bachem AG,
aggregation medi-
–40
1
[30]
Switzerland), lyophilized from 1,1,1,3,3,3-hexafluoro-2-propanol
À1
(
HFIP; 2 mgmL ) and dissolved in dimethyl sulfoxide (DMSO) at a
final concentration of 230 mm, were incubated for 24 h at RT in
sodium phosphate buffer (0.215m, pH 8.0). Aliquots of hAChE
(
2.30 mm, molar ratio 100:1) and hrAChE in the presence of the test
[52]
AutoDock Vina. AutoDockTools (ADT; version 1.5.4) was used to
compound (100 mm) were added. Blanks containing Ab alone,
hrAChE alone, or Ab plus the test compound, and hrAChE plus the
test compound in sodium phosphate buffer (0.215m, pH 8.0) were
prepared. To quantify amyloid fibril formation, the thioflavin T
add hydrogen atoms and partial charges for proteins and ligands
using Gasteiger charges. Flexible torsions in the ligands were as-
signed with the AutoTors module, and the acyclic dihedral angles
were allowed to rotate freely. Because Vina uses rectangular boxes
for the binding site, the box center was defined, and the docking
box was displayed using ADT. For hAChE (PDB ID: 1B41), a grid
box of 20ꢅ26ꢅ22 with grid points separated by 1 ꢆ was posi-
tioned at the active site gorge (x=116.546; y=110.33; z=
À134.181). This box is big enough to include the PAS and the cata-
lytic site. Trp286, Tyr124, Tyr337 and Tyr72 receptor residues were
selected to keep flexible during docking simulation using the Auto-
Tors module. Default parameters were used except “num modes”,
which was set to 40. Before docking our ligands into the hAChE
structure, the AutoDockVina docking protocol was validated using
the four ligand–TcAChE complexes selected from PDB structures:
THA (tacrine)/1ACJ; E20 (donepezil)/1EVE; GNT ((À)-galantamine)/
1DX6 and AA7/2CKM; the three-dimensional structures of the four
ligands were extracted from their respective complexes with AChE.
Finally, the ligands were re-docked using the docking protocol.
TcAChE was used because the X-ray crystal structures for the
hAChE–ligand complexes were not available. The results showed
that the RMSD values of the predicted and experimental poses of
the ligands in the binding pocket of TcAChE are: 0.25 ꢆ for THA,
0.80 ꢆ for E20, 0.20 ꢆ for GNT, and 0.72 ꢆ for AA71536.
[35,36]
(
ThT) fluorescence method was used.
In brief, after incubation,
the samples were diluted to a final volume of 2 mL with 50 mm
glycine/NaOH buffer (pH 8.5) containing 1.5 mm ThT. A 300 s time
scan of fluorescence intensity was carried out (lexc =446 nm, lem
=
4
90 nm), and values at the plateau were averaged after subtraction
of the background fluorescence of the 1.5 mm ThT solution. The
fluorescence intensities in the absence and in the presence of the
inhibitor were compared and the percent inhibition was calculat-
ed.
Determination of the inhibitory potency on Ab1–42 self-aggregation:
HFIP-pretreated Ab1 samples (Bachem AG, Switzerland) were res-
À42
a
olubilized with
(
MeCN/0.3 mm Na CO /250 mm NaOH
2 3
[46]
48.4:48.4:3.2) to have a stable stock solution ([Ab1 ]=500 mm).
À42
Experiments were performed by incubating the peptide in 10 mm
phosphate buffer (pH 8.0) containing 10 mm NaCl at 308C for 24 h
(
[Ab]=50 mm) with and without inhibitor. Stock solutions (1.5 mm)
were prepared by dissolving test inhibitors in MeCN. Blanks con-
taining test inhibitors and ThT were also prepared and evaluated
to account for quenching and interference eventually related to in-
hibitor fluorescence. To quantify amyloid fibril formation, the ThT
[35,36]
fluorescence method was used.
After incubation, samples
were treated as for the determination of the inhibitory potency on
Ab1–40 aggregation induced by hAChE.
Neuroprotection against Ab25–35-induced cytotoxicity
TEM studies: Samples of Ab1–42 (50 mm) with and without (R)-, (S)-
and (R/S)-1 (50 mm) were prepared as reported for the determina-
tion of the inhibitory potency on Ab1–42 self-aggregation. Aliquots
of each sample (2.5 mL) were then adsorbed onto 200-mesh
carbon-coated copper grids (Electron Microscopy Sciences, Hat-
field, USA) until dryness. The grids were then washed with
Culture of the SH-SY5Y cell lines: SH-SY5Y cells were maintained in a
1:1 mixture of F-12 nutrient mixture (Ham12, Sigma–Aldrich,
Madrid, Spain) and Eagle’s minimum essential medium (EMEM)
supplemented with 15 nonessential amino acids, 1 mm sodium
pyruvate, heat-inactivated fetal bovine serum (FBS), 10% penicillin
(100 UmL ), and streptomycin (100 mgmL ). Reagents were ob-
tained from Invitrogen (Madrid, Spain). Cultures were seeded into
flasks containing supplemented medium and maintained at 378C
À1
À1
0
.22 mm-filtered bidistilled water (3ꢅ10 mL), stained with 1% aq
uranyl acetate (Sigma, Milan, Italy) for 5 min, and finally washed
further with 0.22 mm-filtered bidistilled water (3ꢅ10 mL). The grids
were allowed to dry and were then visualized in a Philips CM100
transmission electron microscope (accelerating voltage 80 kV).
in a humidified atmosphere of CO (5%) and air (95%). For assays,
2
SH-SY5Y cells were subcultured in 48-well plates at a seeding den-
5
sity of 1ꢅ10 cells per well. Cells were treated with test compound
before confluence in F12/EMEM with 1% FBS. All the cells used in
Molecular modeling studies
this study were used at a low passage number (<13).
(
R)-1 and (S)-1 were assembled within Discovery Studio version 2.1,
Incubation of test compound: To assess the neuroprotective effect
of (R)-1, (S)-1 and (R/S)-1 against Ab25–35 (30 mm)-induced toxicity,
SH-SY5Y cells were preincubated with test compound at 0.3, 1, 3
software package, using standard bond lengths, bond angles and
without protonation at the pyridine ring. With the CHARMm force-
ChemMedChem 2011, 6, 1990 – 1997
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1995