ACS Chemical Neuroscience
Research Article
mL of H O, 5 mL of 2N NaOH, and 5 mL of H O were carefully
4.2.5. Inhibition of R3 Aggregation. A stock solution of 1.5 mM
R3 (trifluoroacetate salt; Bachem, Bubendorf, Switzerland) was
prepared in TFE and left overnight at room temperature. Incubation
samples were set in nonbinding, flat-bottomed black microplates
(Greiner Bio-One) in triplicate for each concentration and contained
peptide (25 μM), inhibitor (seven concentrations ranging from 30 to
0.1 μM), and ThT (10 μM) in PBS containing 1.7% of 1,1,1-
trifluoroethanol. Fluorescence was read within 4 h at 37 °C with a
Tecan Infinite M1000 Pro instrument. IC50 values were calculated by
means of Prism as the mean of two independent experiments.
4.2.6. Atomic Force Microscopy. Compounds 2, 5, 11, and 12
were dissolved in DMSO at concentrations ranging from 2.0 mg/mL
to 3.3 mg/mL. Aβ42 (Bachem) was dissolved in TFA to obtain a 1
mg/mL stock solution. In each experiment, appropriate peptide
aliquots were deposited in glass vials and dried under a gentle
nitrogen stream. Aggregation was initiated by hydrating the peptide
with PBS premixed with the compound under study. The final
compound and peptide concentrations were 100 μM and 50 μM,
respectively. In the control samples, PBS was premixed with DMSO
volumes equivalent to those present in the compound aliquots
employed for the experiments. Aggregation was performed at room
temperature.
2
2
added to the mixture to decompose the excess of LiAlH , filtering and
4
washing the inorganic residue with Et O. The solution was dried with
2
Na SO , filtered, and evaporated to dryness, yielding an orange oil
2
4
corresponding to the title ethylamino derivative that was chromato-
graphed on SiO eluting with Et O.
2
2
These intermediates were already prepared through different
procedures; thus they were characterized as follows.
1
4
.1.4.1. Tryptamine. Yield: 81%. H NMR (200 MHz, CDCl ) δ:
3
1
2
2
7
0.11 (s, NH of indole), 7.71−7.52 (m, 2H, arom), 7.39−7.20 (m,
H, arom), 7.16−7.03 (m, 1H, arom), 4.88 (broad s, NH ), 3.09−
2
.95 (m, 4H, CH CH -NH ). Anal. Calcd for C H N : C 74.97; H
2
2
2
10 12
2
.55; N 17.48. Found: C 74.81; H 7.95; N 17.23.
.1.4.2. 2-(Benzimidazol-1-yl)ethylamine. Yield: 76%. H NMR
200 MHz, CDCl ) δ: 8.21 (s, 1H, arom), 7.65−7.49 (m, 2H, arom),
1
4
(
7
3
.35−7.21 (m, 2H, arom), 4.97 (broad s, NH ), 4.19−4.01 (m, 2H,
2
N-CH ), 3.16−3.07 (m, 2H, CH -NH ). Anal. Calcd for C H N : C
6
2
2
2
9
11
3
7.06; H 6.88; N 26.07. Found: C 66.91; H 6.79; N 26.31.
.1.4.3. 2-(Benzotriazol-1-yl)ethylamine. Yield: 76%. 1H NMR
4
(
200 MHz, CDCl ) δ: 8.11−7.71 (m, 2H, arom), 7.52−7.31 (m, 2H,
3
arom), 5.01 (broad s, NH ), 4.22 (pseudo s, 2H, N-CH ), 3.45
2
2
(
pseudo s, 2H, CH -NH ). Anal. Calcd for C H N : C 59.24; H
2 2 8 10 4
For AFM inspection, sample aliquots of 20 μL were deposited on a
freshly cleaved mica surface and incubated for 7 min. Samples were
then gently rinsed with Milli-Q water and dried overnight under mild
vacuum. Tapping mode AFM images were acquired in air using a
Dimension 3100 SPM (Bruker, Karlsruhe, Germany) equipped with
6
.21; N 34.54. Found: C 59.01; H 6.56; N 34.16.
4.2. Biological Tests. 4.2.1. Inhibition of Self-Induced Aβ
Aggregation. Aβ40 and Aβ peptides were purchased from EzBiolab,
42
Carmel, IN, USA. In vitro inhibition assay of Aβ aggregation has
4
0
41
been previously reported and adapted to a 96-well plate platform.
Briefly, samples of Aβ40 (30 μM) were coincubated for 2 h at 25 °C
with test molecules (100 μM for single point concentration assay,
seven concentrations ranging from 100 to 0.1 μM for IC50
calculations) in PBS containing 2% v/v of HFIP.
“
G” scanning head (maximum scan size 100 μm) and driven by a
Nanoscope IIIa controller (Bruker). Single-beam uncoated silicon
cantilevers (type TESPA_V2, Bruker) were used. The drive frequency
was 300−320 kHz, and the scan rate was 0.5 Hz.
4
.2.7. Primary Cultures. Cerebellar granule cells were prepared
Inhibition of Aβ42 aggregation was assayed by incubating the
peptide (30 μM) alone (as the control) or with test compounds (5 or
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from 8-day-old Sprague Dawley rats as described previously. Cells
were studied from the 6th to the 12th day in vitro. The experimental
procedures and care of the animals were performed in compliance
with the Directive of the EU Parliament and Council of September
1
00 μM) in PBS at 37 °C for 48 h.
After incubation in 96-well black, nonbinding microplates (Greiner
Bio-One GmbH, Frickenhausen, Germany), thioflavin T (25 μM) was
added and fluorimetric reads (ex 440, em 485 nm) were performed in
a multiplate reader Infinite M1000 Pro (Tecan, Cernusco sul
Naviglio, Italy). Experiments were run in triplicate. Results were
expressed by statistical analysis while IC50 values were obtained by
nonlinear regression using Prism software (GraphPad Prism version
2
2, 2010 (2010/63/EU) and were approved by the Italian Ministry of
Health (COD. 75F11.N.6DX) in accordance with D.M. 116/1992.
All efforts were made in order to minimize animal suffering and the
number of animals necessary to obtain reliable results.
4
.2.8. Cell Survival Assay. Cerebellar granule cells in 48-well plate
were treated with Aβ , aggregated in PBS for 24 h, in the absence and
4
2
5
.00 for Windows, GraphPad Software, San Diego, CA, USA).
.2.2. Inhibition of Cholinesterases. The classical Ellman’s
method, modified to a 96-well plate platform, was used as already
in the presence of each tested compound in the same conditions
described in section 4.2.6. The final compound and Aβ42
concentrations after addition to the well plate were 10 μM and 5
4
65
described. AChE from electric eel (463 U/mL), BChE from horse
serum (13 U/mL), acetyl- and butyrylthiocholine, and dithiobis(2-
nitrobenzoic acid) were purchased from Sigma-Aldrich, Milan, Italy.
Experiments were run in triplicate, and inhibition values were
obtained by nonlinear regression using Prism software.
μM, respectively. Cells were maintained in 5% CO at 37 °C. The cell
2
viability was assessed by the MTT assay 48 h after the treatment.
MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide] (Sigma-Aldrich, Germany) was added to the medium at a
concentration of 0.25 mg/mL (MTT) into each well, and the
multiwell plates were incubated for 3 h at 37 °C. After the removal of
the medium, formazan crystals were dissolved in DMSO and the
values of optical density (OD) were measured spectrophotometrically
at 570 nm using a BioTek ELx800 (Winooski, VT, USA) microplate
reader. The survival rates of viable cells were calculated by comparing
the optical absorbance of treated samples with that of the untreated
controls. All experiments were repeated at least three times
independently, and data are expressed as the mean ± SEM.
4
.2.3. Inhibition of Human Monoamine Oxidases. The
fluorimetric assays of MAO A and B following the oxidation of
kynuramine to 4-hydroxyquinoline (ex 310, em 400 nm in NaOH)
45
were performed as described. All enzymes and reagents were
purchased from Sigma-Aldrich. For inhibition kinetics, four
concentrations of 2 (ranging from 0 to 20 nM) and five
concentrations of kynuramine (from 2 to 30 μM) were used. Direct
spectrophotometric measurement of 4-hydroxyquinoline absorption
47
at 316 nm was performed as described. Inhibition values and kinetic
4
.2.9. In Vitro Blood−Brain Barrier Permeation Assay (PAMPA-
parameters were calculated by means of Prism.
BBB). Prediction of the brain permeation of compounds 2, 5, 8, 11,
and 12 was evaluated using a parallel artificial membrane permeation
4
.2.4. Inhibition of PHF6 Aggregation. The fluorimetric assay,
40,50
using thioflavin T as the chromophoric reagent, has already been
assay (PAMPA-BBB), following a well-established procedure.
45
described. Briefly, samples of PHF6 (JPT Peptide Technologies
GmbH, Berlin, Germany) (50 μM), inhibitor (10 μM), and ThT (10
μM) were prepared in triplicate in PBS containing 3.3% of 1,1,1-
trifluoroethanol and read within 3 h at 30 °C with a Tecan Infinite
M1000 Pro instrument. The plateau values (as % of residual activity
compared to control) were used for the calculation of residual
aggregation or in nonlinear regression by means of Prism to calculate
the IC50 values as the mean of two independent experiments.
Briefly, a semiautomated pipetting system (BenchSmart 96, Mettler
Toledo) and a microplate spectrophotometer (SpectraMax Plus 384
microplate reader, Molecular Devices) were employed for pipetting
and UV reading, respectively. All commercial drugs and reagents were
purchased from Sigma-Aldrich. The porcine brain lipid (PBL) was
acquired from Avanti Polar Lipids, while Millex filter units (PVDF
membrane, pore size 0.45 μm) were obtained from Millipore. For the
assay, the 96-well acceptor microplate (PTFE, Millipore) was filled
4
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ACS Chem. Neurosci. 2021, 12, 447−461