V.S. Zanon et al.
Journal of Inorganic Biochemistry 191 (2019) 183–193
administered drug candidates, states that poor absorption or permea-
tion is expected when MW > 500, HBD > 5, HBA > 10 or Clog
P > 5 [62]. For Central Nervous System (CNS) penetration, the drugs
physical properties have a smaller range than general therapeutics, CNS
penetration being likely if MW < 400, HBD ≤ 3, HBA ≤ 7 and Clog
P ≤ 5 [63]. The results indicate compounds HLa, HLd and HLe satisfy
the criteria of the Lipinski's rule of 5 for CNS.
3.11.2. Molecular dynamics simulations
After the docking studies, the best poses of each ligand were sub-
mitted to 20.0 ns of MD simulations in order to verify their dynamic
behavior and analyze the interactions observed inside the enzyme.
Therefore studies of the variation of total energy and RMSD were per-
formed. The plots of variation of total energy during the MD simula-
tions for the 5 simulated systems (data not shown) point to stabilization
after 10 ns of simulation, with an average energy around
6
−1
−
2.80 × 10 kcal·mol
for all systems, suggesting structural stabili-
3.10. Neurotoxic effects
zation. The temporal RMSD calculations were performed on all atoms of
each complex to 2500 frames at every 20 ps, during the 20.0 ns of si-
mulation. Considering that the complexes could fluctuate in the box,
each frame was adjusted by the least squares method to its previous one
for the calculation of the standard deviation. As shown in Fig. 7, the
systems achieve equilibrium around the initial 7000 ps of simulation
with maximum deviations below 0.24 nm (2.4 Å) and 0.18 nm (1.8 Å)
for the protein and ligand, respectively. These results suggest that the
ligands accommodate well inside EeAChE during the 20.0 ns of simu-
lation, showing stabilization of the system and confirming the results
obtained from the total energy calculations previously described.
Analysis of Fig. 7 shows that HLa was the ligand with the smallest
fluctuation during the simulation time, stabilizing bellow 0.05 nm
Primary mixed brain cultures were treated with different con-
H
centrations of HLa–e, tacrine and of the monomers of [Cu
2
(N Ld)
4
]Cl
4
(
which were defined based on their AChE inhibitory IC50 values). Cell
viability was assessed after 24 and 48 h. The cells remained viable after
treatment with all compounds at concentrations ranging from 0.045 to
0
.45 μM, including with the copper complex (Fig. S45, A–G), indicating
that these compounds are not neurotoxic and should be good candi-
dates as drugs in the AD treatment. Neurons and astrocytes were
identified by immunostaining using the β-Tubulin III and GFAP anti-
bodies, respectively, attesting the efficiency of the mixed brain culture
method (Fig. S46).
(
0.5 Å), in clear contrast to the other ligands. This suggests a greater
stabilization inside the enzyme and, therefore, more affinity, corro-
borating experimental data and the docking results described above.
3.11. Molecular modeling
3
.11.1. Docking studies
The best re-docking result obtained for donepezil inside HssAChE
4. Conclusions
(
shown in Fig. S47) presented a Root Mean Square Deviation (RMSD)
value for the superposition of the non-hydrogen atoms of 1.81 Å. Ac-
cording to the literature a RMSD value below 2.0 Å is considered ac-
ceptable [65], therefore these results validate the docking protocol used
for the enzyme.
The AChE inhibitory activity of a series of chelating molecules de-
1
rived from 1,2-ethanediamine, N -(7-chloro-4-quinolinyl) and salicy-
2
+
laldehydes, HLa–e, and of their novel Cu
complexes have been in-
vestigated. Analytical data, X-ray crystallography and EPR studies
Table 4 presents the energy results and the residues contributing for
the stabilization of the best poses of donepezil, ligands HLa–e, and the
established that in the solid state the complexes are dimeric cationic
H
species of formulae [Cu
2
(N La–e)
4
]Cl
4
, whereas in solution they exist
H
2+
H
+
H
complexes [Cu(N Ld)
2
]
and [Cu(N Ld)(Ld)]
,
inside EeAChE,
in the form of monomers of the type [Cu(N La–e)
2
]Cl /[Cu(La–e)
2
H
during the docking calculations (Figs. 5 and 6).
(N La–e)]Cl stabilized by solvent molecules. All ligands and the copper
complex of HLd were shown to selectively inhibit AChE in the
4.61–9.31 μM range. Results of docking and molecular dynamic studies
suggest that the ligands are able to bind inside AChE in the same way
known for AChE inhibitors, such as donepezil. Docking studies also
As can be observed, all ligands presented close negative values of
−1
energy, with the best result observed for HLa (−9.55 kcal·mol ) and
−
1
the worse for HLc (−6.67 kcal·mol ). These results fit quite well to
the experimental results shown in Table 2. In comparison with done-
pezil, all ligands presented similar energy values. This suggests that
they are also capable of binding to EeAChE. The negative values and the
large number of stabilizing residues suggest that all compounds have a
good affinity for the gorge of EeAChE. Also it is possible to see that all
ligands docked with similar orientations, in the same pocket, between
residues from the catalytic anionic site (CAS) (Glu202, Ser203 and
His447) and the peripheral anionic site (PAS) (Tyr124, Trp286) of
EeAChE, similarly to donepezil [66–68]. Particularly and in agreement
H
2+
H
+
showed that the complexes [Cu(N Ld)
2
]
and [Cu(N Ld)(Ld)] are
able to effectively bind to EeAChE. The dynamic of this binding, how-
ever, has not been investigated in this work and will be the subject of a
future study. Considering that the high serum copper levels present in
AD patients can trigger oxidative stress and cause neurotoxicity, we
2
+
have investigated the Cu
chelating potential of HLd in HEPES at
pH 6.6 and 7.4, in μM concentrations. UV–Vis spectrophotometric ti-
trations confirmed formation of the 1:2 complex. Taking into account
H
2+
2+
with the experimental results (Table 2) complexes [Cu(N Ld)
2
]
and
that neither HLd nor its Cu
complex was neurotoxic in mixed brain
H
+
[
Cu(N Ld)(Ld)] presented better energy values (more negative) than
cultures and that HLd satisfies the criteria of the Lipinski's rule of 5 for
CNS, our results suggest that the chelating compound HLd is promising
as a multifunctional drug, by inhibiting AChE and, via complexation of
all ligands, except for HLa, thus suggesting that these complexes can
effectively bind to the active site of EeAChE.
Table 4
H
]2+ and [Cu(N Ld)(Ld)]+, inside EeAChE.
H
Docking results for the best poses of donepezil, compounds HLa–e, and the complexes [Cu(N Ld)
2
Molecules
Binding energies
Interacting residues
kcal·mol−
1
)
(
Donepezil
HLa
−7.90
−9.55
−8.61
−6.67
−7.92
−7.56
−8.90
−8.80
Trp286, Leu289, Gln291, Ile294, Phe338, Tyr341
Gly120, Gly121, Tyr124, Glu202, Ser203, Ser293, Ile294, Phe295, Phe297, Tyr337, Phe338, Tyr341, His447, Gly448.
Trp86, Gly120, Gly121, Tyr124, Ser125, Ile294, Phe295, Phe338, Tyr341.
HLb
HLc
Trp86, Gky121, Tyr124, Ser203, Trp286, Leu289, Ser293, Phe297, Tyr337, Phe338, Tyr341.
Trp86, Gly120, Gly121, Tyr124, Ser203, Trp286, Phe297, Tyr337, His447.
HLd
HLe
Trp86, Gly120, Gly121, Tyr124, Ser203, Trp286, Ile294, Phe295, Phe297, Tyr337, Phe338, His447.
Trp286, Ile294, Phe295, Phe297, Phe338, Tyr341, Gly342, Tyr377.
H
H
2+
[
[
Cu(N Ld)
2
]
+
Cu(N Ld)(Ld)]
Tyr72, Thr75, Leu76, Trp286, Leu289, Glu292, Phe295, Phe295, Tyr341, Gly342.
190