ACS Combinatorial Science
Structure−activity relationship of these compounds can be
described as two parts viz the compounds bearing 4-chloroaryl
part (series 1) and pyridyl part (series 2). Interstingly, series 1
compounds were significantly active toward MAO-B inhibition
compared to series 2 compounds. However, only few derivaties
of both series showed moderate inhibitions toward MAO-A and
AChE. None of the compounds of series 2 were active toward
AChE inhibition. Considering MAO-B, the series 1 compounds,
ABH1−ABH3, ABH5, and ABH6, with the benzylidene moiety
bearing the substituents such as -NMe , -F, -Cl, benzyloxy, and
2
thiophenyl, respectively, showed effective inhibitory activities,
whereas the series 2 compounds (ABH7, ABH9, and ABH12−
ABH14) showed weak inhibition. Among the substituents
studied, benzyloxy group (ABH5) showed the greatest
inhibitory effect. Therefore, 4-chloroaryl part (series 1) can be
considered as a good pharmacophore unit for MAO-B inhibition
rather than pyridyl part. Considering MAO-A, the only
compound ABH5 of series 1 showed effective inhibitory effect,
suggesting that the benzyloxy substituent effectively interacts
with MAO-A. However, ABH8, ABH10, and ABH11 of series 2
showed moderate inhibitory activities, also suggesting that
pyridyl part can be an effective pharmacophore unit for MAO-A
inhibition. In contrast, none of all the compounds were not
effective toward AChE inhibition, except ABH5 and ABH3.
Therefore, no substituents and parts might effectively intereact
with AChE.
Kinetics experiments were conducted at five substrate
concentrations and inhibition studies were at three inhibitor
concentrations, that, ∼ 1/2 × IC , IC , and 2 × IC . K value
50
50
50
i
and inhibitor type were determined by using Lineweaver−Burk
22
(
LB) plot and secondary plot. Kinetics studies of ABH5 were
performed on MAO-A and MAO-B inhibitions. LB plots and
secondary plots showed that ABH5 was a competitive inhibitor
Figure 3. Recovery of MAO-A (A) and MAO-B (B) inhibitions by
ABH5, using dialysis experiments.
for MAO-A and MAO-B (Figure 2A and C), with K values of
i
0
.96 ± 0.19 and 0.024 ± 0.0077 μM, respectively (Figure 2B and
4EY7, respectively. With the purpose of inspecting their
24−26
D). These results suggest that ABH5 binds to the active site of
free enzyme by competing with the substrate and is potent,
selective, and competitive inhibitor for MAO-A and MAO-B.2
multitarget activity,
the enzymes were processed using
dinger
this step allows to refine and optimize the crystal
the protein preparation wizard available from the Schro
̈
27,28
suite:
3
The reversibility study was determined by dialysis method.
structures, correcting the protonation states and carrying out
energy minimization. In this respect, nine water molecules
within MAO-A and eight water molecules within MAO-B were
Dialysis experiments were performed by reacting MAO-B and
the inhibitor or reference inhibitors at approximately twice of
the IC50 in 0.1 M sodium phosphate buffer for 30 min and
dialyzed for 6 h with a buffer change. Residual activities were
calculated for undialyzed (A ) and dialyzed (A ) experiments
compared to controls (i.e., without inhibitor). Reversibility
studies on MAO-A and MAO-B inhibitions were conducted for
ABH5, the most potent compound. The inhibition of MAO-A
by ABH5 was recovered from 38.4 (value of A ) to 85.9% (value
of A ). The recovery value was similar to that of the reversible
reference toloxatone, from 38.0 to 83.7% and higher than that of
the irreversible reference clorgyline (recovery from 29.2 to
3
29
kept and not deleted. Next, the ligand structures to be docked
30
were prepared using the LigPrep tool that allows generating
the ionization state at physiological pH as well as all the possible
tautomers. The obtained files were, thus, used for docking
simulations by employing Grid-based Ligand Docking with
U
D
31
Energetics (GLIDE). The enclosing box was centered on the
cognate ligand center of mass of the three PDB structures, with
an edge of 10 Å × 10 Å × 10 Å and 28 Å × 28 Å × 28 Å for the
inner and outer boxes, respectively. The default force Field
U
D
32
OPLS_2005 and standard Precision (SP) docking protocol
with default settings were employed for docking simulations. To
corroborate the validity of docking studies, redocking
simulations were performed on the cognate ligands in their
binding sites. Cognate ligands HRM, SAG, and Donepezil for
MAO-A, MAO-B, and AChE, respectively, moved back to the
original positions with root mean square deviations (RMSD)
accounting for all the heavy atoms equal to 0.762, 0.390, and
2.0%). For MAO-B, inhibition by ABH5 was recovered from
5.6 to 80.0%. The recovery value was similar to that of the
3
reversible reference lazabemide, from 27.4 to 86.9% and higher
than that of the irreversible reference pargyline (recovery from
2
4.8 to 35.1%). These experiments showed that inhibitions of
MAO-A and MAO-B by ABH5 were recovered to the reversible
reference levels, suggesting the compound played as a reversible
inhibitor (Figure 3).
Structure-based studies were employed to analyze the binding
modes of ABH2, ABH3, and ABH5 toward MAO-A, MAO-B,
and AChE, whose X-ray solved structures were taken from the
Protein Data Bank (PDB) with the entries 2Z5X, 2V5Z, and
Docking scores for ABH2, ABH3, and ABH5 over the three
biological targets MAO-A, MAO-B, and AChE are provided in
Table 3. The values of ABH5 were the best for the three
enzymes.
D
ACS Comb. Sci. XXXX, XXX, XXX−XXX
Palakkathondi, Ashique
