P. Güller, Z. Dag˘alan, U. Güller et al.
Journal of Molecular Structure 1239 (2021) 130498
homeostasis, and have been the target of retinal pathologies, brain
oedema, glaucoma and epilepsy [14]. CAIs are known to be used in
ease, idiopathic intracranial hypertension, neurological disorders,
osteoporosis, gastric and duodenal ulcers, etc. [13, 19–21].
bined and dialyzed against the 0.05 M Tris-SO4 buffer at pH:7.4,
then they were stored at -20 °C up to use in inhibition studies.
For isolation of AChE enzyme, the pH of hemolysate (25 ml)
was adjusted to 7.8 with 0.1 M K2HPO4 solution and applied to
DE-52 anion exchange chromatography column as previously de-
scribed by Güller et al., 2020 [26]. Active eluates were combined
and dialyzed against the 20 mM KH2PO4 (pH 7.5) buffer. All these
process were carried out at about + 4°C by using ice blocks.
In order to calculate specific activity quantitative protein assay
was carried out according to the method of Bradford 1976 at 595
Following the strategy that AChEIs and CAIs are effective in the
treatment of some diseases, it was aimed to clarify the inhibition
profiles of benzylidenemalononitrile derivatives on AChE and CA
isozymes through both in vitro and in silico approaches and inves-
tigate the antibacterial properties of these derivatives.
2. Experimental
2.3.3. Inhibition assays
In order to investigate the inhibition effects of benzylidene-
malononitrile derivatives (Fig. 1), firstly, enzyme activities were as-
sayed at the various concentrations of compounds. Control enzyme
activity (in the absence of the compound) was taken as 100%. For
each derivative, an activity%- [compound] graphs were drawn us-
ing conventional polynominal regression software (Microsoft Of-
fice 2010, Excel). Benzylidenemalononitrile derivatives concentra-
tions that produced 50% inhibition (IC50) were calculated from the
equation of these graphs. Secondly, for molecules, that showed in-
hibition effects, inhibition types and constants of the breakdown
of enzyme-inhibitor complexes (Ki) were determined by drawing
Lineweaver Burk plots. For this purpose, at three different inhibitor
concentrations, enzyme activities were measured with five various
substrate concentrations.
2.1. Chemicals
ꢀ
DE-52 anion exchange gel, dialysis bag, 5,5 -dithiobis-(2-
nitrobenzoic acid) (DTNB), acetylthiocholine iodide, Sepharose-4B,
p-nitrophenyl acetate (PNF), L-tyrosine and all other chemicals
were procured from Sigma Chem. Co. and E. Merk AG. The chemi-
cals used for the synthesis of benzylidenemalononitrile derivatives
including organic solvents were purchased from Sigma-Aldrichs
and used without any purification. All test microorganisms (Staphy-
lococcus aureus, Escherichia coli and Klebsiella pneumonia) used in
the determination of antibacterial activity were isolated from clin-
ical samples from Ig˘dır State Hospital microbiology laboratory. The
expired waste human blood was obtained from the Erzurum Turk-
ish Red Crescent, Turkey.
2.2.5. In silico studies
2.2. General procedure for the synthesis of benzylidenemalononitrile
derivatives
Possible docking modes between molecules and isozymes were
studied using the AutoDock4 [28]. The crystal structures of hCA-I
(PDB code: 3LXE) [29] hCA-II (PDB code: 5AML) [30], and hAChE
(PDB code: 4EY7) [31] were used in docking calculations and their
tives were obtained by using ChemDraw software as the sdf file
and turned into pdb file by using Avogadro software.
Benzylidenemalononitrile derivatives were synthesized in previ-
ous study [22]. Benzaldehyde (0.25 mmol) and malononitrile (0.25
mmol) were added in a round bottom flask with 3 mL MeOH /
H2O (2: 1) and the mixture was magnetically stirred for 15 min-
utes at room temperature. After the completion of the reaction, the
solvent removed under reduced pressure and the product was pu-
rified by recrystallization from dichloromethane/hexanes (3:1) at
room temperature. Finally, the samples were prepared for 1H NMR
analysis. NMR spectra were recorded using a 400 MHz Bruker NMR
instrument (1H NMR at 400 MHz) in deuterated chloroform unless
stated otherwise and the Topspin (Topspin 2.1) software was used
in NMR.
After pdb files were obtained, ligand and protein were prepared
by using Autodock tool. To prepare the protein, water molecules
and other unnecessary atoms were deleted, polar H atoms were
added, missing atoms were checked, and Kollman charge was
˚
added. The grid box dimension (60 × 60 × 60 A) and grid spac-
˚
ing (0.375 A) were adjusted manually to keep the ligand flexible
around the protein active site. Grid boxes were centred as x= -
5.425, y= 30.918, z= 50.152 for hCA-I, x= -0.183, y= -3.555, z=
7.192 for hCA-II. In docking procedure of AChE, grid boxes were
centred as x= 1.165, y= -63.640, z= -24.967 for active site and x=
-2.892, y= -40.113, z= 14.866 for the allosteric site. The Lamarckian
genetic algorithm was used to determine the appropriate binding
positions, orientations, and conformations of ligands. To prepare
ligand, rotatable torsions in the ligands were generated with the
Autodock tool. Acetazolamide (AZA) was used as standard inhibitor
for hCA isozymes and tacrine was used as standard inhibitor for
hAChE. The results files were analysed using Discovery Studio Vi-
sualizer.
2.3. Biochemical studies
2.3.2. Enzyme assays
Esterase activity of CA was measured according to the method
described by Verpoorte, Mehta [23]. The carbonic anhydrases catal-
yse the hydrolysis of p-nitrophenylacetate to p-nitrophenol or p-
nitrophenylate ion which gives maximum absorption at 348 nm.
The AChE activity was assayed at 436 nm with
a spec-
trophotometer, according to Worek, Mast [24] method, a modified
method of the Ellman’s procedure.
Before molecular docking studies, the validity of the protocol
was analysed through re-docking of co-crystallized ligand into the
receptors [32]. To test the scoring power, a dataset of known in-
hibitors and non-inhibitors of enzymes was constructed, and Re-
ceiver operating characteristic (ROC) curves were drawn. Active lig-
ands and decoys were generated using the DUD-E web service for
2.2.3. Isolation of enzymes from human erythrocytes
To prepare hemolysate, erythrocyte pellets were haemolysis by
stirring with 5 volume of ice water and cell membrane debris was
removed by centrifugation at 10 000 rpm for 15 min.
For isolation of CA isozymes, the pH of hemolysate (25 ml) was
adjusted to 8.7 with solid Tris and applied to the Sepharose 4B-L-
tyrosine-sulfanylamide affinity column as previously described by
Arslan, Nalbantog˘lu [25]. CA-I and CA-II isozymes were eluted se-
quentially from the column with different elution buffer. All ex-
periments were carried out at 4°C. The active eluates were com-
2.3.1. Antibacterial assay
All benzylidenemalononitrile derivatives were dissolved in
dimethyl sulfoxide (DMSO), reported as not being antibacterial
2
Güller, P?nar
