Design, synthesis and biological evaluation of novel nitroaromatic compounds as potent glutathione reductase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.07.002

Discovery of GR inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, the synthesis and GR inhibitory capacities of novel nitroaromatic compounds (NCs) (1-3) were reported. Some commercially available mo

Full text of DOI:10.1016/j.bmcl.2011.07.002

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5398–5402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel nitroaromatic
compounds as potent glutathione reductase inhibitors
Reßsit Çakmak a,b, Serdar Durdagi c, Deniz Ekinci d, , Murat Sßentürk e, Giray Topal b,f,
⇑
⇑
a Batman University, Science and Art Faculty, Chemistry Department, Batman, Turkey
b Dicle University, Ziya Gökalp Education Faculty, Chemistry Department, Diyarbakır, Turkey
c Institute for Biocomplexity and Informatics, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
d Ondokuz Mayıs University, Agriculture Faculty, Agricultural Biotechnology Department, Samsun, Turkey
e
_
Ag˘rı Ibrahim Çeçen University, Science and Art Faculty, Chemistry Department, Ag˘rı, Turkey
f Batman University, Technical Education Faculty, Batman, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Discovery of GR inhibitors has become very popular recently due to antimalarial and anticancer activities.
In this study, the synthesis and GR inhibitory capacities of novel nitroaromatic compounds (NCs) (1–3)
were reported. Some commercially available molecules were also tested for comparison reasons. The
novel NCs were obtained in high yields using simple chemical procedures and exhibited much potent
inhibitory activities against GR at low micromolar concentrations with Ki values ranging from 0.211 to
Received 20 May 2011
Revised 17 June 2011
Accepted 4 July 2011
Available online 13 July 2011
4.57 lM as compared with well-known agents. Inhibition mechanism was assessed as being due to occlu-
Keywords:
Glutathione reductase
Antimalaria
Nitroaromatic
In silico docking
sion of the active site entrance by means of the NCs. Molecular docking results have shown that docking
poses of ligands are able to construct binding interactions with the essential amino acids.
Ó 2011 Elsevier Ltd. All rights reserved.
Glutathione (g-
L
-glutamyl-
L
-cysteinylglycine; GSH) has many
sensitivity.6 Indeed, GSH is known to protect the parasite from oxi-
dative damage and recently, GSH was shown to be involved in the
Fenton-based degradation of toxic heme by continuously recycling
the PFIX(Fe3+) into PFIX(Fe2+) in the malaria parasite.7 GSH is found
almost exclusively in its reduced form and high intracellular GSH
levels depend on the efficient reduction of glutathione disulfide
(GSSG) by glutathione disulfide reductase (GR). Thus, GR inhibitors
were developed to fight for malaria.8–10
Considering above stated information, synthesis and discovery
of novel glutathione reductase inhibitors are of particular interest
due to their possible antimalarial capacities. Therefore, in this
study, we analyzed the GR inhibitory capacities of several mole-
cules, mostly nitroaromatic compounds (NCs), three of which have
been newly synthesized by our group (i.e., compounds 1–3, see
Fig. 1).
The purification of the erythrocyte GR was performed by the
simple two step method by means of 20,50-ADP Sepharose-4B affin-
ity gel chromatography and Sephadex G-200 gel filtration chroma-
tography. The enzyme was purified 2633-fold with a specific
activity of 19.72 EUmgꢀ1 and overall yield of 32%.
First we tested simple nitrobenzene (NB) on the enzyme activ-
ity and compared the inhibition data with those of the newly syn-
thesized derivatives. The simple nitrobenzene showed rather weak
important functions. For example, it is an antioxidant, involved in
the detoxification of xenobiotics and serves as a cofactor in isomer-
ization reactions.1 Glutathione has an important role in the synthe-
sis and degradation of proteins, regulation of enzymes, formation of
the deoxyribonucleotid precursors of deoxyribonucleic acid (DNA)
and protection of cells against free radicals and reactive oxygen
species.2 Glutathione reductase (GR) catalyzes the reduction of glu-
tathione disulfide (GSSG) at the expense of NADPH. By maintaining
a high ratio of [GSH]/[GSSG], the enzyme enables several vital func-
tions of the cell such as the detoxification of reactive oxygen species
as well as protein and DNA biosynthesis.3
Malaria is one of the most common infectious diseases and an
enormous public-health problem. It is an important cause of death
and illness in tropical and subtropical countries. It causes 600 mil-
lion clinical cases and one to three million deaths each year, mostly
among young children.4 Mortality has risen in recent years due to
the increase of resistance to antimalarial treatments.5 It was ob-
served that an elevation of the glutathione (GSH) content in Plasmo-
dium falciparum leads to an increased resistance to antimalarial
agents, while GSH depletion in resistant strains restores the
⇑
Corresponding authors. Tel.: +90 362 312 1919; fax: +90 362 357 6034 (D.E.);
inhibitor capacity with the Ki value of 274.1
novel NCS (1–3). Compound 3 behaved as the strongest inhibitor
with Ki value of 0.211 M. The compounds 1, 2 exhibited similar
lM as compared with
tel.: +90 412 248 80 36; fax: +90 412 248 82 57 (G.T.).
E-mail addresses: dekinci80@gmail.com (D. Ekinci), gtopal@dicle.edu.tr (G.
Topal).
l
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.002

R. Çakmak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5398–5402
5399
HO
O
H
N
O
NO2
O
O
NH
NO2
O
HN
OH
H
NO2
Cl
O
2
NB
1
OH
HOOC
O
O
NO2
CO2Me
NH2
NH
NH2
MeO2C
NO2
O
HOOC
N
H
HOOC
H2N
3
5
4
6
Figure 1. Structures of the tested compounds.
inhibitions with Ki values of 4.16 and 4.57
l
M, respectively (Table
Glutathione reductase is a dimeric FAD-containing enzyme with
a redox-active disulfide at its active site. The two cysteine residues
are designated as the distal cysteine, which can interact with FAD
domain, and proximal cysteine, which does not. GR catalyzes the
reduction of oxidized glutathione (GSSG) to reduced glutathione
(GSH) at the expense of NADPH.13 GR inhibitors, such as isot-
hiazol-3-one, can disrupt the GSH-GSSG balance and cellular
reduction potentials.14
1). Thus, the nature of the groups in para- to the cyclic NO2 moiety
strongly influences GR inhibitory activity (Fig. 1). The weakest
inhibitors were compounds 4, 5 and 6. We tested 4 and 5 just for
comparison reasons as they have very similar structures to the
groups in para- to the cyclic NO2 moiety in 3. The only difference
between 3 and 4 is the nitroaromatic scaffold in compound 3.
However, compound 3 has approximately 150 times higher inhibi-
tion effect than 4. Compound 6 has much lower action among NCs
although it has nitroaromatic moiety, probably due to the steric
hindrance (see Supplementary data, Fig. S1). It is important to note
that newly synthesized compounds have much stronger inhibitory
effect than widely used agents against protozoan infections.11
Kinetic investigations (Lineweaver Burk plots, data not shown)
indicate that only compound 1 acted as noncompetitive inhibitor
while other derivatives showed competitive inhibition (Table 1).
These results suggest that protein sulphydryl groups are the target
for inhibition by the large organic nitrate derivative.
Interactions of various chemicals with specific enzymes are
very useful for medicinal chemists and pharmacologists. A promis-
ing approach to develop new drugs against parasitic diseases is to
target enzymes of a unique metabolic pathway that are present
exclusively in the pathogens. In trypanosomatids, the nearly ubiq-
uitous glutathione redox system is replaced by the trypanothione
system.12,13 Whereas the glutathione system, composed of gluta-
thione and the flavoenzyme glutathione reductase (GR, EC
1.6.4.2), is present in nearly all eukaryotes and prokaryotes, try-
panosomatids possess trypanothione ([N1,N8-bis (glutathionyl)
spermidine], T(SH)2) as the main lowmolecular-mass thiol that is
kept in its reduced state by trypanothione reductase (TR, EC
1.6.4.8).12 This specific trypanothione system protects the parasites
from oxidative damage. With TR as this pathway’s key enzyme
having been shown to be essential, it is a promising target for
the development of new anti-trypanosomal drugs.
Grellier et al.15 showed the antiplasmodial activity of a series of
homologous NCs, which were either strong or weak inhibitors of
erythrocyte GR. They selected the derivatives of 2-(59-nitrofurylvi-
nyl)-quinoline-4-carbonic acid, which possess a broad spectrum of
bactericidal and antiparasitic activities, and inhibit yeast GR16 and
Trypanosoma congolense trypanothione reductase.16 It was found
that compounds were uncompetitive inhibitors toward both GR
substrates, NADPH and GSSG; that is, they decreased enzyme kcat
and did not affect the steady-state bimolecular rate constants of
substrate reaction with GR (kcat/Km). The character of inhibition
is consistent with the binding of electron-deficient aromatic com-
pounds at the interface of two subunits of GR, at the vicinity of his-
tidines-75,759 and phenylalanines-78,789.17
Schistosomiasis is one of the most important human parasitic
infections in the world, affecting over 200 million people in devel-
oping countries with 280,000 deaths/year in sub-Saharan Africa
alone.18 The therapy for schistosomiasis is based on a single drug
that is administered to millions of people yearly. A reliable alterna-
tive does not exist at the moment. A treatment strategy that relies
on just a single drug is at risk given the high probability that drug-
resistant parasites will emerge. Recently, a possible solution to this
urgent problem has been proposed, that is, targeting the antioxi-
dant pathway of the parasite. In platyhelminths, like Schistosoma
and Echinococcus, thiol redox homeostasis is completely depen-
dent on the enzyme thioredoxin-glutathione reductase (TGR),18
which directs the NADPH reducing equivalents to both GSH and
Table 1
IC50 and Ki values obtained from regression analysis graphs for GR in the presence of different inhibitor concentrations
Compound
IC50 value (lM)
Ki value (lM)
Inhibition type
Nitrobenzene (NB)
546.8
15.4
13.3
0.344
53.7
61.2
23.4
274.1
4.16
4.57
0.211
42.7
54.6
17.4
Competitive
Noncompetitive
Competitive
Competitive
Competitive
Competitive
Competitive
N
a-Boc-Ne-2-chloro benzyloxycarbonyl-L-lysine-p-nitrophenylester (1)
N-p-Nitrobenzyl-(R)-2-amino-1-buthanol (2)
N-p-Nitrobenzoyl-
Glutamic acid (4)
Lysine (5)
L-glutamic acid (3)
Nifedipine (6)

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R. Çakmak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5398–5402
Table 2
Molecular docking binding scores and binding interactions of compounds 1–6 within the hGR active site
Ligand No.
Glide/IFD binding score (kcal/mol)
H-bonds
VDW Close contacts
1
2
3
4
5
6
ꢀ7.18
ꢀ6.32
ꢀ9.06
ꢀ6.64
ꢀ6.60
ꢀ5.82
I198, H2O-569, Y197, K66,
L337, G381, T379
I289, K66, I198, Y197, A199, C63, G290, H2O-494
C63, T339, D331, L337, T57, S30, H2O-496, H2O-982
H2O-496, H2O-982, D331, C58, L337, S30, T339
K66, H2O-501, H2O-500, L338,
Y197, G196, I367, C63, T57, T369, L338, L337, I289
F226, I367, A336, G381, L338
A288, G62, C63, I289, A288, Y197, R291, K66
L338, C63, L337, D331, T339
D331, T57, C63, L337, L338, C58
K66, L338, Y197, R291, G62, I198
Residues/water molecules participating in hydrogen bonds and close van der Waals contacts (<4 Å) with the inhibitors are shown.
thioredoxin.16 On the other hand, in humans and other vertebrates
the same function is fulfilled by two distinct enzymes that initiate
two parallel pathways, namely thioredoxin reductase (TR) and glu-
tathione reductase (GR).
discovered a GR inhibition mechanism for the simple semicarba-
zone derivatives. Thus, NCs, with their NO2 group and through
the polar moieties found in pendant tails attached to them, may
interact with the entrance of the GR active site in a manner which
may resemble the binding of semicarbazone. The xanthene and
semicarbazone binding sites to GR are the same, as determined
by X-ray crystallography. Indeed, these two classes of compounds
bind at the entrance of the active site and interact with a large
number of amino acid residues both from the hydrophilic and from
the hydrophobic halves of it.
As we were unable for the moment to crystallize a GR in com-
plex with one of the NCs, we performed in silico docking studies
in order to investigate the interactions between NCs and amino
acid residues within the GR active site. To this end, fully flexible
docking methodology for both receptor residues (1GRA pdb file
was used for the target structure) and docked ligands was used
by Glide XP (extra precision)—Induced Fit Docking (IFD) algorithm
which was implemented with the Prime module under Schroding-
er molecular modeling package.19
NCs are relatively rare in nature and have been introduced into
the environment mainly by human activities. This important class
of industrial chemicals is widely used in the synthesis of many di-
verse products, including dyes, polymers, pesticides, and explo-
sives. The nitro group, which provides chemical and functional
diversity in these molecules, contributes to the recalcitrance of
these compounds to biodegradation. The electron-withdrawing
nature of the nitro group, in concert with the stability of the
benzene ring, makes NCs resistant to oxidative degradation. Recal-
citrance is further compounded by their acute toxicity, mutagenic-
ity, and easy reduction into carcinogenic aromatic amines. Some
NCs have also been shown to be drug candidates for example in
hypoxia.18
It is apparent that NCs have many important roles. In addition,
GR inhibitors have gained great attention due to their impacts on
malaria and similar infectious diseases. Thus, we synthesized novel
NCs and measured their potencies at GR. In addition, we compared
and discussed the novel NCs GR inhibitory propensities with the
simple precursor and commercially available compounds.
NCs (1–3, 6) contain amino, acid, methoxy and alcohol moieties,
typically found in GRIs. Nevertheless, recently, new studies have
The compounds 1–6 were docked at the binding site of the
target (hGR). Glide/IFD docking scores of docked inhibitors at
hGR targets and corresponding binding interactions were shown
in Table 2. We have performed docking studies of compounds
1–6 within the hGR active sites, by using as templates hGR adducts
for which the X-ray crystal structures have been reported in
Figure 2. Superimposition of substrate (atoms are represented with sticks) in the crystal structure of hGR (pdb code: 1GRA) and the top docking pose of compound 3 (atoms
are represented with CPK format).

R. Çakmak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5398–5402
5401
complexes with inhibitors (the hGR-xanthene adduct). The results
of the docking experiments are presented in Table 2 for hGR, and
are shown in some details in Figures 2 and 3. Relatively a high cor-
relation between experimental and calculated binding energies is
found (r2 = 0.69; predicted Ki values were calculated using the
the GSSG binding site and the NADPH binding sites in the hGR cav-
ity. As reported in the literature, hGR and TX enzymes are succep-
tible to NO2 containing molecules.15-17 In the current study, we
aimed to asses the effects of natural compound analogues and
nitrobenzene containing molecules, which are also used as drugs,
on human GR enzyme in addition to the amino acids constituting
the skeleton of these compounds. Therefore, the reported data in
this study may assist to design new compounds with enhanced
affinity and eventually more selectivity for hGR or TX, since the re-
gions towards the exit of the cavity are those with the highest var-
iability in amino acid residues.17–22
Consequently, NCS used in this study affect the activity of hu-
man erythrocyte GR due to the presence of the different functional
groups present in their aromatic scaffold. Our findings here indi-
cate thus another class of possible GR of interest, in addition to
the well-known chloroquine and aminoquinoline derivatives bear-
ing bulky in their molecules. Indeed, new nitroaromatics investi-
gated here showed very effective GR inhibitory activity compared
to well known agents. These findings point out that novel substi-
tuted nitroaromatics may be used as leads for generating potent
GR inhibitors. Thus, this approach may also be useful in the design
D
G = RTlnKi equation, (T = 300 K)), see Supplementary data, Figure
S1.
As seen from data of Table 2 and Figures 2 and 3, there are many
amino acid residues/water molecules participating in hydrogen
bonds and close van der Waals, <4 Å contacts with the inhibitors
1–6 when bound to the enzyme active sites, for hGR. Most of these
amino acids include Tyr197 (one of the gate keeper residues of this
enzyme).12 We thus concentrated on one derivative, the strongest
inhibitor, compound 3, for which we generated detailed binding
modes, based on the docking results (Figs. 2 and 3). Indeed, as seen
from Figures 2 and 3, the binding of the compound 3 within the
hGR active site is done without interaction with the NADPH bind-
ing side, by means of an extended network of hydrogen bonds and
van der Waals interactions involving Ile 289, Lsy 66, Ile 198, Tyr
197, Ala 199, Cys 63, Gly 290, water 494. Thus, the new inhibitors
reported here exploit binding pockets which are found between
Figure 3. A. Top docking pose of compound 3 at the active site of the receptor. B. Binding interactions were detailed (for clarity only hydrogen bonds were shown in the
figure). For details of the interaction see also Table 2.

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R. Çakmak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5398–5402
manual): (i) Constrained minimization of the receptor with an RMSD cutoff of
0.18 Å. (ii) Initial Glide docking of each ligand using a soft potentials (0.8 van
der Waals radii scaling of non-polar atoms of ligands and receptor using partial
charge cutoff of 0.15). (iii) Refinement of the docking poses was done using the
Prime module of Schrodinger. Residues within 5.0 Å of ligand poses were
minimized in order to form suitable conformations of poses at the active site of
the receptor. (iv) Glide redocking of each protein–ligand complexes.
and exploitation of novel antimalaria and anticancer candidates.
However, these features of the compounds are beyond the scope
of this study and merits further investigations.
Acknowledgment
20. (a) Carlberg, I.; Mannervik, B. J. Biol. Chem. 1975, 250, 5475; (b) Bradford, M. M.
Anal. Biochem. 1976, 72, 248; (c) Laemmli, U. K. Nature 1970, 227, 680; (d)
Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 57, 685.
The authors greatly acknowledge the Scientific and Technical
Research Council of Turkey (TUBITAK) for financial support (Project
No: 110T507) for (G.T. and R.C.).
21. Detailed synthetic procedures for the preparation of all derivatives 1–3 can be
found in: Synthesis of
nitrophenylester (1): 3.15 g (7.6 mmol) N
nyl- -lysine and 1.35 (9.7 mmol) g p-nitrophenol were dissolved in 40 mL
Na
-Boc-N
e
-2-chloro benzyloxycarbonyl-
L-lysine-p-
a
-Boc-N -2-chloro benzyloxycarbo
e
L
References and notes
ethylacetate and 1.9 (9.3 mmol) g Dicyclohexylcarbodiimide (DCC) was added
to this solution at 0 °C. After 5 h, the occured dicyclohexylure was filtered and
the solvent was evaporated. The remaining crude product was dissolved in
methylene chloride and extracted with 0.5 M potassium carbonate at 0 °C. The
organic phase was washed with 0.1 M HCl and water then, dried over
anhydrous sodium sulfate. The solution was filtered and evaporated. The
crude product was crystallized from ethanol–water mixture (1:1). (Light grey
crystal, 5.5 g, 90%), 1H NMR (CDCl3) d: 1.37–1.45 (m, 15H), 3.03 (br s, 2H), 4.04–
4.16 (m, 1H), 5.09 (s, 2H), 7.36–7.57 (m, 8H), 8.31 (d, 1H). One of the NH
protons was not observed. 13C NMR (CDl3) d: 24.6, 28.5, 29.3, 32.2, 54.4, 55.3,
63.0, 79.0, 116.2, 123.3, 125.8, 127.7, 129.6, 130.0, 130.1, 132.7, 135.0, 145.5,
155.8, 156.2, 171.5. Anal Calcd for C25H30N3O8Cl (521.16): C, 55.23; H, 5.41; Cl,
6.79; N, 8.05; O, 24.52. Found: C, 55.47; H, 5.23; Cl, 6.41; N, 7.98; O, 24.38. mp:
92 °C.
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Synthesis of N-p-nitrobenzyl-(R)-2-amino-1-buthanol (2): 4.46 g (50 mmol)
(R)-2-amino-1-buthanol, 1.3 (12.3 mmol)
g sodium carbonate and 2,7 g
(12.5 mmol) p-nitrobenzylbromide were dissolved in 100 mL benzene and
taken into 250 mL three necked round bottom flask equipped with dean stark
apparatus. This solution was refluxed stiringly for 12 h under nitrogen
atmosphere in oil bath at 110 °C. Then, the solution was filtered and the
solvent was evaporated. The remaining crude product was crystallized from
benzene–ethanol mixture (1:1) and washed with dry diethyl ether. (White
crystal, 1.82 g, 70%), 1H NMR (CDCl3) d: 0.95 (t, 3H), 1.44–1.62 (m, 2H), 1.98 (br
s, 1H), 2.61–2.65 (m, 1H), 3.37–3.70 (m, 2H), 3.94 (q, 2H), 7.53 (d, 2H), 8.19 (d,
2H) 13C NMR (CDl3) d: 10.35, 24.22, 50.30, 59.98, 62.81, 123.70, 128.65, 147.08,
148.22. Anal Calcd for C11H16N2O3 (224.26): C, 58.91; H, 7.19; N, 12.49; O,
21.40. Found: C, 58.77; H, 7.25; N, 12.68; O, 21.28. mp: 113 °C.
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glutamic acid was dissolved in 15 mL 1 N NaOH. Then 1.5 (18.3 mmol) g
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added into this solution, respectively. The purple solution was stirred for 5 min
and filtered. Then, 3 N HCI was added dropwise until the filtrate become
yellow. The solution was continued to be acidified with dilute hydrochloric
acid by controlling pH. A white-yellow precipitate occurred. The mixture was
kept for 1.5 h at room temperature and filtered several times in vacuum. The
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19. Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R., Farid, R. J. Med. Chem. 2006,
49, 534. In Silico docking studies: Crystal structures of GRs in complex with
inhibitors (1GRA pdb file) was used for the molecular docking calculations of
compounds 1-6 within the active sites of the hGR. Explicit water molecules
from the X-ray structures were kept for all the calculations. Before the docking
simulations, the target structure was submitted to the protein preparation
module of the Schrodinger molecular modeling package.19 Compounds 1–6
were constructed using the Schrodinger’s Maestro module and then geometry
optimization was performed for these ligands using Polak–Ribiere conjugate
glutamic acid was obtained. White product was filtered and crystalized from
ethanol. (0.94 g, 64%), 1H NMR (CDCl3) d: 1.93–2.16 (m, 2H), 2.36–2.50 (m, 2H),
4.40–4.46 (m, 1H), 8.12 (d, 2H), 8.33 (d, 2H), 9.02 (d, 1H), 12.35 (br s, 2H) 13C
NMR (CDl3) d: 26.3, 30.9, 52.7, 124.0, 129.5, 140.0, 149.6, 165.5, 173.5, 174.2.
Anal Calcd for C12H12N2O7 (296.23): C, 48.65; H, 4.08; N, 9.46; O, 37.81. Found:
C, 48.83; H, 4.21; N, 9.58; O, 37.78. mp: 192 °C.
22. In vitro inhibition studies: In order to determine the effects of the nitro
compounds on human GR, compounds 1–6 were added into the reaction
medium. The enzyme activity was measured and an experiment in the absence
of inhibitor was used as control (100% activity). The IC50 values were obtained
from activity (%) versus inhibitor concentration plots. To determine Ki
constants in the media with or without inhibitor, the substrate (GSSG)
concentrations were 0.015, 0.04, 0.07, 0.10, and 0.15 mM. Inhibitor solutions
gradient (PRCG) minimization (0.0001 kJ Åꢀ1 molꢀ1
, convergence criteria).
Protonation states of ligands and residues were tested using LigPrep and
Protein Preparation modules under Schrodinger package19 at neutral pH
(experimentally the compounds have been tested at pH of 7.4). The Glide-XP
(extra precision) (v. 5.0) combined with Induced Fit Docking (IFD) have been
used for the docking calculations. IFD uses the Glide docking program to
account the ligand flexibility and the refinement module, and the Prime
algorithm to account for flexibility of the receptor.19 Schrodinger’s IFD protocol
model uses the following steps (the description below is from the IFD user
were added to the reaction medium, resulting in
3 different fixed
concentrations of inhibitors in 1 ml of total reaction volume. Lineweaver–
Burk graphs were drawn by using 1/V versus 1/[S] values and Ki constant were
calculated from these graphs.20 Regression analysis graphs were drawn using
inhibition% values by a statistical package (SPSS-for windows; version 10.0) on
a computer (student t-test; n = 3).20