Kinetic and conformational studies of adenosine deaminase upon interaction with oxazepam and lorazepam

Article abstract of DOI:10.2174/092986610790226076

Oxazepam and lorazepam inhibit the adenosine deaminase (ADA) differently. In the case of lorazepam temperature increment causes an increase in the inhibition potency whereas higher temperature reduces the inhibitory effect of oxazepam; which proposes the overall profounder structural changes in the case of lorazepam relative to those caused by oxazepam.

Full text of DOI:10.2174/092986610790226076

Protein & Peptide Letters, 2010, 17, 197-205
197
Kinetic and Conformational Studies of Adenosine Deaminase Upon Inter-
action with Oxazepam and Lorazepam
1
,2,*
1
3
4
A.A. Moosavi-Movahedi , H. Sepassi Tehrani , M. Amanlou , M.N. Soltani Rad ,
5
6
1
1
7
8
G.H. Hakimelahi , F.-Y. Tsai , G. Ataie , A.A. Saboury , F. Ahmad , A. Khalafi-Nezhad ,
N. Poursasan and A. Sharifizadeh
1
1
1
2
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; Foundation for Advancement of Science
3
and Technology in Iran, Tehran, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran; Department of Chemistry, Faculty of Basic Sciences, Shiraz University of Technology,
Shiraz, Iran; TaiGen Biotechnology, 7F, 138 Hsin Ming Rd., Neihu Dist., Taipei, Taiwan; Center of General Educa-
tion, Chang Gung University, 259 Wen-Hwa, Kwei-Shan, Taoyuan 333, Taiwan; Centre for Interdisciplinary Research
4
5
6
7
8
in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India; Department of Chemistry, College of Sci-
ences, Shiraz University, Shiraz, Iran
Abstract: Oxazepam and lorazepam inhibit the adenosine deaminase (ADA) differently. In the case of lorazepam tempera-
ture increment causes an increase in the inhibition potency whereas higher temperature reduces the inhibitory effect of ox-
azepam; which proposes the overall profounder structural changes in the case of lorazepam relative to those caused by oxaze-
pam.
Keywords: Adenosine deaminase, oxazepam, lorazepam, kinetics, structures.
INTRODUCTION
cerebral blood flow. Therefore, the decrease in enzyme ac-
tivity would potentiate the sedative actions of adenosine in
interneuronal communication of the central nervous system
Adenosine deaminase (ADA), (E.C. 3.5.4.4.), is an
important enzyme which is involved in the purine metabolic
pathway and in the maintenance of a competent immune
system. ADA is a monomeric protein (34.5 kDa), that cata-
lyzes the irreversible hydrolytic deamination of adenosine
and 2’-deoxyadenosine nucleosides to their respective
inosine derivatives and ammonia with a rate enhancement of
[
9].
ADA can hydrolyze the free amino substituent on the 6-
position of a variety of substituted purine nucleosides. The
enzyme hydrolytic capabilities have been exploited to con-
vert lipophilic 6-substituted purine nucleosides to products,
which show anti-HIV (human immunodeficiency virus) ac-
tivity [10, 11].
1
2
2
ꢀ10 relative to the nonenzymatic reaction [1]. Catalysis
+2
requires a Zn cofactor [2]. The enzyme is widely distrib-
uted in vertebrates, invertebrates and mammals including
humans. The enzyme is present in virtually all human tis-
sues, but the highest levels are found in the lymphoid system
such as lymph nodes, spleen, and thymus [3]. Aberrations in
the expression and function of ADA have been implicated in
several disease states such as severe combined immunodefi-
ciency (SCID), which is characterized by impaired B- and T-
cell based immunity resulting from an inherited deficiency in
ADA [4, 5 and 6]. Higher levels of ADA in the alimentary
tract and decidual cells, developing fetal-maternal interface
put ADA among those enzymes performing unique roles
related to the growth rate of cells, embryo implantation, and
other undetermined functions [7, 8]. ADA is widely distrib-
uted in the brain, and one important function of this enzyme
is probably associated with regulation of the extracellular
level of adenosine and 2’-deoxyadenosine in contact with
cerebral blood vessels. The inhibition of adenosine
deaminase in brain would allow an accumulation of adeno-
sine, which would produce vasodilation and increase in
ADA is a glycoprotein, sequenced in 1984 [12], that con-
sists of a single polypeptide chain of 311 amino acids. The
primary amino acid sequence of ADA is highly conserved
across species [13]. ADA has a (ꢀ/ꢁ) barrel structure motif
and the active site of ADA resides at the C-terminal end of
the ꢁ barrel, in a deep oblong-shaped pocket; a penta-
+2
coordinated Zn cofactor is embedded in the deepest part of
the pocket. The zinc ion is located deep within the substrate
binding cleft and coordinated in a tetrahedral geometry to
His 15, His 17, His 214, and Asp 295. A water molecule,
which shares the ligand coordination site with Asp 295, is
polarized by the metal giving rise to a hydroxylate ion that
replaces the amine at the C6 position of adenosine through a
stereo-specific addition–elimination mechanism [14, 15].
Mutation studies of amino acids in the proposed active site
near the zinc-binding site in adenosine deaminase confirmed
the essential role of these residues in catalysis [16, 17, and
1
8].
It is well cited in the literature that some drugs with neu-
roregulatory effects influence the level of adenosine
deaminase [19]. Based on such a hypothesis it was predicted
that drugs with anxiolytic, anticonsulvant and sedative char-
*
Address correspondence to this author at the Institute of Biochemistry and
Biophysics, University of Tehran, Tehran, Iran; Tel: 9821-66403957; Fax:
821-66404680; E-mail: moosavi@ibb.ut.ac.ir
9
0
929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

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98 Protein & Peptide Letters, 2010, Vol. 17, No. 2
Moosavi-Movahedi et al.
acteristics (such as the members of the benzodiazepine fam-
ily), might also be capable to regulate the levels of the en-
zyme adenosine deaminase.
trations of the two drugs. The excitation wavelength was
adjusted to 290 nm and emission spectra were recorded for
all of the samples in the range of 300 to 500 nm. Samples of
1
7
.2 ꢀM ADA were in 50 mM standard phosphate buffer, pH
.5. All spectra were normalized for protein concentration.
To investigate the structure and activity of the enzyme,
we have compared the effect of two widely used drugs, ox-
azepam and the lorazepam [Scheme 1], which differ only in
a chlorine atom on the C-ring of lorazepam.
The mixture was incubated for three minutes. The “three
minutes”, is the time required for the equilibrium between
the ligands and the protein. Within three minutes the system
was found to be stable, before that i.e. t < 3 min; the system
is far from equilibrium, and as far as the steady-state as-
sumptions are in effect (in derivation of equations), the re-
sults obtained would not be eligible. At longer times, how-
ever no significant changes in the results were observed; that
confirm the fact that 3 minutes is a reasonable time span for
the incubation of the ligands and the enzyme.
O
O
NH
NH
HO
HO
B
C
B
C
A
A
N
N
Cl
Cl
Cl
Oxazepam
Lorazepam
Circular Dichroism (CD) Measurements
Scheme 1. Two members on the benzodiazepine family of drugs;
lorazepam and oxazepam; the drugs differ only in the additional O-
chlorophenyl moiety in the lorazepam, which is absent in the case
of oxazepam (note the ring (c)).
CD spectra were recorded on a JASCO J-715 spectropo-
larimeter ™. The solutions for far-UV investigations con-
tained two constant concentrations of ligands as well as pro-
tein (0.25 mg/ml), compared to those obtained in the absence
of these ligands as control. The ligand-enzyme incubation
time was three minutes in each case. “The three minutes” is
the time at which we ascertain that the system has reached
equilibrium as mentioned earlier. The results were expressed
MATERIAL AND METHODS
Materials
2
-1
Adenosine deaminase (type IV, from calf intestinal mu-
cosa), adenosine, and the drugs oxazepam and lorazepam
were obtained from Sigma. The other related chemicals, of
the highest grade, were obtained from different industrial
sources. The solutions were prepared in doubly distilled wa-
ter.
in molar ellipticity [ꢀ] (deg cm dmol ) based on a mean
amino acid residue weight of 111 (MRW). The molar ellip-
ticity was determined as [ꢀ] = (100ꢁꢀobs/cl); where ꢀobs is the
ꢁ
observed ellipticity in degrees at a given wavelength, c is the
protein concentration in mg/ml and l is the length of the light
path in cm. The noise in the data was smoothed using the
JASCO J-715 software. This software uses the fast Fourier-
transform noise reduction routine that allows enhancement of
most noisy spectra without distorting their peak shapes. The
JASCO J-715 software was used to predict the secondary
structure of the protein according to the statistical method
[22-25].
Methods
Enzyme Assay
Enzymatic activities were assayed by UV-Vis spectro-
photometry with a Shimadzu-3100™ instrument, based on
The Kaplan Method to follow the decrease in absorbance at
Docking Simulations
2
65 nm resulting from the conversion of adenosine to inosine
[
20]. This method uses the change in extinction coefficient
Docking simulations were performed with AutoDock
-
1
-1
of adenosine (8400M cm ), on conversion to inosine by the
catalytic activity of the enzyme. The concentration of en-
zyme in the assay mixture, 50 mM sodium phosphate buffer,
pH 7.5, was 1.5 nM with a final volume of 1 ml [20, 21].
Activities were measured using at least six different concen-
trations of lorazepam and five different concentrations of
oxazepam. The ligand-enzyme incubation time was three
minutes in each case. The assays were at least in triplicate.
The adenosine concentration range used is between 0.25–2.5
Km. Care was taken to use experimental conditions where
the enzyme reaction was linear during the first minute of the
reaction. The experiments in each case were also carried out
in the absence of drugs as the control. The rates were meas-
ured, individually, at 27 °C and 37 °C.
3
.0.5 using a Lamarckian genetic algorithm (LGA) [26] dur-
ing standard flexible docking procedure. Three dimensional
coordinates of ADA structure (PDB ID: 1krm) was obtained
from RCSB. After removing co-crystalled ligand (the transi-
tion-state analogue, 6-hydroxy-1,6-dihydropurine riboside
(
HDPR)) from protein complex in the PDB file using a plain
text editor, a short minimization (100 steepest descent steps
using AMBER95 force fields with a gradient convergence
value of 0.05 kcal/mol Å) was performed using HyperChem
7
™ in order to release any internal strain. AutoDockTools
package was used to prepare docking files. The grid maps
were calculated using AutoGrid (part of AutoDock package;
version 3.0.5). A grid map of 40, 40, and 40 points in x, y,
and z directions, with grid-point spacing of 0.375 Å was
built, centered on the center of the mass of the zinc atom on
the catalytic site of the protein (1krm). 100 independent runs
with the step sizes of 0.2 Å for translations and 5° for orien-
tations and torsions were performed. The clustering tolerance
for the root-mean square positional deviation (RMSD) was
0.5 Å, and the crystallographic coordinates on the co-
crystalled ligand were used as the reference structure. De-
Fluorescence Spectroscopy
Fluorescence spectroscopy was performed using a Varian
fluorescence spectrophotometer, Carey Eclipse model 100 ™
and a 1-cm path length fluorescence cuvette; thermostated to
maintain the temperature at 27 and 37 °C. Substrate addition
followed after incubation of enzyme with different concen-

Kinetic and Conformational Studies of Adenosine Deaminase
Protein & Peptide Letters, 2010, Vol. 17, No. 2 199
fault settings were used for all other parameters. At the end
of docking, ligands with the most favorable free energy of
binding were selected as the resultant complex structures. All
calculations were carried out on PC-based machines running
Linux x86 as operating systems. The resultant structure files
were analyzed using Rasmol [27] visualization programs.
inhibitor gives a straight line with an abscissa-intercept of –
ꢀKi (Figs. (1c)), where Ki is the inhibition constant and ꢀ is
the interaction factor between the substrate and inhibitor
i
sites (K = 0.317 mM, ꢀ = 4.02).
The experiment was further conducted at the same condi-
tions of phosphate buffer 50 mM with pH=7.5 and the same
concentrations of oxazepam, but this time at 37°C as the
independent variable. The value of Michaelis constant (K’m)
is unchanged by increasing the oxazepam concentrations, but
the Vmax values are decreased Fig. (2a). This confirms the
non-competitive inhibition of adenosine deaminase by ox-
RESULTS
Kinetic Studies of Oxazepam
Fig. (1a) shows the double reciprocal Lineweaver-Burk
plot for the ADA adenosine system in which five different
concentrations of oxazepam (0.05–0.25 mM) are incubated
i
azepam (K = 0.476 mM), indicating that the inhibitor does
(
three minutes) with the enzyme-substrate complex at 27 °C.
not interfere with the binding of substrate to the active site.
The value of the inhibition constant or the Ki is determined
via secondary plot by plotting 1/Vmax app against the different
concentrations of oxazepam at 37 °C and obtaining the cor-
responding abscissa-intercept Fig. (2b).
These plots show a set of straight lines, which intersect on
the left hand side of the vertical axis, and a little further from
the horizontal axis, which confirms mixed inhibition. The
apparent maximum velocity (Vmax app) and apparent Micha-
elis constant (K’
m
) values as well as the slope values of these
/ Vmax app) can be obtained at different
Kinetic Studies of Lorazepam
straight lines (K’
m
fixed concentrations of the inhibitor as shown in Fig. (1b). A
secondary plot of the slope against the concentration of in-
hibitor gives a straight line with an abscissa-intercept of –Ki
Fig. (3) shows the double reciprocal Lineweaver-Burk
plot for the ADA adenosine system in which seven different
concentrations of lorazepam (0.05–0.35 mM) are incubated
(three minutes) with the enzyme-substrate complex at 27°C.
(
Fig. (1b)) and also another secondary plot of the reciprocal
apparent maximum velocity against the concentration of
a)
b)
c)
Figure 1. (a) The double reciprocal Lineweaver-Burk plot for the ADA adenosine system in which five different concentrations of oxazepam
0.1–0.25 mM; Absence of oxazepam (ꢁ), 0.1 mM (ꢂ), 0.15 mM ( ), 0.20 mM (ꢃ), 0.25 mM (ꢁ) ) are incubated (three minutes) with the
enzyme-substrate complex at 27 °C. (b) Secondary plot of the slope (K’ /Vmax app) against the concentration of inhibitor, which gives the -Ki
from the abscissa-intercept. (c) Secondary plot of 1/Vmax app versus concentration of inhibitor, which gives -ꢀKi from the abscissa intercept.
(
m

2
00 Protein & Peptide Letters, 2010, Vol. 17, No. 2
Moosavi-Movahedi et al.
a)
a)
b)
b)
Figure 3. (a) The double reciprocal Lineweaver-Burk plot for the
ADA adenosine system in which seven different concentrations of
lorazepam (0.05–0.35 mM; Absence of oxazepam (ꢀ), 0.05 mM
Figure 2. (a) The double reciprocal Lineweaver-Burk plot for the
ADA adenosine system in which five different concentrations of
( ), 0.1 mM (ꢀ), 0.15 mM ( ), 0.20 mM (ꢁ), 0.25 mM (ꢀ), 0.30
*
mM (+), 0.35 mM (ꢂ)) are incubated (three minutes) with the en-
zyme-substrate complex at 27°C. (b) Secondary plot of the slope
(1/Vmax app) against the concentration of inhibitor, which gives
the -Ki from the abscissa-intercept.
oxazepam (0.05–0.25 mM; Absence of oxazepam (ꢀ), 0.05 mM ( ),
*
0
.1 mM (ꢀ), 0.15 mM ( ), 0.20 mM (ꢁ), 0.25 mM (ꢀ) ) are incu-
bated (three minutes) with the enzyme-substrate complex at 37°C.
b) Secondary plot of the slope (1/Vmax app) against the concentra-
(
Structural Studies
tion of inhibitor, which gives the -Ki from the abscissa-intercept.
Fluorescence Technique
The experiment was also performed at 37 °C Fig. (4), the
techniques used were the same as those applied to the oxaze-
pam. This time at both temperatures non-competitive inhibi-
Herein, fluorescence technique is used for determination
of temperature-induced conformational changes occurring on
the ADA structure at the tertiary level. Fig. (5) shows the
fluorescence spectra of the native as well as those of other
states of enzyme structure, formed upon incubation of the
protein with three different concentrations of oxazepam at 27
and 37 °C.
i i
tion were observed (K = 0.250 mM at 27˚C and K = 0.163
mM at 37˚C). These results are summarized in Table 1.
Table 1. Kinetic Parameters and Estimated Docking Energy (kcal/mol) of Adenosine Deaminase Upon Interaction with Loraze-
pam and Oxazepam
-
1
-1
Drugs
Estimated
K
i
(mM) at 27°C
(calculated)
K
i
(mM) at 27°C
K
i
(mM) at 37°C
V
max (mM.min
at 27°C
)
V
max (mM.min
at 37°C
)
Docking energy
(kcal/mol)
Lorazepam
Oxazepam
-7.85
-7.95
0.148
0.177
0.250
0.317
0.163
0.476
0.018
0.023
0.029
0.029
ꢁ
= 4.02

Kinetic and Conformational Studies of Adenosine Deaminase
Protein & Peptide Letters, 2010, Vol. 17, No. 2 201
a)
b)
Figure 4. (a) The double reciprocal Lineweaver-Burk plot for the ADA adenosine system in which seven different concentrations of loraze-
pam (0.05–0.35 mM; Absence of oxazepam (ꢀ), 0.05 mM ( ), 0.1 mM (ꢀ), 0.15 mM ( ), 0.20 mM (ꢁ), 0.25 mM (ꢀ),
*
0
.30 mM (+), 0.35 mM (ꢂ)) are incubated (three minutes) with the enzyme-substrate complex at 37°C. (b) Secondary plot of the slope
(1/Vmax app) against the concentration of inhibitor [I], which gives the -Ki from the abscissa-intercept.
a) b)
Figure 5. (a) The fluorescence spectra of ADA (1.2 ꢃm) at pH =7.5 and T=27 °C in the presence of different fixed concentrations of oxaze-
pam (from top to bottom: 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM). All spectra are normalized for protein concentration. the mix-
ture was incubated for three minutes. (b) The spectra obtained under the identical conditions, but at 37°C.
The fluorescence technique has revealed that the increase
in the temperature causes a slight quenching due to the mi-
gration of tryptophan residues and their exposure to the
aqueous solvent. The increase of temperature show a shift of
emission intensity of about 10 nm but the fluorescence
quenching seems to be the comparable. The increase in the
concentration of the inhibitor causes a more dramatic shift
and decrease in emission intensity; which is more pro-
nounced in the case of lorazepam (Fig. (6)); in comparison to
that of the oxazepam, which is in full consistency with the
stronger potency of lorazepam as an inhibitor. Interestingly,
similarity can be seen to the findings of the circular dichro-
ism (CD) investigations.
in the case of lorazepam Fig. (7b), Table 2 with respect to
that of observed in the case of oxazepam Fig. (7a), Table 2.
DISCUSSION
Due to higher lipophilicity of lorazepam, the hydropho-
bic interactions with ADA are generally stronger at both
temperatures and lorazepam is generally a more potent in-
hibitor at both temperatures (Scheme (2) and (3)).
In addition in the case of lorazepam, the increase in tem-
perature abolishes the steric hindrance caused by the large
van der Waals radius of the O-chlorophneyl moiety, which
leads to the greater inhibitory potency and hence a reduced
Ki at 37°C. The mixed system in the case of oxazepam could
be attributed to the lower lipophilicity (higher hydrophilicity)
that can lead to a better establishment of the electrostatic
interactions between the enzyme and the oxazepam. The
increment in the temperature from 27°C to 37°C is expected
to lead in abolishment of such electrostatic interactions as a
result of which the system of inhibition would be noncom-
CD Investigations
To find out the nature of the induced structural changes
i.e. secondary or tertiary, spectra of the far UV-CD were
recorded at 27 °C for both drugs. The results confirmed a
more pronounced secondary structure conformational change

2
02 Protein & Peptide Letters, 2010, Vol. 17, No. 2
Moosavi-Movahedi et al.
a)
b)
Figure 6. (a) The fluorescence spectra of ADA (1.2 ꢀm) at pH =7.5 and T=27 °C in the presence of different fixed concentrations of loraze-
pam (from top to bottom: 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, 0.35 mM), the mixture was incubated for three min-
utes. (b) The spectra obtained under the identical conditions, but at 37 °C.
a)
b)
Figure 7. (a) The UV–CD spectra for the native structure of ADA (0.25 mg/ml) at pH =7.5 and 27 °C in the presence of a two different con-
centrations of oxazepam (from top to the bottom: The arrow shows the order of increase in oxazepam concentration ; In the absence of oxaze-
pam (A), 0.125 mM (B), 0.250 mM (C). (b), The UV–CD spectra for the native structure of ADA (0.25 mg/ml) at pH =7.5 and 27 °C in the
presence of a three different concentrations of lorazepam (from top to the bottom: In the absence of lorazepam (A), 0.125 mM (B), 0.250 mM
(C), 0.350 mM (D).
Table 2. Circular Dichroism Informations Regarding the Secondary Structure of ADA upon Incubation (3 Minutes) with Different
Concentrations of Oxazepam, and Lorazepam at 27°C
Secondary
ADA
ADA+0.125mM
[Oxazepam]
ADA+0.250mM
[Oxazepam]
ADA+0.125mM
[Lorazepam]
ADA+0.250mM
[Lorazepam]
ADA+0.350mM
[Lorazepam]
Structure at 27°C
ꢀ
ꢁ
Helix
Sheet
33.74%
12.04%
16.05%
38.17%
30.87%
13.51%
17.15%
38.46%
24.19%
18.25%
18.25%
38.27%
37.70%
7.90%
37.70%
7.90%
23.9%
0.00%
Turn
33.50%
20.90%
33.50%
20.90%
36.90%
29.90%
Random Coil
petitive. It is also well cited in the literature that the inhibi-
tion potency increases with the hydrophobicity of the ligand,
which proposes a profounder involvement of the hydropho-
bic interactions in the noncompetitive inhibition of a variety
of enzymes [28, 29].
The data of macromolecular docking have indicated that
both ligands situate themselves at the same site within the
enzymatic macromolecular structure (Scheme (4)).
The sole difference is the slight localization of the (C)
ring at the site of interaction between the enzyme and the

Kinetic and Conformational Studies of Adenosine Deaminase
Protein & Peptide Letters, 2010, Vol. 17, No. 2 203
3
00-500 nm, lorazepam seems to be more able to adapt into
the enzyme catalytic pocket. Moreover, the higher stability
of lorazepam–ADA complex may depend from larger num-
ber of H-bond formed (Scheme 3).
Scheme 4. Interaction of ADA by oxazepam (green) and lorazepam
(yellow) as revealed by the data of macromolecular docking
Taking the fact in to consideration that lorazepam is a far
more potent therapeutic than oxazepam, it exerts its intended
effect at a far lower concentration (Table 3) [30].
The potent concentration for the selective action at the
benzodiazepine receptor; is ultimately low in the steady-state
serum consideration to inhibit the ADA. In other word to
inhibit the ADA, copious concentrations of lorazepam
should be administrated which is of course toxic. On the
other hand oxazepam, in its intended use, is a far less potent
drug, allowing a much higher steady state concentration to
be achieved, considering the mentioned facts and the Ki of
both ligands which is quite similar; it seems reasonable to
propose that oxazepam is a better candidate for the therapy
in patients with elevated levels of the ADA; whereas loraze-
pam is advantageous in those who are ADA-deficient.
Scheme 2. Interaction of ADA by oxazepam (green) and lorazepam
(yellow) as revealed by the data of macromolecular docking.
As confirmed by the partition coefficient data [31], it is
evident that both drugs have near similar degrees of hydro-
phobicity; yet lorazepam is more hydrophobic and can estab-
lish a better interaction with the enzyme, which is validated
by both the kinetic and structural results. The mixed inhibi-
tion system, observed in the case of oxazepam at 27 °C con-
forms to the fact that oxazepam is more hydrophilic (has a
smaller octanol/water partition coefficient) and can establish
a minute degree of interaction with the polar residues of the
enzyme; the increase in the temperature leads to the in-
creased hydrophobicity; which in turn leads to the observa-
tion of noncompetitive inhibition in the case of oxazepam at
3
7 °C. Lorazepam however due to it's higher hydrophobicity
exhibits noncompetitive inhibition at both temperatures.
These results were confirmed by the theoretical studies con-
ducted on both drugs and are consistent to the aforemen-
tioned structural studies (Schemes 2 and 3).
Docking investigations have shown that the calculated K
i
values and the estimated docking energy are less for loraze-
pam in comparison with oxazepam (Table 1); which implies
higher inhibitory potency and tighter binding, respectively.
Scheme 3. LIGPLOT for lorazepam, depicting five hydrophobic
contacts and three hydrogen bonds.
The results are in good agreement with the experimental
data mentioned earlier at both temperatures (Table 1). Fur-
thermore the LIGPLOT schemes of the interaction between
ADA and ligands show a higher number of interactions in
the case of lorazepam in comparison with that of oxazepam
ligands. Therefore, it might be plausible to propose that the
more hydrophobic ligands would be better candidates for
ADA inhibition. From the fluorescence data in the range of

2
04 Protein & Peptide Letters, 2010, Vol. 17, No. 2
Moosavi-Movahedi et al.
Table 3. Steady State Therapeutic and Toxic Concentration for Lorazepam and Oxazepam
-
1
-1
Drug
[Therapeutic] mgL
[Toxic] mgL
[Therapeutic] ꢀM
[Toxic] ꢀM
Lorazepam
Oxazepam
0.3
1
1
5
0.93
3.48
3.11
17.4
(
5 hydrophobic bonds and 3 hydrogen bonds versus 2 and 3
impaired T-cell functions in ADA-SCID patients. Blood, 2008,
11(8), 4209-4219.
Gan, T.E.; Dadonna, P.E.; Mitchell, B.S. Genetic expression of
adenosine deaminase in human lymphoid malignancies. Blood,
1987, 69 (5), 1376-1380.
Hong, L.; Mulholland, J.; Chinsky, J.M.; Knudsen, T.B. Develop-
mental expression of adenosine deaminase during decidualization
in the rat uterus. Biol. Repord., 1991, 44(1), 83-93.
1
in the case of oxazepam)(Scheme 2 and 3). Therefore it
might further be suggested that in addition to lipophilicity;
the mode of interaction between the ligands and the enzyme
is also important.
[
7]
[8]
[9]
Oxazepam and lorazepam are effective on their target
benzodiazepine receptor at sub-micromolar concentrations,
which is caused by their structure activity relationships
Phillis, J.W.; Wu, P.H. The role of adenosine and its nucleotides in
central synaptic transmission. Prog. Neurobiol., 1981, 16(3-4),187-
2
39.
(
SAR); whereas indicated by the experimental as well as
[10]
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theoretical results, these drugs are effective on ADA at mi-
cromolar concentrations. Therefore for inhibition of ADA at
higher concentrations of these drugs should be administrated
to achieve the saturation as implicated by the Michaelis-
Menton equation.
[11]
The ligands (drugs); oxazepam and lorazepam do not
cause any adverse effect; i.e. inhibition of adenosine
deaminase (ADA) at clinical dose, however the increase of
ligand concentration caused an inhibition. Taking the non-
nucleoside structure of the ligands (oxazepam and loraze-
pam) into consideration; it might be plausible to block or
alleviate the effect of ligands on their intended benzodi-
azepine receptor and there it might provide an insight into
the design of a new class of non-nucleoside inhibitors of the
ADA with reduced central nervous system (CNS) toxicity
and higher ADA inhibitory potency.
[
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1
2101-12106.
[
Chang, Z.; Nygaard, P.; Chinualt, A.C.; Kellems, R.E. Deduced
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for catalytic function. Biochemistry, 1991, 30(8), 2275-2280.
Wilson, D.K.; Quiocho, F.A. A pre-transition-state mimic of an
enzyme: X-ray structure of adenosine deaminase with bound 1-
deazaadenosine and zinc-activated water. Biochemistry, 1993,
[14]
3
2(7), 1689-1694.
[
15]
Moosavi-Movahedi, A.A.; Safarian, S.; Hakimelahi, G.H.; Ataei,
G.; Ajloo, D.; Panjehpour, S.; Riahi, S.; Mousavi, M.F.; Mardan-
yan, S.; Soltani Rad, M.N.; Khalafi-Nezhad, A.; Sharghi, H.;
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ACKNOWLEDGEMENTS
The financial support given by the University of Tehran,
Tehran University of Medical Sciences (TUMS) and the Iran
National Science Foundation (INSF) are gratefully acknowl-
edged.
6
13-624.
[
[
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Received: December 16, 2008
Revised: January 26, 2009
Accepted: March 09, 2009