Cysteine based novel noncompetitive inhibitors of urease(s)-Distinctive inhibition susceptibility of microbial and plant ureases

Article abstract of DOI:10.1016/j.bmc.2006.05.078

Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver-Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum K_i value of 2 μM for N,N-dimethyl l-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.

Full text of DOI:10.1016/j.bmc.2006.05.078

Bioorganic & Medicinal Chemistry 14 (2006) 6737–6744
Cysteine based novel noncompetitive inhibitors of urease(s)—
Distinctive inhibition susceptibility of microbial and plant ureases
a, ,ꢀ,ꢁ
b,ꢁ
c
d
Zareen Amtul, * Naheed Kausar, Cristian Follmer, Richard F. Rozmahel,
a
b
e
Atta-Ur-Rahman, Syed Arif Kazmi, Mohammed Saleh Shekhani,
Jason L. Eriksen, Khalid M. Khan and Mohammad Iqbal Choudhary
f
a
a,
*
a
International Center for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
b
Department of Chemistry, University of Karachi, Karachi 75270, Pakistan
Department of Physical Chemistry, Institute of Chemistry, Universidade Federal do Rio de Janeiro, 21941-590, Brazil
c
d
London Regional Cancer Centre, University of Western Ontario, London, Ont., Canada N6A 5B8
e
Invitrogen Drug Discovery Solution, 501 Charmany Drive, Madison, WI 53719, USA
f
Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA
Received 18 April 2006; revised 26 May 2006; accepted 26 May 2006
Available online 21 July 2006
Abstract—Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and
synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis,
PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease
activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very
weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives
are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Linewe-
aver–Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum K value
i
of 2 lM for N,N-dimethyl L-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial
ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many path-
ological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
clear Ni active site with a modified amino acid side
chain-containing a carbamylated lysine residue that
5
–7
Ureases (urea amidohydrolase; EC 3.5.1.5) are nickel-
dependent enzymes that catalyze the hydrolysis of urea
to form ammonia and carbon dioxide. Ureases have
been isolated from a wide variety of organisms, includ-
ing plants, fungi, and bacteria.
of the enzymes from Klebsiella aerogenes, Bacillus
pasteurii, and Helicobacter pylori have revealed a dinu-
bridges the deeply buried metal atoms. Although only
bacterial ureases have had their 3D crystallographic
structure successfully resolved, the high sequence simi-
larity of all ureases indicates they are variants of the
same ancestral protein, and are consequently likely to
possess similar tertiary structures and catalytic
1
,2
3
,4
Structural studies
8
mechanisms.
The primary physiological role of urease is to allow the
organism to use externally and internally generated urea
as a nitrogen source (for reviews, see 4 and 9). Urease
has also been described as defense protein against preda-
Keywords: Bacillus pasteurii urease; Jack bean urease; Noncompetitive
inhibitor; Cysteine derivatives; Enzyme kinetics.
*
Corresponding authors. Fax: +92 21 4819018 19; e-mail addresses:
zareen_amtul@fulbrightweb.org; hej@cyber.net.pk
Present address: Department of Biochemistry, University of Western
Ontario, Canada.
3
,10,11
tors and phytopathogenic organisms.
Urease is
ꢀ
known to be a major cause of pathologies induced by
H. pylori, which allows the bacteria to survive at the
low pH of the stomach during colonization and, there-
ꢁ
These authors contributed equally to this work.
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.078

6738
Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
fore, plays an important role in the pathogenesis of gas-
8
tric and peptic ulcers (including cancer). In infections
teine methyl ester (COOCH ; K = 0.12 mM) which is
3 i
more potent than cysteine (COOH; K = 95 mM). A
i
with Proteus mirabilis and Yersinia enterocolitica, urease
has been implicated in urolithiasis (stone formation) and
contributes to the development of acute pyelonephritis
and infection-induced reactive arthritis, respective-
12- to 9500-fold increase in urease inhibitory potency
of cysteine was observed for cysteine methyl ester and
cysteamine, respectively. This observation makes car-
boxyl group a limiting factor while designing any cys-
teine based inhibitors. On the other hand, it had also
been reported that thiol compounds such as ethanethiol
and 2-propanethiol, that do not contain any carboxyl
8,12,13
ly.
In agriculture, high urease activity causes signif-
icant environmental and economic problems by
releasing abnormally large amounts of ammonia into
the atmosphere during urea fertilization. Moreover, it
induces plant damage primarily by depriving plants of
their essential nutrients and secondarily by ammonia
1
8
group, display a very weak inhibitory activity.
Taken these findings together and in order to find new
potential inhibitors for microbial ureases, we decided
to design CysDs, comprised of carboxyl and thiol
groups alone as well a chimera of both groups. A series
of 10 different L-cysteine (alkylated, acidic, and alco-
holic) derivatives was designed (Table 1) synthesized
(Fig. 1) and checked for the kinetic parameters of ure-
ase inhibition (Table 2). Thiol derivatives were found
to be more potent than thioether derivatives in inhibit-
ing the activity of both P. mirabilis urease (PMU) and
B. pasteurii urease (BPU) (Fig. 2A). Compounds 4 and
7 were 2–4-fold potent inhibitors of PMU than BPU,
whereas compounds 1–3 selectively inhibited BPU over
PMU with 1.5–2-fold magnitude. In general, PMU was
more susceptible to thioether CysDs than BPU. Neu-
tralization of the terminal SH group in CysDs (by
CH I, CH COOH treatment; Fig. 1) or SH-terminal
8
,14
toxicity, increasing the pH of the soil.
Given the diverse functions of this enzyme, its inhibition
by potent and specific compounds could provide an
invaluable addition for the treatment of infections
caused by urease-producing bacteria. Although several
potent inhibitors of this enzyme have been reported.
1
5–17
as the first line of treatment for infections caused
by urease-producing bacteria, more effective inhibitors
with safe and more potent profile are considered neces-
sary for the control of urease-related ailments and to ex-
plore any novel aspects of mechanism of action of
ureases isolated from different sources.
Based on the previous data of urease inhibition proper-
1
ties of thiol-compounds, 10 different cysteine deriva-
8
3
3
extension (by C H , CH COOH; Fig. 1) altered the
2
5
3
tives (CysDs) with cysteine-like scaffold ( ),
inhibitory effects, suggesting a determinant role of
substituted thiol-terminal residues in binding to urease,
although not necessarily to the active site. Based on the
preliminary structure–activity relationships, we specu-
late that the electronegative sulfhydryl group of CysDs
acts as a ligand chelator (probably to Ni atoms, similar
to other thiol compounds) to form subsequent com-
plexes with the enzyme. Formation of the initial EÆI
complex may involve coordination of the –SH- anion
were designed and synthesized from synthetic combina-
torial library. CysDs were then tested for urease-inhibi-
tory activity against two bacterial and one plant-derived
enzyme, as well as for the inhibition of growth of bacte-
ria and yeast. Structure–activity relationship (SAR) of
the active compounds identified a novel pharmacophore
model for urease inhibitors.
that has pK value around 8.7 (as proposed for other
a
2
. Results and discussion
anions; e.g., phosphates). This hypothesis agrees with
the observation in the present study that K is pH-de-
i
Several thiol compounds have been reported to be com-
petitive inhibitors of ureases. Todd and Hausinger
pendent. Sequence similarity between PBU and PMU
indicates 60.3% identity of amino acid residues for
these microbial ureases. In addition, all residues in-
(1989) demonstrated the susceptibility of K. aerogenes
urease to thiol compounds with K values in the range
of 0.01 mM (cysteamine) to >100 mM (2-propanethiol
i
volved in catalytic site, that is, D362, H274, H248,
K219, H136 and H138 (in BPU), are conserved. The find-
ings with CysDs indicate the other unnoticed impor-
tant structural differences at or near the active sites
of these very conserved bacterial ureases that affect
their susceptibility to CysDs implicating that rational
development of inhibitors for these enzymes must to
consider these aspects.
1
8
and 3-mercaptopropionate). In addition, the absorp-
tion spectrums in the presence of such inhibitors are sim-
ilar to those seen with known thiolate–nickel complexes,
suggesting the mechanism of inhibition might involve an
1
8,19
interaction with Ni atoms in catalytic site.
cysteamine decreases linearly as pH is increased, with a
The K for
i
slope of 0 at pH values above its pK (8.1), indicating
a
that the deprotonated thiol is acting as the inhibitory
species. Thiol compounds that contain an anionic car-
boxyl group are typically poor inhibitors at pH 7.75,
BPU inhibition by 4, 7, 8, and 10 was dependent on the
pH of the pre-incubation mixture (Fig. 2B), with a de-
crease in K values at pH 8.2. In contrast, compound 1
i
such as cysteine (K ꢀ95 mM) and 3-mercaptopropio-
showed an increasing K at pH 8.2. Out of these com-
i
i
nate (K > 100 mM).
i
pounds, thiol-CysDs (7, 8, and 10) are more potent in
inhibiting BPU at pH 8.2 than the respective thioether
CysDs (2, 3, and 4). This difference in their inhibition
might be explained by the thiolate formation at pH 8.2
as described for K. aerogenes urease inhibition by thiol
Cysteine derivatives containing a carboxyl group have
been previously shown as poor inhibitors of ureases.
For instance, cysteamine (K = 0.01 mM) with no car-
i
1
8
boxyl group is more potent urease inhibitor than cys-
compounds.

Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
6739
Table 1. Design of Cysteine Derivatives (CysDs) (with cysteine-like scaffold) and their observed urease inhibitory effects
0
00
Chemical groups
R, R and R
Urease inhibitory effects
0
0
Sulfur derivatives: thioethers versus thiols R = H, CH
3
or CH
2
CH
3
Very important [compare 1–6 (thioethers) with 7–10 (thiols)]
Important (compare 2 ! 3 ! 4 with 7 ! 9 and 7 ! 10)
Not so important (compare 1 ! 2 with 5! 6)
0
Oxygen derivatives
R = COOH, CH
2 2 3
OH, CH OCH
Amine derivatives: primary amines
versus tertiary amines
R = H or CH
3
CH2OH
CH2OCH2CH3
COOH
A
COOH
N
B
D
CH3
CH3
CH3
CH3
H
H
N
H
H
N
NH2
CH3
CH3
SCH3
SCH3
SCH3
SCH3
3
4
1
2
E
O
COOH
COOH
NH2
CH2OH
C
R'
H
NH3+ Cl-
F
H
A, B
CH3
H
N
H
CH3
N
R
SH
SCH2CH3
SCH2CH3
6
S
R''
5
Pharmacophore
A
CH2OCH3
CH3
CH3
CH2OH
CH3
H
COOH
N
C
N
CH3
CH3
B
H
H
N
SH
CH3
9
SH
SH
D
8
7
CH2OCH2CH3
CH3
N
H
A= Pd-C, H
2
O, HCOH, H2- atmposphare
CH3
B=THF, NaAlH
2
(OCH
CO3, CH
CO3,
2
CH
I, refluxo, 24 h.
I, refluxo, 24 h.
COOH, r.t., 3 h
COOH, r.t., 3 h
2 3 2
OCH ) , r.t, 5 h.
SH
C= acetone, K
D= acetone, K
2
2
3
10
C
2
H
5
E= NaOEt, CH
3
I, CH
3
F= NaOEt, C
2
H I, CH
5
3
Figure 1. Schematic synthesis of a series of 10 different L-cysteine (alkylated, acidic, and alcoholic) derivatives starting from commercially available
amino acid, L-cysteine hydrochloride hydrate.
A comparison of the inhibitory activity of CysDs con-
taining carboxyl group (2, 5, and 7) with those contain-
ing alcohol group (3, 6 and 8, respectively), that is,
comparing (2 and 3, 5 and 6, 7 and 8) did not show
any significant difference in their inhibition pattern at
either pH values (Fig. 2B). This is in contrast to the
reduction in the inhibitory effect of CysDs when its
acidic hydrogen (carboxylic or alcoholic hydrogen) is
removed by the formation of ether groups (e.g., carbox-
ylic CysDs to ether derivatives, compare: 2 ! 4, 7 ! 9,
CysDs then there must be some other functional groups
which are modulating the inhibitory properties of Cys-
Ds. In this scenario sulfhydryl group seems to be very
promising not only in overcoming the undesirable effects
of carboxyl group but also to enhance the urease inhib-
itory properties of CysDs. Our data suggest that the
presence of thiol group is more critical to urease inhibi-
tion than carboxyl group (Table 1).
Although CysDs inhibited the bacterial enzymes potent-
ly (BPU IC50 = 10–110 lM and PMU IC50 = 22–
100 lM) than jack bean urease (JBU), of the 10 com-
pounds tested only three were inhibitors of JBU (2, 7,
and 10). For instance compound 2 is more effective
against the plant enzyme than BPU (Fig. 3). The present
7
! 10 and from alcoholic CysDs to ether derivatives,
compare: 8 ! 9, 8 ! 10, and 3 ! 4). Since the inhibito-
ry activity of CysDs decreases in the following order
2–3 > 4 and 7 > 9 and 7 > 10. If we assume that carboxyl
group is there to impede the urease inhibitory activity of

6740
Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
Table 2. Inhibition effect (IC50 ± SEM, lM) of CysDs on microbial ureases (BPU and PMU) and plant urease (JBU) and the influence of two
different pH (pH 5.0 and 8.2) on the BPU inhibition by CysDs
Compound
IC50 (pH 7.5), lM
K
i
(BPU), lM
pH 8.2
PMU
JBU
BPU
pH 5.0
5
5
Cysteine
Cysteamine
Hydroxyurea
—
—
—
—
—
—
1 · 10 ± 450
20 ± 0.9
—
1 · 10 ± 410
15 ± 1.6
—
150 ± 5
100 ± 2.5
140 ± 7
COOH
H
NH2
50 ± 3.5
50 ± 1.5
N.I.
78 ± 3.4
9 ± 1.4
36 ± 3.5
SCH3
COOH
CH3
CH3
H
N
78 ± 6.3
110 ± 7.5
6 ± 0.75
8 ± 1.5
SCH3
CH OH
2
CH3
CH3
H
H
N
63 ± 4.1
N.I.
N.I.
95 ± 5.0
5 ± 0.5
15 ± 0.41
SCH3
CH
2
OCH
2
CH
3
CH
3
N
100 ± 4
50 ± 6.5
7 ± 0.65
40 ± 4
CH
3
SCH
3
COOH
3
5 ± 1.5
N.I.
N.I.
30 ± 2.5
29 ± 1.6
11 ± 2.5
18 ± 1.6
14 ± 1.5
H
H
NH
2
SCH
2
CH
3
CH OH
2
CH3
CH3
N
40 ± 3
17 ± 3
SCH CH
2
3
COOH
CH3
CH3
5
0 ± 2.5
58 ± 2.5
10 ± 0.02
9 ± 2.0
2.0 ± 0.25
H
H
H
N
SH
CH OH
2
CH3
N
22 ± 3.1
N.I.
N.I.
25 ± 1.2
36 ± 4.8
13 ± 1.2
19 ± 4.8
2.0± 0.31
2.0 ± 0.15
CH3
SH
CH
2
OCH
CH
CH
3
3
3
5 ± 1.5
N
3
SH
CH OCH CH
2
2
3
CH3
31 ± 2.5
100 ± 5.5
20 ± 1.0
55 ± 1.0
2.0 ± 0.25
H
N
CH3
SH
Values given are means ± SEM of three independent experiments.
N.I., no inhibitory activity. IC50 values are comparable with hydroxyurea (IC50 = 100 lM).

Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
6741
A
0.2
.15
B
0.06
0.05
0.04
0.03
BPU
PMU
pH 5.0
pH 8.2
0
Thioethers
Thiols
*
*
*
*
0.1
0
.02
0
.05
0
**
*
*
*
*
0.01
0
OHU
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Cys Derivatives
Cys Derivatives
Figure 2. Inhibitory effects of CysDs on microbial ureases. (A) The susceptibility of Bacillus pasteurii urease (BPU) and Proteus mirabilis urease
(
PMU) to CysDs was evaluated. Microbial enzymes were incubated in the presence of 5 ll of CysDs (10–100 lM concentration) at 37 ꢁC, pH 7.5, for
3
1
3
0 min and then residual enzyme activity was monitored as described. Values are highly significant, except 2, 3, and 10. (B) Effect of pH (5.0 or 8.2)
on the activity of BPU, determined by the inhibitory constant, K
i
. Values given are mean ± SEM of three independent experiments. In (A) statistical
analyses indicated significant effects except compounds 2, 3, and 10.
observations of different kinetic behavior of bacterial
and plant ureases are consistent with the structural dif-
ferences between the two types of urea-splitting enzymes
the COOH-group core could serve as the driving force
for the second complementary interaction, effectively
bridging two remote binding domains separated by a
spacer of suitable length. The potency of this series
was improved through the incorporation of more elec-
tronegative and less bulky groups. Moreover, bifunc-
tional inhibitors of this type are expected to exhibit
enhanced activity and specificity resulting from a
favored intramolecular interaction.
2
0,21
already reported in the literature.
The data of kinetic
parameters for the CysD inhibitors are summarized in
Table 2.
The mode of inhibition of BPU activity by the CysDs
2
2
was analyzed by Lineweaver–Burk (LWB). Inhibition
was found to be noncompetitive (also denoted as mixed
inhibition) at both pH values (5.0 or 8.2), Fig. 4; data
shown for only compounds 6 and 8. CysDs were found
to decrease Vmax ranging from 30.5% to 70.4% without
producing an appreciable change in the Km values.
The mean Km values were found to be between
In addition to establishing the mechanism of urease inhi-
bition at the molecular level, we were also interested in
finding the effects of CysDs on the growth of urease-pro-
ducing bacteria. CysDs inhibited the growth of both BP
and PM, however not as strongly as their inhibitory ef-
fect seen upon cell-free ureases (Fig. 5). Antibacterial
activity of CysDs was assessed in the presence of
0.5 mM NiCl (NiCl is needed to produce activated nick-
el–urease complex). It is possible that the CysDs bind
free metal and the resulting CysDs–Ni complex might
be less potent in inhibiting bacterial growth compared
with the corresponding metal-free CysDs, because the
inhibitors seem to bind to the nickel site of the urease,
as has been established above.
5
.04 ± 0.10 and 8.93 ± 0.20 lM. The inhibition pattern
observed suggests a modification of any functional
group or a conformational change in the environment
near the catalytic site rather than in the active site, thus
causing a decrease in Vmax in the presence of inhibitor
without any change in Km values of urease.
N,N-Dimethyl L-cysteine (7; Ki value 2.0 lM) and
S,N,N-trimethyl L-cysteine (2; K value 6.0 lM) were
i
found to be the most active ones, most probably due
00
0
to the small R, R , and R groups (Fig. 1). Based on
the above data, we propose that the initial binding of
This observation suggests that the inhibitory effects of
CysDs on PMU and BPU activity might be related to
their ability to inhibit bacterial growth, consistent with
the previous findings that there is a direct relationship
between the growth and urease inhibitory actions of
0
.2
2
1
such compounds.
BPU
JBU
0
.15
In order to evaluate the physiological tolerance of Cys-
Ds, various dose levels of the inhibitors were assessed
for their mutagenic potential using Saccharomyces cere-
visiae strains optimized for this purpose. But none of the
inhibitors showed any considerable mutagenic effect or
did not interfere with the cell-proliferation of the yeasts
at nuclear level, except for 1, 3, and 4, which were found
to be toxic against both strains in the range of 500–
0
.1
0
.05
0
OH
2
7
10
Cys Derivatives
1000 lM. This may be correlated to their cytotoxic ef-
fects rather than DNA damage, since according to the
principle of the assay, any agent that is cytotoxic
(>65% inhibition) to the yeast in the RS322Yplate
Figure 3. Inhibitory effects of CysDs 2, 7 and 10 on jack bean urease
JBU) and BPU as described in Fig. 2A. Values given are mean-
s ± SEM of three independent experiments.
(

6742
Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
Inhibitor (μM)
2
1
1
0
0
0
,0 Inhibitor (μM)
2,0
1,5
1,0
0
1
2
3
3
4
6
6
,00
,0
0,00
1,0
2,0
3,0
3,5
4,5
6,0
6,5
,6
,2
,8
,4
,0
,0
,0
,5
,5
,0
,5
0
0
,5
,0
CysD6 pH 5.0
CysD6 pH 8.2
-
0,2
-0,1
0,0
1
0,1
-0,2
-0,1
0,0
0,1
-
-1
1
/urea (μM )
1/urea (μM )
Inhibitor (μM)
0,00
Inhibitor (μM)
,00
1,0
2
1
,0
,5
0
1
2
3
3
,0
,0
,0
,5
1
,5
,0
,5
,0
2,0
3,0
3,5
4,5
6,0
6,5
1
0
0
4,5
1,0
0,5
6
6
,0
,5
CysD8 pH 8.2
CysD8 pH 5.0
0,0
-0,2
-0,1
0,0
0,1
-0,2
-0,1
0,0
0,1
-
1
-
1
1
/urea (μM )
1
/urea (μM )
Figure 4. Lineweaver–Burk plots for BPU in the presence of CysDs. Lineweaver–Burk plots representing reciprocal of initial enzyme velocity versus
reciprocal of urea concentrations in the absence and presence of inhibitors 6 (above) and 8 (below) (concentrations mentioned in the legend box) at
both pH 5.0 (left) and 8.2 (right). Each point represents means ± SEM of three independent observations (p < 0.05). K
inhibitor was found to be 120 lM.
m
of BPU in the absence of
itive (mixed) fashion. CysDs and other similar com-
pounds may provide useful probes for the study of
the active site of urease and other hydrolytic enzymes,
their catalytic activity, intimate details of the molecular
geometry at the active-site, and the kinetics of the
binding site of the urease. Additional detailed studies
with more potent derivatives of CysDs will further clar-
ify the mechanism of action of this enzyme in urea
hydrolysis, and provide new template structures for
the design of new inhibitors with an optimized pharma-
cological profile.
1
1
1
.6
.4
.2
1
*
* *
BPU
PMU
0
0
0
0
.8
.6
.4
.2
0
* * *
*
* *
OHU
1
2
3
4
5
6
7
8
9
10
Cys Derivatives
Figure 5. Minimum inhibitory concentrations (MICs) in the presence
of 0.1–1.5 mM CysDs after 24 h of incubation. The progression in the
turbidity of the broth was used as a measure of bacterial growth at
4. Experimental
4
.1. Chemical synthesis
600 nm in 96-well plates. Statistical analyses indicated significant
effects except compounds 2, 3 and 10.
The CysDs (1–10) were synthesized starting from com-
mercially available amino acid, L-cysteine hydrochloride
2
3–29
(
mutated species) and not cytotoxic (<35% inhibition) to
hydrate (Fig. 1). Elemental microanalysis (C, H, N
percentages), infrared (IR) and mass spectrometry, pro-
the yeast in the LF15 plate (wild type), or whose IC50
gives a 3-fold differential in the concentration response
assay will be considered genotoxic.
1
ton nuclear magnetic resonance ( H NMR), and carbon
1
3
13 nuclear magnetic resonance ( C NMR) have been
used to determine the structures of compounds.
3. Conclusions
4.2. Enzymes
The present study concludes that CysDs inhibit bacte-
rial and plant ureases in a concentration-dependent
manner and shows that these compounds are more
effective at inhibiting microbial ureases (BPU and
PMU) than the plant urease (JBU). The kinetic data
suggest that these novel inhibitors act in a noncompet-
Commercially available preparation of B. pasteurii ure-
ase (100,000 U/g, pH 8.2; Sigma) and Jack bean urease
type C-3 (12,000,000 U/g, pH 7.0; Sigma) was used in
all experiments without further purification. P. mirabilis
urease was partially purified as described. The specific
activity of the enzyme was 100,000 U/g protein.
3
0

Z. Amtul et al. / Bioorg. Med. Chem. 14 (2006) 6737–6744
6743
4
.3. Urease inhibition assay
correlation coefficient, slope, intercept, and their stan-
dard errors are presented as means ± SEM and obtained
by the linear regression analysis using this program. All
statistical comparisons were performed by two-tailed
t-test.
In order to evaluate the inhibitory properties of CysDs
on the urease activities, the purified enzymes were incu-
bated in the presence of 5 ll of CysDs (0.001–0.2 mM
concentration) at 37 ꢁC, pH 7.5, for 30 min in 96-well
plates. Urease activity was determined by measuring
3
1
Acknowledgments
ammonia production using the indophenol method.
One unit of urease was defined as the amount of en-
The authors are grateful to the Higher Education Com-
mission (HEC), Pakistan, for the financial assistance un-
der ‘Research Grant for Universities’ program and
University Grants Commission (UGC), Pakistan, for a
predoctoral scholarship to Z.A.
zyme that releases one lmol ammonia per minute, at
3
of urease activity (IC ) was determined using the
7 ꢁC, pH 7.5. The concentration that inhibited 50%
5
0
TM
EZ-Fit 5.0, software program (Perrella Scientific,
Inc. USA). The K values (the dissociation/inhibition
i
constant of the urease–CysDs complex into free BPU
and CysDs) were also calculated using three different
methods. First, the slope of each LWB plot was plotted
against concentrations of different CysDs. Second,
1/Vmaxapp was calculated at each concentration of dif-
ferent inhibitors and then plotted against CysD con-
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