Identification of novel urease inhibitors by high-throughput virtual and in vitro screening

Article abstract of DOI:10.1021/ml100068u

Ureases are important in both agriculture and human health. Bacterial ureases are directly involved in many farm-field problems and pathological conditions. Here, we report a structure-based virtual screening of an in-house compound bank of about 6000 molecular entities by computational docking and binding free energy calculations followed by in vitro screening. Applied protocol leads to the identification of novel urease inhibitors, which can serve as starting points for structural optimization.

Full text of DOI:10.1021/ml100068u

pubs.acs.org/acsmedchemlett
Identification of Novel Urease Inhibitors
by High-Throughput Virtual and in Vitro Screening
Obaid-ur-Rahman Abid,† Tariq Mahmood Babar,† Farukh Iftakhar Ali,† Shahzad Ahmed,†
Abdul Wadood,‡ Nasim Hasan Rama,*,† Reaz Uddin,‡ Zaheer-ul-Haq,*,‡ Ajmal Khan,‡ and
M. Iqbal Choudhary‡
†Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan, and ‡Dr. Panjwani Center for Molecular
Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
ABSTRACT Ureases are important in both agriculture and human health. Bacte-
rial ureases are directly involved in many farm-field problems and pathological
conditions. Here, we report a structure-based virtual screening of an in-house
compound bank of about 6000 molecular entities by computational docking and
binding free energy calculations followed by in vitro screening. Applied protocol
leads to the identification of novel urease inhibitors, which can serve as starting
points for structural optimization.
KEYWORDS Urease inhibitors, high-throughput screening, in vitro screening,
computational docking, binding free energy calculations
rease (EC 3.5.1.5; urea amidohydrolase) is a metal-
containing enzyme that catalyzes the hydrolysis of
urea into ammonia and carbon dioxide.1 It is present
Over the past decade, high-throughput virtual screening
(HTVS) has emerged as an attractive and a valuable alter-
native to high-throughput screening (HTS). The protocol of
structure-based virtual screening (SBVS) can be built up in
many ways. Two conditions, however, are essential for SBVS:
a target structure and a database of ligands. In our case, the
target structure was Bacillus pasteurii (BP) urease, in com-
plex with acetohydroxamic acid (PDB code 4UBP),11 which
was retrieved from the Protein Data Bank.
The current status of urease inhibitors is quite limited. All
available urease inhibitors are not satisfactorily substantial to
the binding cavity and hence not able to optimally interact
with the crucial amino acid residues and the metal ions
present in the urease active site. Therefore, a search of new
and more effective urease inhibitors to diversify the current
urease inhibitors is essential.
U
in a wide variety of organisms, including plants, algae, fungi,
and bacteria.2-4 Urease-producing bacteria have a negative
effect on human health. They are directly involved in urinary
tract infections.5 Urease is known to be a major cause of patho-
logies induced by Helicobacter pylori, as it allows the bacteria
to survive at the low pH of stomach during colonization,
which leads to gastric and peptic ulcers and, in some cases,
may lead to cancer.6 In agriculture, high urease activity causes
significant environmental and economic problems by relea-
sing abnormally large amounts of ammonia into the atmos-
phere during urea fertilization.3,5,7 Besides the importance
of urease in making nitrogen available to plants and the
impact of microbial ureases in agriculture and medicine, the
structure and catalytic mechanism of this enzyme are of aca-
demic interest, because of its large enhancement (1014-fold)
of the rate of urea hydrolysis and the presence of active site
nickel, which is unique among hydrolytic enzymes.8
The structure, number, and type of subunits, molecular
weight, and amino acid sequence of urease depend on its
origin. The urease from jack bean (Canavalia ensiformis) is a
homohexameric molecule (R6), whereas the bacterial urea-
ses are heteropolymeric and contain three subunits, R, β,
and γ. Despite the difference in the molecular structure, the
amino acid sequences of the active site are largely similar in
all known ureases, with same catalytic mechanism. The active
sites are always located in R subunits and contain the bi-
nuclear nickel center, in which the Ni-Ni distances range
between 3.5 and 3.7 Å.9,10 The studies on novel urease inhi-
bitors are essential for the possible development of a highly
needed therapy for urease-mediated bacterial infections.2,3,5,6
Recently, we have developed an in-house bank of over
6000 compounds of synthetic or natural origins. These struc-
turally characterized compounds fulfill the druglike criteria,
according to Lipinski's rule of five, with the exceptions of a
few naturally occurring compounds. The compounds were
prepared for docking in two steps. First, the “washing” module
of Molecular Operating Environment (MOE)12 was applied
to generate physiologically relevant protonation states of com-
pounds. Second, three-dimensional (3D) coordinates were
generated by Concord implemented in Sybyl 7.3.13
Computational docking methods GOLD14 and MOE-Dock12
were implemented. The protein structure was prepared by
Received Date: December 18, 2009
Accepted Date: May 3, 2010
Published on Web Date: May 10, 2010
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deletion of water molecules, 3D protonation, and energy
minimization by using the MOE. Energy minimization was
carried out by implementing the MMFF94x force field and
distance-dependent dielectric potential with the rms gradi-
ent of 0.05. During minimizations, all heavy atoms in the
protein were kept fixed. Histidine residues were considered
as neutral according to Musiani et al.15
Docking calculations were performed by using default
parameters (unless explicitly mentioned) with GOLD scores
and London dG scores as scoring functions for GOLD and
MOE-Dock, respectively. In both cases, an active site region
was defined by a sphere of radius 10 Å around the ligand in
the complex structure of BP urease.
Table 1. Initial Hit IDs with Their Respective Scores (i.e., Gold
Fitness Scores and London dG Scores) and Their Inhibitory Acti-
vities against Urease Enzyme
compd ID Gold fitness score London dG score IC50 ( SEMa (μM)
AAB429
AAB539
AAD355
8d
56.28
41.38
55.93
60.59
47.20
NDb
-13.4425
-11.0070
-15.6482
-10.8738
-09.0359
NDb
110.0 ( 3.06
238.50 ( 2.04
31.20 ( 0.47
34.10 ( 1.24
150.23 ( 2.10
21.0 ( 0.01
14i
thiourea
a SEM, standard error of the mean. b ND, not determined.
First, structures of 6000 in-house compounds were ana-
lyzed to check the redundancy. Consequently, the repeated
compounds were removed from the database. Then, it was
ensured that each class of compounds was represented at
least once. This selection procedure made certain the diver-
sity of the database.
To test the efficiency of applied docking protocol, three
urease inhibitors (i.e., biscoumarin, imidazol, and hydroxa-
mic acid derivatives) were included in a randomly selected
3000 compounds (decoy set) as positive controls. Two sepa-
rate docking runs were carried out, one with MOE-Dock and
the other with the GOLD docking program. There are three
possible scoring functions in MOE-Dock, while there are two
with GOLD.
In addition to scoring functions, there were several other
parameters that needed to be set for attaining appropriate
binding poses and rankings. For example, we saw that out
of a few available placement methods in MOE-Dock, only
Triangle Matcher was quite satisfactory in reproducing the
binding mode of one of the known inhibitors (acetohydroxa-
mic acid), which was retrieved from PDB ID 4UBP.
Similarly, each scoring function, implemented in docking
programs, was evaluated to observe the ability of placing
the correct docking pose in the top rankings. The London dG
scoring function implemented in MOE-Dock was able to
identify all three known inhibitors within the top 1% of the
whole decoy set tested. With the GOLD docking, a gold fit-
ness function was found to produce similar enrichments like
London dG in MOE-Dock.
After setting the docking and scoring protocol, a SBVS
experiment was set up against urease using compounds
from the in-house database. Each molecule of the database
was docked, and the top 10 conformations of each com-
pound were retained for further analysis.
In the first postdocking filtering strategy, 200 top-ranked
compounds were selected by both GOLD and MOE-Dock
scoring functions. There were few identical compounds ran-
ked identically in the top 200 list by both scoring functions.
To filter out further, another selection criterion was app-
lied. A visual inspection of all selected compounds was per-
formed to filter out similar compounds, hence improving the
diversity in the selected compounds. We also analyzed
the docked orientation of each selected compound inside
the binding site of BP urease enzyme. Docked conformation
analysis helped to select only those compounds that were
able to depict key interactions with two nickel ions and
additionally with the crucial active site residues.15,16 Speci-
fically, coordination with nickel ions and hydrogen bonding
with His323, His324, Arg339, Gly280, Cys322, Ala366, and
Ala170 were considered. In addition, special attention was
given to the fact that compounds showed some enhance-
ment in the potency with respect to urease inhibition when
possessing a petite hydrophobic group. It is because of that
that the active site of urease is located within the cavity or the
crevice in the internal territory and is surrounded by hydro-
phobic amino acid residues of the urease enzyme. The hydro-
phobic character of compounds may play a role in the random
walk process of the compounds to the active site and in the
hydrophobic binding, near the active site of urease.17 After
the aforementioned selection criteria were applied, 14 struc-
turally distinct compounds were shortlisted and submitted
for in vitro screening against urease inhibition by the stan-
dard mechanism-based in vitro urease assay. In this assay,
reaction mixtures comprised of 25 μL of enzyme (jack bean
urease) solution and 55 μL of buffer containing 100 mM urea
were incubated with 5 μL of test compounds (0.5 mM con-
centration) at 30 °C for 15 min in 96-well plates. The urease
activity was determined by measuring ammonia production
using the indophenol method as described by Weatherburn.18
Percentage inhibitions were calculated from the formula 100 -
(ODtestwell/ODcontrol) ꢀ 100. Thiourea was used as the stan-
dard inhibitor of urease. The results are reported in Table 1.
All of the 14 hit compounds (Table S1 in the Supporting
Information) were experimentally tested against urease
enzyme by using a standard urease inhibition assay. Five
compounds among 14 hits showed urease inhibitory acti-
vities with IC50 values ranging between 34.10 ( 1.24 and
238.16 ( 2.04 μM, as shown in Table 1. The observed acti-
vities for compounds AAB429,19 AAB539,20 AAD355,21 8d,
and 14i were 110.0 ( 3.06, 238.16 ( 2.04, 31.2 ( 0.47,
34.10 ( 1.24, and 150.23 ( 5.65 μM, respectively.
From the binding mode of compound AAB429, it was
observed that hydroxyl oxygen on the purine ring of this
compound coordinates with both nickel ions, like the hydro-
xyl oxygen in the acetohydroxamic acid. Similarly, one of the
nitrogen atoms in purine ring coordinates with one of the
nickel ions, reminiscentof the carbonyl oxygens in the aceto-
hydroxamic acid (Figure S1a in the Supporting Information).
This compound mimicked the binding mode of aceto-
hydroxamic acid. In addition, there are three hydrogen bonds
formed between compound AAB429 and important active
site residues, that is, Asp224, His323, and Cys322.
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Scheme 1. Synthesis of 4,5-Disubstituted 2,4-Dihydro-3H-1,2,4-
triazole-3-thione 8a-na
a Reagents and conditions: (i) Dry methanol, concentrated H2SO4,
reflux ∼10-12 h. (ii) Dry methanol, NH2-NH2 H2O (80%), reflux ∼8-
3
Figure 1. Interactions of compound 8d (magenta) with the nickel
ions and key residues of BP urease.
10 h. (iii) Pure CS2, 33% NH4OH, dry ethanol, stir at room temperature
∼10-14 h. (iv) Pb(NO3)2, stir at room temperature overnight. (v) Dry
ethanol, reflux ∼10-15 h. (vi) 4 N NaOH, reflux 15-18 h, neutralize
with 4 N HCl.
Just like compound AAB429, the docked conformation of
compound AAD355 (Figure S1b in the Supporting Informa-
tion) also coordinates with the Ni atom and forms four hydro-
gen bonds with important active site residues. The hydrogen
bonding was observed between the hydroxyl and carbonyl
oxygens of the compounds and the binding site residues
Arg339, His323, Kcx220 (modified lysine), and Ala170. Both
compounds have distinct features in the ring substituent
near the Ni atoms. Those features are responsible for the
difference in the activity. For example, with the compound
AAD355, the aromatic ring has three hydroxyl substituents;
one interacts with the Ni atom, whereas the other two form
hydrogen bonds with the Ala170 and Arg339. This extra
stability due to additional hydrogen bonding plays a signifi-
cant role in enhanced bioactivity of compound AAD355. In
effect, the extra hydrogen bonding in AAD355 renders the
interaction of this compound with the crucial Ni atom more
favorably than the AAB429.
In the binding mode of compound AAB539, the arene-
cation interaction between the phenyl ring and one of the
nickel atoms was observed. The hydrogen bond between the
carbonyl oxygen and the binding site residue Arg339 was
observed. These weak interactions to the binding pocket of
urease might be one of the reasons for the least activity
exhibited by this compound than its other counterparts, that
is, AAB429 and AAD355 (Figure S1c in the Supporting
Information).
From the docking conformation of compound 8d (Figure 1),it
was observed that the sulfur moiety of the triazole ring of the
compound coordinates with both of the nickel atoms in the
active site. Similarly, in compound 14i, the carbonyl oxygen
coordinates with nickel atoms, bridging them and also
mediating few hydrogen bonds with the active site residues.
Unexpectedly, this compound showed less activity. Although
the reason remains unclear, however, a possible explanation
for this discrepancy is the requirement of the carboxylate
group at the position where it is interacting with the Ni ion
and the compound 14i lacks this feature (Figure S3 in
Supporting Information).
compd
R
R0
8a
8b
8c
8d
8e
8f
2-(Br)C6H4-CH2
2-(Br)C6H4-CH2
4-(Br)C6H4-CH2
4-(Br)C6H4-CH2
3-(Br)C6H4-CH2
3-(Br)C6H4-CH2
2-(Br)C6H4-CH2
3-(Br)C6H4-CH2
4-(Br)C6H4-CH2
3,4-(OCH3)2C6H3
2,4-(Cl)2C6H3-CH2
2,4-(Cl)2C6H3-CH2
3-(OH)-4-(OCH3)C6H3
2,4-(Cl)2C6H3-CH2
4-(CH3)C6H4
4-(OCH3)C6H4
4-(CH3)C6H4
4-(OCH3)C6H4
4-(Br)C6H4-CH2
4-(CH3)C6H4
3-(OCH3)C6H4
3-(OCH3)C6H4
3-(OCH3)C6H4
4-(CH3)C6H4
4-(OCH3)C6H4
4-(CH3)C6H4
4-(CH3)C6H4
3-(OCH3)C6H4
8g
8h
8i
8j
8k
8l
8m
8n
Three compounds (i.e., AAB429, AAB539, and AAD355)
out of five hits were of commercial origin. We were not able
to further investigate them due to inaccessibility at the moment
of the present study. We are in the process of procuring them
from commercial sources. The remaining two compounds
(i.e., 8d and 14i) were part of the in-house-synthesized com-
pounds and were thus further investigated.22,23
To optimize the ligand interactions with the binding site,
we decided to synthesize different derivatives of compounds
8d and 14i. For compound 8d, 13 analogues (series A) were
synthesized, whereas for the second hit 14i, 21 different
derivatives (series B) were synthesized (Table S2 in Support-
ing Information). Scheme 1 presents the general synthesis of
the compounds in series A, whereas Schemes 2 and 3
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Scheme 2. Synthesis of 3-(Halobenzyl)isocoumarins 13a-i, Their
Respective Keto Acids 14a-i, and 3,4-Dihydroisocoumarins 16a-ia
In series B, there are two types of compounds; one is
isocoumarin, while the other is keto acid. The isocoumarins
in series B showed a strong interaction between their car-
bonyl oxygen and the His and Arg residues of the active site.
This strong interaction could be the basis of the activity of this
class of compounds. The docking analysis of keto acids resulted
in an interesting interaction of these compounds with the
binding pocket of urease. Like acetohydroxamic acid, most of
these compounds interact with urease in a famous bidentate
fashion with the nickel ions. The carboxylate oxygens of these
compounds bridge the two nickel ions.11 With the docking
analysis, we found that more favorable interactions point to
keto acids than the isocoumarin. This might be one of the
reasons of turning keto acids into more actives than the
isocoumarins (Figures S2a,b in the Supporting Information).
An acceptable trend between calculated binding energies and
experimental activities within the individual series was ob-
served for synthesized compounds (Table S2 in Supporting
Information).
a Reagents and conditions: (i) Dry benzene, stirring. (ii) Heat at 200 °C for
4 h. (iii) 5% KOH/ethanol, reflux 5 h. (iv) NaBH4/1% NaOH, over-
night stirring. (v) Ac2O, reflux for 1 h.
Furthermore, an attempt was made to develop a trivial
structure-activity relationship for the synthesized com-
pounds. The compounds 8a-n in the A series and 13a-f,
i, 14a-c,e-i, 16a,c,e,f,i, and 17a,d in series B were
screened for their urease inhibitory activities, and results
are presented in Table S2 in Supporting Information. In the
A series, the tested compounds 8h and 8f were found to be
more potent than the standard, and compounds 8b, 8c, and
8n exhibited an activity comparable with the standard,
whereas compounds 8a, 8g, 8j, and 8l exhibited very good
activities. Compounds 8e, 8i, and 8k were only moderate.
The most active compounds 8h and 8f both have a bromine
atom as a substituent at the meta position of the benzyl part,
whereas they have methoxy or methyl substituents at para
or meta positions in the phenyl part. Compounds 8b and 8c
have a bromine atom with substituents at ortho and para
positions in the benzyl part and methoxy or methyl with
substituents at the para position of the phenyl part, whereas
8n has chlorine substituents at ortho and para positions in
the benzyl part and a methoxy substituent at the meta
position in the phenyl part exhibited urease inhibition
comparable with the standard. These results revealed that
a bromine substituent at the meta position in the benzyl part
and methoxy or methyl at para or meta positions in the
phenyl part play an important role in the enhancement of
activity. The compounds 8h and 8f may serve as lead
compounds for further investigation. In the B series, the
tested compounds 13e, 14b, and 14e exhibited moderate
urease inhibition activity, whereas the compounds 13a-d,f,
i, 14a,c,f-i, 16c,e,f, and 17a,d exhibited a low activity.
Compounds 16a, 16i, and 17d exhibited no activity. Among
the isocoumarins (13a-f,i), 13e exhibited more potent
urease inhibition as compared to others. It might be due
to a chlorine substituent at the meta position in the benzyl
part of 13e. The rest of the compounds have chlorine either
at ortho (13d) or para (13f) positions. Similarly, a bromine or
fluorine substituent at ortho (13a and 13g), meta (13b and
13h), and para (13c and 13i) positions in the benzyl part is
not required for optimum activity. In keto acids 14a-c,e-i,
compounds 14b and 14e are more active with bromine and
Scheme 3. Synthesis of 3-(Halobenzyl)-1-thioisocoumarins 17a-ia
a Reagents and conditions: (i) Lawesso's reagent, dry toluene, reflux for 8
h. Key: a, 2-(Br)-C6H4-CH2; b, 3-(Br)-C6H4-CH2; c, 4-(Br)-C6H4-CH2; d,
2-(Cl)-C6H4-CH2; e, 3-(Cl)-C6H4-CH2; f, 4-(Cl)-C6H4-CH2; g, 2-(F)-C6H4-
CH2; h, 3-(F)-C6H4-CH2; and i, 4-(F)-C6H4-CH2
describe the synthesis of compounds included in series B.
The detailed procedure of synthesis in Schemes 1-3 can be
found in the Supporting Information.
All synthetic compounds were then submitted for bio-
logical screening against urease inhibition through the standard
urease assay18 (Table S2 in Supporting Information). To analyze
the binding modes, we used GOLD and MOE-Dock programs,
and to calculate the binding energies, we used the AutoDock
method, which is famous for its quality predictions of binding
energy calculations.
Compounds of series A and B showed interesting biologi-
cal results. As expected, the compounds of A series are more
active than the series B. In the case of series A, most of the
analogues coordinate with nickel ions by a thiol moiety of
triazole, along with forming hydrogen bonds with important
active site residues. In series B, the carboxylate group of
keto acids coordinates with one of the nickel ions, whereas in
isocoumarins, the arene-cation interaction between the
phenyl ring and the nickel ion was observed. Other moieties
in these compounds showed hydrogen bonding with crucial
active site residues.
From the docking results, the similar docking poses were
observed for the synthesized derivatives. Docking analysis
identified the essential thiol group in series A, at the position
of interaction with the Ni ion, as a key factor for the activity,
since it mimicks the role of the hydroxyl group, as present in
one of the most active hits (i.e., AAD355).
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chlorine substituents at the meta position in the benzyl part
as compared to ortho (14a and 14d) and para (14c and 14f)
positions. Dihydroisocoumarins 16c,e,f and thioisocoumar-
ins 17a,d exhibited low activity, whereas 16a,i and 17d
were inactive. These results revealed that chloro and bromo
substitutions at the meta position in the benzyl part play an
important role in the enhancement of the activity. Com-
pound 14e may serve as a lead compound for further
investigation.
Despite identifying compounds with good inhibitory acti-
vities, we still observed some compounds with no activity
(IC50 > 1000 μM in Table S2 in Supporting Information). The
reasons for lack of activity of these compounds are still under
investigation.
In summary, we have carried out a virtual screening of
an in-house database with the aim to find novel urease
inhibitors. Initially, we identified five novel compounds as
urease inhibitors. Further investigation of two classes of
inhibitors was performed. The biological activities and
binding energies of these compounds were carried out
against the urease. From this study, 34 new inhibitors were
identified with acceptable correlation between biological
activities and binding energies. The binding modes of the
synthesized compounds were analyzed with the docking
programs and were found to be consistent, as reported in
earlier studies.15
Moreover, we also succeeded in identifying some com-
pounds showing inhibitory potency better than the standard
thiourea. Future studies will lead to a better understanding of
structure and activity relationships of these compounds. The
results presented so far led us to conclude that these com-
pounds can be used as starting points in lead optimization
stages.
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SUPPORTING INFORMATION AVAILABLE Structuresof the
initial hit compounds, three commercially available hits and their
diagrams showing interactions with urease receptor, graphical
illustration of the inhibitory activities at various concentrations of
the most active compound, and compound spectral information
and complete synthesis schemes. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. E-mail: nhrama@qau.edu.pk (N.H.R.) or zaheer_qasmi@
hotmail.com (Z.H.).
(19) Catalog No. 102049, Lot No. 3478E; M. B., LLC: Aurora, OH.
(20) Lot and Filling Code 1108805; Fluka Chemie GmbH: Buchs,
Germany.
(21) Tlegenoy, R. T. K. S. U., 742012, 24-microaion, 5-1, Nukus City,
Uzbekistan.
Author Contributions: Molecular modeling was done by A.W.
In vitro testing was done by A. K.
(22) Amtul, Z.; Rasheed, M.; Choudhary, M. I.; Rosanna, S.; Khan,
K. M. Kinetics of novel competitive inhibitors of urease enzymes
by a focused library of oxadiazoles/thiadiazoles and triazoles.
Biochem. Biophys. Res. Commun. 2004, 319, 1053–1063.
(23) Serwar, M.; Akhtar, T.; Hameed, S.; Khan, K. M. Synthesis,
urease inhibition and antimicrobial activities of some chiral
5-aryl-4-(1-phenylpropyl)-2H-1,2,4-triazole-3(4H)-thiones.
ARKIVOC 2009, 7, 210–221.
Funding Sources: We gratefully acknowledge funds from HEC
(Project #20-946/R&D/07/607, September 28, 2007), Islamabad-
45320, Pakistan.
ACKNOWLEDGMENT We are grateful to Prof. Dr. Bernd M.
Rode (University of Innsbruck, Innsbruck, Austria) for providing us
technical support.
r
2010 American Chemical Society
149
DOI: 10.1021/ml100068u ACS Med. Chem. Lett. 2010, 1, 145–149
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