1,2-Benzisoselenazol-3(2H)-one Derivatives As a New Class of Bacterial Urease Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00986

Urease inhibitors are considered promising compounds for the treatment of ureolytic bacterial infections, particularly infections resulting from Helicobacter pylori in the gastric tract. Herein, we present the synthesis and the inhibitory activity of novel and highly effective organoselenium compounds as inhibitors of Sporosarcina pasteurii and Helicobacter pylori ureases. These studied compounds represent a class of competitive reversible urease inhibitors. The most active compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen), displayed K_ivalues equal to 2.11 and 226 nM against S. pasteurii and H. pylori enzymes, respectively, indicating ebselen as one of the most potent low-molecular-weight inhibitors of bacterial ureases reported to date. Most of these molecules penetrated through the cell membrane of the Gram-negative bacteria Escherichia coli (pGEM::ureOP) in vitro. Furthermore, whole-cell studies on the H. pylori J99 reference strain confirmed the high efficiency of the examined organoselenium compounds as urease inhibitors against pathogenic bacteria.

Full text of DOI:10.1021/acs.jmedchem.6b00986

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Article
1,2-Benzisoselenazol-3(2H)-one derivatives
as a new class of bacterial urease inhibitors
Katarzyna Macegoniuk, Ewa Grela, Jerzy Palus, Ewa Rudzinska-
Szostak, Agnieszka Grabowiecka, Monika Biernat, and #ukasz Berlicki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00986 • Publication Date (Web): 14 Aug 2016
Downloaded from http://pubs.acs.org on August 16, 2016
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1,2-Benzisoselenazol-3(2H)-one derivatives as a new class of bacterial
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urease inhibitors
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Katarzyna Macegoniuka, Ewa Grelaa, Jerzy Palusb, Ewa RudzińskaꢀSzostaka, Agnieszka
Grabowieckaa, Monika Biernatc, Łukasz Berlickia*
aDepartment of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of
Technology, Wybrzeże Wyspiańskiego 27, 50ꢀ370 Wrocław, Poland,
bDepartment of Organic Chemistry, Faculty of Chemistry, Wrocław University of Technology,
Wybrzeże Wyspiańskiego 27, 50ꢀ370 Wrocław, Poland,
cDepartment of Microbiology, Medical University of Wroclaw, Tytusa Chałubińskiego 4, 50ꢀ
368 Wroclaw, Poland
* Corresponding author
Eꢀmail: lukasz.berlicki@pwr.edu.pl
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ABSTRACT
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Urease inhibitors are considered promising compounds for the treatment of ureolytic bacterial
infections, particularly infections resulting from Helicobacter pylori in the gastric tract.
Herein, we present the synthesis and the inhibitory activity of novel and highly effective
organoselenium compounds as inhibitors of Sporosarcina pasteurii and Helicobacter pylori
ureases. These studied compounds represent a class of competitive reversible urease
inhibitors. The most active compound, 2ꢀphenylꢀ1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone (ebselen),
displayed ꢀꢁ values equal to 2.11 and 226 nM against S. pasteurii and H. pylori enzymes,
respectively, indicating ebselen as one of the most potent lowꢀmolecularꢀweight inhibitors of
bacterial ureases reported to date. Most of these molecules penetrated through the cell
membrane of the Gramꢀnegative bacteria E. coli (pGEM::ureOP) in vitro. Furthermore,
wholeꢀcell studies on the H. pylori J99 reference strain confirmed the high efficiency of the
examined organoselenium compounds as urease inhibitors against pathogenic bacteria.
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Keywords
Ebselen, cysteine, biological activity, covalent inhibitors, diselenides
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INTRODUCTION
Urease, a large heteropolymeric enzyme belonging to
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superfamily of
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amidohydrolases and phosphotriesterases,1 catalyzes the hydrolysis of urea present in the
seeds of leguminous plants, fungi, bacteria, algae, soil and even in the human gut and kidneys.
Consequently, these reactions cause significant alkalinity, reflecting the release of large
quantities of ammonia.2ꢀ5 The increase in pH is directly associated with the development of
numerous health complications in humans, which depend on colonization site by ureaseꢀ
producing microorganisms and primarily include urinary and gastrointestinal tract infections.3
Approximately 50% of the world’s population are carriers of H. pylori, a Gramꢀnegative
microaerophilic bacterium that survives in the acidic environment of the stomach (pH 2)
through a distinctive mechanism for resistance against host defenses.6 Cytoplasmic urease as
well as urease bound to the surface of the bacterial cell are the main virulence factors of H.
pylori, as ureolytic activity enables this bacterium to overcome the low pH of the stomach and
facilitates the colonization of the mucus layer.7ꢀ9
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The firstꢀline therapy for H. pylori consists of a combination of amoxicillin and
clarithromycin with the proton pump inhibitor — omeprazole. However, the increase in H.
pylori resistance to these antibiotics has made this therapy a lessꢀattractive option in recent
years.10ꢀ12 Indeed, other treatment strategies against H. pylori infection, including the use of
bismuth salts combined with a proton pump inhibitor or a combination of other classes of
antibiotics (e.g., fluoroquinolones, aminopenicillins, and tetracyclines), have emerged.10,12,13
Promising adjuvant therapies for the treatment of the diseases resulting from urea hydrolyzing
pathogenic microorganisms might be based on urease inhibitors.14ꢀ17 Many classes of
compounds show considerable inhibitory activity against urease.18ꢀ19 Unfortunately, most of
these compounds (e.g., phosphoramides,20,21 hydroxamic acids,22ꢀ25 heterocyclic compounds,26
quinones,27ꢀ29 heavy metal ions,30ꢀ32 and thiols33,34) exhibit unfavorable features, such as
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relatively low hydrolytic stability or toxicity, which alter clinical use. Benzimidazoles, such as
omeprazole, rabeprazole or lansoprazole, which are approved for the treatment of gastric
disorders associated with H. pylori infection, also exhibit urease inhibitory activity.35
Rabeprazole has demonstrated the most efficient inhibition of H. pylori urease, with a
reported ꢂꢃꢄꢅ of 0.29 µM.36 This compound also possesses antibacterial activity against H.
pylori strains.37,38 The in vivo effect on monoꢀtherapy, however, is not sufficient for H. pylori
eradication, and benzimidazoles are widely prescribed based on the other biological activities
associated with these compounds, including the irreversible inhibition of H+/K+ꢀATPase
proton pumps, which reduce gastric acid secretion in parietal stomach cells.39
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Notably, a similar profile of biological activity was observed for the organoselenium
compound, 2ꢀphenylꢀ1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone (ebselen). A previous study showed that
ebselen inhibits gastric secretion and shows antiulcer properties.40 These properties were
attributed to the inhibition of H+/K+ꢀ ATPases.41 Moreover, this compound possesses potent
antimicrobial properties.42 Indeed, the bactericidal effects of ebselen against multiꢀresistant
pathogens reflect the competitive inhibition of bacterial thioredoxinꢀreductase (TrxR).
Ebselen reacts with the dithiol Cys135– Cys138 present in the E. coli TrxR active site with a
dissociation constant of 0.14 µM.43 Thus, ebselen and its derivatives can be considered as a
class of antibiotics that target microorganisms lacking glutathione in which the thioredoxin
system acts as hydrogen donor in DNA synthesis.44 These microbial targets include
Mycobacterium tuberculosis, methicillinꢀresistant Staphylococcus aureus and virulent H.
pylori strains. Moreover, the metabolism and toxicity of ebselen have been examined in
detail, showing that this compound could be safely used in humans.45,46 All of the aboveꢀ
mentioned properties of 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone derivatives suggest that these
compounds are promising candidates for H. pylori treatment. The known cysteineꢀbinding
properties of this class of compounds suggested the potential inhibition of urease, a highly
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required element for H. pylori eradication, which would contribute to the improvement of
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human life quality.
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In the present study, we describe the biological evaluation of novel selenoorganic
bacterial urease inhibitors based on a 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone scaffold and related
diselenides. The successful synthesis of the designed compounds facilitated the kinetic
evaluation of their potency against ureases purified from S. pasteurii (native enzyme) and H.
pylori (recombinant enzyme). This research was complemented by H. pylori wholeꢀcell
urease studies.
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RESULTS AND DISCUSSION
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Design. The design of specific urease inhibitors is a challenging task, as little space available
in the enzyme active site restricts strictly structural modifications of inhibitors and the
movable loop of variable conformation, located at the entrance of the active site, hamper
structureꢀbased approaches. An effective strategy could involve compounds binding to the
cysteine residue present at the entrance of the active site (Cys322 in S. pasteurii urease or
Cys321 in H. pylori urease). Although this cysteine residue is not directly involved in the
catalysis of enzymatic reaction, the placement of this residue enables effective inhibition. This
likelihood has previously been postulated,47 and structural studies directly confirmed the
binding of cysteineꢀreactive compounds to Cys322 of S. pasteurii urease.48 1,2ꢀ
Benzisoselenazolꢀ3(2H)ꢀone (Figure 1, structure 1) is a promising lead structure that could
react with the –SH group of Cys322 to produce adducts, which cover the entrance to the
active site of urease. Moreover, synthetic derivatives with various nitrogen atom substitutions
are readily available synthetically. Substituents containing phenyl group (e.g., ebselen,
structure 2) or carboxylic acid and related ester have been designed. This set of substituents
could enable exploration of possible hydrophobic and/or hydrogen bond interactions with the
protein. Moreover, analogous diselenides, which form the same adducts upon reaction with
cysteine but could exhibit different reactivity, were also included in the screening.
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Figure 1. The structures of 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone (1) and its most widely studied
derivative ebselen (2).
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Chemistry. Derivatives of 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone (compounds 4) were synthesized
using bis(carboxyphenyl) diselenide (3) as the starting material by known methodology
(Scheme 1).49 The formation of 2ꢀ(chloroseleno)benzoyl chloride in the reaction of compound
3 with SOCl2 (with catalytic amount of DMF) was followed by a reaction between this
intermediate compound and amines (threeꢀfold excess) or amino acids (threeꢀfold excess) or
amino acid methyl ester hydrochlorides (in the presence of Nꢀmethylmorpholine). Related
diselenides (compounds 5 and 6) were also prepared from acid 3 using a previously published
method (Scheme 2).49 The first synthetic route was initiated after obtaining bis[(2ꢀ
chlorocarbonyl)phenyl] diselenide from the reaction of 3 with SOCl2 in refluxing benzene.
Subsequently, the intermediate was reacted with amines or amino acids (threeꢀfold excess) to
form products 5. The second option was based on the coupling of compound 3 with amino
acid methyl ester hydrochlorides using DCC/HOBt in the presence of Nꢀmethylmorpholine
(NMM).50
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Scheme 1a
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aReagents and conditions: (a) SOCl2, cat. DMF, reflux; (b) amine (excess) or amino acid
(excess) or amino acid methyl ester hydrochloride/NMM, cat. DMAP.
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Scheme 2a
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aReagents and conditions: (a) SOCl2, benzene, reflux; (b) amine or amino acid (excess), cat.
DMAP; (c) HOBt, DCC, NMM, amino acid hydrochloride methyl ester, THF.
Inhibition studies. The values of kinetic parameters (ꢀꢆ and ꢇꢈꢉꢊ) of highly purified S.
pasteurii urease (CCM 2056TM) and recombinant H. pylori urease were determined by fitting
the initial reaction velocities measured at a range of urea concentrations to the Michaelisꢀ
Menten equation using nonlinear regression. The values obtained for S. pasteurii urease were
ꢀꢆ of 4.92 ± 0.31 mM, ꢇꢈꢉꢊ of 4.224 ± 0.0601 µM sꢀ1 with specific activity of 2451 U/mg,
and the values obtained for the H. pylori urease were ꢀꢆ of 0.28 ± 0.017 mM, ꢇꢈꢉꢊ of 1.312
± 0.074 µM sꢀ1, with specific activity of 1654 U/mg. The activities of organoselenium
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compounds were initially evaluated against the model bacterial enzyme S. pasteurii urease.
The nonlinear progress curves for urease revealed that this class of compounds exhibited
timeꢀdependent inhibition of urease activity (Figure S1). The linear relationships of 1/ꢇꢅ,
1/ꢇꢋ and 1/ꢌꢉꢍꢍ confirmed the slow binding according to mechanism B. This mechanism is
characterized by progressive inhibition of enzyme activity resulting from slow conformational
changes of the rapidly forming initial enzymeꢀinhibitor complex, ꢎꢂ. These changes resulted
in the formation of an isomerized form of enzymeꢀinhibitor complex (ꢎꢂ∗), with much higher
binding affinity between the inhibitor and the new enzyme conformation state and a
significantly lower inhibition constant.51 All inhibitors demonstrated competitive and
reversible types of inhibition as confirmed using LineweaverꢀBurk plots (Figure S2) and a fast
dilution assay (data not shown). The competitive reversible mode of inhibition indicates that
the inhibitor exclusively binds to the free enzyme and decreases the amount of substrate
binding to the enzyme. The maximum velocity of the reaction was unchanged, while the
apparent affinity of the substrate to the binding site decreased. The same inhibition kinetics
were observed for all studied compounds, and the similarities in their reactivity profiles
suggested that the biological action at the molecular level should be similar for this set of
inhibitors. The slow binding mechanism B is consistent with the assumed mode of inhibition
involving the reaction of 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀones and related diselenides with the
cysteine residue present at the entrance of the active site.
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The lead structure examined in the present study, 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone (1),
exhibited high inhibitory activity towards S. pasteurii urease with a steadyꢀstate inhibition
constant (ꢀ∗) of 24.70 nM (Table 1), suggesting the potential blocking of the active site
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entrance. Subsequently, we tested derivatives with substituents containing phenyl rings
(compounds 2, 4a-b). All of these compounds showed high activity, however ebselen (2)
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showed a particularly interesting result of ꢀ∗ equal to 2.11 nM, ranking this compound as one
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of the most active inhibitors of bacterial ureases.
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Table 1. Inhibitory activity of the 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀoneꢀbased compounds
containing modifications on the nitrogen atom and diselenide derivatives against purified
native urease from S. pasteurii.
ꢀ∗ [nM]
No
Compound
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H
24.70 ± 0.91
2.11 ± 0.18
139.2 ± 8.6
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Ph
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12.10 ± 0.87
4b
883.2 ± 5.0
1007 ± 96
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936 ± 75
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2410 ± 190
ꢀ(CH2)2COOH
ꢀ(CH2)3COOH
1 210 ± 120
806 ± 71
4g
4h
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ꢀCH2COOMe
84.0 ± 6.2
32.6 ± 2.6
4i
4j
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9
164.2 ± 9.3
76.3 ± 5.2
26.1 ± 2.1
4k
4l
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4m
Ph
9.44 ± 0.76
4.62 ± 0.32
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ꢀCH2COOH
10 300 ± 890
14 900 ± 1200
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5d
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ꢀCH2COOMe
ꢀ(CH2)3COOMe
15 100 ± 1400
6.67 ± 0.54
3.48 ± 0.29
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H
319 000 ± 24000
528 ± 44
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3 300 ± 400
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Further attempts to improve the affinity of organoselenium compounds to the enzyme
through the examination of various Nꢀsubstituted 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone molecules
(4c-m) and related diselenides (5, 6), were undertaken (Table 1). To determine the structureꢀ
activity relationship, the effect of different functional groups, such as carboxylate (4c-h, 5c-
e), ester (4i-m, 6) and hydroxyl groups (5b), was investigated. In the group of 1,2ꢀ
benzisoselenazolꢀ3(2H)ꢀone molecules, the incorporation of substituents bearing carboxylic
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acid groups significantly decreased activity (ꢀ∗ in the range of 806–2410 nM for inhibitors
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4c-h) compared with unsubstituted compound 1 (ꢀ∗ = 24.70 nM). In addition to the
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carboxylic acid derivatives, we also evaluated their ester counterparts (4i-m). These
modifications successfully enhanced the inhibitory potency compared with analogous amino
acids by more than one order of magnitude in cases of substituted 1,2ꢀbenzisoselenazolꢀ
3(2H)ꢀone molecules (4c versus 4j and 4e versus 4l). Within the group of inhibitors 4,
structures 4j and 4m were the most potent with ꢀꢁ∗ values equal to 32.6 and 26.1 nM,
respectively. Methyl esters with other substituents exhibited slightly lower inhibitory
activities (ꢀꢁ∗ in the range of 76.3–164.2 nM for 4i, 4k and 4l). As expected, the kinetic
characteristics depended on the enantiomeric form of the inhibitor, and (R)ꢀenantiomer (4m,
ꢀ∗ = 26.1 nM) was slightly more effective than (S)ꢀenantiomer (4l, ꢀ∗ = 76.3 nM).
ꢁ
ꢁ
Strong antiꢀSPU activity was also observed for diselenide compounds 5 and 6. The
diselenide analog of ebselen (5a) exhibited inhibitory constants at the low nanomolar range
(ꢀ∗ = 9.44 nM). Analogous compound with ester group instead of amide group (structure 7)
ꢁ
showed nearly two orders of magnitude lower inhibitory activity (ꢀꢁ∗ = 528 nM), suggesting
the involvement of the NH fragment in interactions with the enzyme. Interestingly, related
carboxylic acid 3 showed poor activity, with an inhibition constant of more than four orders
of magnitude higher than that of phenyl amide 5a. In addition, diselenide molecules
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containing substituents with carboxylic acid groups (5c-e) showed low enzyme affinities (ꢀ∗
ꢁ
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in the range of 15100–46800 nM). However, analogous methyl esters were much more potent,
and the difference between particular esters and acids approached four orders of magnitude
(5c versus 6a and 5e versus 6b). Differences in the inhibitory activity between acids and
methyl esters were observed for both 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀoneꢀbased molecules and
diselenides, but the effect was much more profound for the second group of compounds.
Compounds 5b, 6a and 6b showed the highest enzyme affinity among the examined
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diselenides (ꢀ∗ of 4.62 nM, 6.67 nM and 3.48 nM, respectively) and achieved potency of
ꢁ
activity similar to most active 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone molecules.
The extensive screening of 25 organoselenium compounds for inhibitory properties
against S. pasteurii urease revealed the structureꢀactivity profiles of the inhibitors, indicating
the most valuable compounds, such as 2, 5a-b and 6a-b. We evaluated the inhibitory activity
of these compounds towards recombinant H. pylori urease (Table 2). The progress curves for
the urease reaction and kinetic analyses of the substrate in the presence of the studied
compounds revealed fast binding and the competitive reversible mode of inhibition. The
inhibition constants for these compounds against H. pylori urease increased compared with
the described inhibition of S. pasteurii urease but still exhibited significant activity at a
nanomolar range (with the exception of compound 5b). Ebselen (2) showed high inhibitory
activity against the H. pylori enzyme (Ki = 226 nM), however the most active compound (6b
with Ki = 184 nM) was found among diselenide derivatives. Diselenides 5a and 6a showed
activities in a similar range, with ꢀ values equal to 651 and 915 nM, respectively. In contrast
ꢁ
with SPU, weak inhibition was observed for compound 5b with a hydroxyl group (ꢀ = 3259
ꢁ
nM).
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Table 2. Inhibitory activity of the most active organoselenium compounds against the
recombinant H. pylori. urease.
6
7
8
9
Compound
ꢀ [nM]
ꢁ
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226 ± 16
2
651 ± 56
5a
3259 ± 271
915 ± 65
5b
6a
184 ± 12
6b
AHA
96 000 ± 17 000
Molecular modeling. It was recently shown that the inhibitory activities of compounds
reactive with thiols reflect reactions with the Cys322 residue, located at the entrance of the
urease active site and conserved among bacterial ureases.48 Therefore, we modeled the
ebselenꢀurease adduct resulting from the reaction of ebselen with Cys322 (Figure 2). The
fragment derived from the ebselen molecule fit the region near the active site, and its
positioning completely blocked the entrance to the active site. The hydrogen amide (from
ebselen fragment) and carbonyl (from Cys322) formed hydrogen bond, while π−π stacking
interactions were observed between the phenyl substituent and His323. These two interactions
are consistent with structureꢀactivity relationships observed for the studied organoselenium
compounds, where inhibitor 1 (without phenyl ring) and inhibitor 7 (without NH amide) were
much less potent than ebselen (2).
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Figure 2. Modeled structure of the ebselenꢀurease adduct. The residues forming the active site
are shown as sticks, nickel ions are shown as blue spheres, hydrogen bonds are indicated as
solid green lines, and π−π interactions are indicated as dashed green lines.
Whole-cell studies. Exceptionally high inhibitory properties of the studied organoselenium
compounds against purified ureases were verified using a wholeꢀcell in vitro model. Urease
inhibition was studied using the H. pylori J99 reference strain and transformed E. coli Rosetta
cells producing H. pylori urease. Urease in H. pylori cells is present both intraꢀ and
extracellularly; therefore, transformed E. coli cells were used as a model for the determination
of compound diffusion into Gram(ꢀ) cells and inhibition of cytoplasmic urease. For both
wholeꢀcell urease models, similar susceptibilities were observed. In H. pylori J99, compounds
entered the cells more slowly, as preincubation was required to achieve activity. The group of
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compounds containing ebselen (2) and its derivatives with phenyl substituents (4a-b) showed
high inhibitory properties in H. pylori cells. Similarly, high activities were observed for
ebselen derivatives containing methyl ester functionality (4i-m). Corresponding diselenides
(compounds 5 and 6) were generally less active, except for the ebselen analog 5a. For the
most active compounds, the calculated ꢂꢃꢄꢅ was reduced by approximately two orders of
magnitude compared with the wellꢀestablished urease inhibitor acetohydroxamic acid (AHA).
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Table 3. Wholeꢀcell inhibition of H. pylori recombinant urease in the Rosetta host strain and
native urease in H. pylori J99 grown in Brucella broth. ꢂꢃꢄꢅꢅ – parameter assayed without
ꢏ ꢐ
preincubation with inhibitory compounds. ꢂꢃ – parameter assayed using cells preincubated
ꢄꢅ
with inhibitors for 2 h.
Compound E. coli (pGEM::ureOP)
H. pylori J99
ꢂꢃꢄꢅꢅ [µM] ꢂꢃ [µM] ꢂꢃꢄꢅꢅ [µM] ꢂꢃ [µM]
ꢏꢑ
ꢏꢑ
ꢄꢅ
ꢄꢅ
48.6 ± 6.4 1.28 ± 0.27
NI
6.92 ± 0.35
1
69 ± 11
NI
3.80 ± 0.43 45.4 ± 2.4 3.69 ± 0.08
2
25.3 ± 3.9
22.7 ± 2.6
196 ± 24
NI
NI
NI
5.04 ± 0.22
5.03 ± 0.74
611 ± 147
4a
4b
4c
4d
4e
4f
4g
4h
4i
79 ± 10
NI
186 ± 24
301 ± 42
388 ± 47
NI
38.4 ± 5.0 390.2 ± 9.7 25.4 ± 5.6
95 ± 13
319 ± 39
38.0 ± 4.7
37.0 ± 4.7
NI
NI
NI
NI
69.5 ± 2.4
50.8 ± 6.4
NI
103 ± 16
98 ± 22
107.7 ± 9.8 9.2 ± 1.3 3.47 ± 0.65 2.12 ± 0.02
75 ± 11 19.0 ± 3.3 75.1 ± 3.4 3.91 ± 0.69
4j
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96 ± 16
127 ± 20
121 ± 18
87 ± 11
NI
25.8 ± 3.3
35.2 ± 5.3
NI
NI
4.07 ± 0.99
3.3 ± 1.9
4k
4l
40.9 ± 4.4 75.7 ± 2.4 3.14 ± 0.29
4m
5a
5b
5c
10.9 ± 1.6 38.7 ± 3.9
6.7 ± 2.3
115 ± 43
357.9 ± 6.4
182.6 ± 9.0
421 ± 19
83.5 ± 1.2
11.8 ± 2.0
NI
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108 ± 16
NI
NI
NI
NI
NI
NI
NI
NI
NI
163 ± 18 121.5 ± 8.1
85 ± 11
NI
25.9 ± 3.5
35.4 ± 5.1
85 ± 10
20.1 ± 2.8
794 ± 98
NI
5d
5e
NI
6a
6b
3
NI
NI
NI
NI
7
154 ± 31
96 ± 17
156 ± 32
AHA
NI – not inhibitory
CONCLUSIONS
A newly developed class of highly potent lowꢀmolecularꢀweight bacterial urease inhibitors
has been described. The availability of organoselenium inhibitors based on two scaffolds and
with various substituents enabled the identification of one of the most potent urease inhibitors,
ebselen, with
ꢀ values equal to 2.11 and 226 nM against S. pasteurii and H. pylori enzymes,
ꢁ
respectively. Molecular modeling results combined with experimentally proven mechanism B
of slow binding kinetics indicate an interaction with Cys322 present at the loop near the active
site entrance. As urease inhibitors targeting the cysteine residue remain poorly explored and
only a few examples have been reported.27,52ꢀ54 the described class of compounds present new
opportunities for the discovery of highly active urease inhibitors. The studied group of
compounds also showed promising antiꢀurease profiles in wholeꢀcell studies. The known
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properties of 1,2ꢀbenzisoselenazolꢀ3(2H)ꢀone derivatives such as gastric juice antiꢀsecretory,
antiꢀinflammatory, antibiotic activities towards H. pylori, and low toxicity combined with the
newly discovered antiꢀurease activity suggest that these compounds are favorable candidates
for H. pylori treatment.
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EXPERIMENTAL SECTION
General. All chemicals were purchased from commercial suppliers (Aldrich, Merck, POCh)
at the highest available purity and used without further purification unless otherwise stated.
Ultrapure, deionized water was obtained using an Easy Pure II UV Thermo Scientific water
purification system (resistivity 18.2 Mꢁ cm). NMR measurements were performed on a
spectrometer operating at 600.58 MHz for H and 151.03 MHz for 13C at 298 K. The NMR
1
spectra were recorded in a MeODꢀd4 or DMSOꢀd6. Chemical shifts (δ, ppm) are reported
relative to the solvent peak: MeODꢀd4 – 3.31 [1H] and 49.86 [13C], DMSOꢀd6 – 2.50 [1H] and
39.52 [13C]. RPꢀHPLC analyses were carried out using the Analytical column 150 mm × 4.6
mm, Kinetex 100A (C18, 5 ꢂm), at a flow rate of 1.0 mL/min. Solvent A: 99.95% H2O,
0.05% TFA. Solvent B: 99.95% CH3CN, 0.05% TFA. The following gradients were used: A:
t=0 min (40% B), t=2 min (40% B), t=13 min (100% B), t=15 min (40% B); B: t=0 min (0%
B), t=2 min (0% B), t=13 min (90% B), t=15 min (0% B). The eluted peaks were detected
using a UV detector at 220 and 254 nm. The retention times are reported in minutes, and the
HPLC purity was reported at 220 nm. All compounds showed purity at least 95%.
Chemistry. The synthesis of compounds 1, 3, 4a, 4c-f, 4i-j, 4l, 5a, 5c, and 7 has been
previously described (the purity, analytical HPLC, high resolution ESIꢀMS for these
compounds are shown in Table S2).49,55,56
General method A. Amino acid or amine (16.5 mmol) was suspended or dissolved in
acetonitrile (25 mL), 4ꢀdimethylaminopyridine (0.2 mmol) was added to the reaction mixture
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in one portion, followed by the slow dropwise addition of a solution of 2ꢀ
(chloroseleno)benzoyl chloride49 (5 mmol) in acetonitrile (20 mL) at room temperature. The
reaction mixture was monitored using TLC (ethyl acetate). When the reaction was complete
(2–14 days), the solvent was removed in vacuo. HClaq (0.5 M, 40 mL) and diethyl ether (10
mL) were added to the oily residue, and the mixture was stirred until the product solidified.
The filtered product was washed (2 × 25 mL of water) and crystallized (methanol/water, 3:2
or ethanol/water, 3:2).
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General method B. Amino acid methyl ester (5.5 mmol) was suspended in anhydrous THF
(50 mL) and cooled to –14°C, followed by the dropwise addition of Nꢀmethylmorpholine
(18.7 mmol). The mixture was stirred for 30 min. Subsequently, 4ꢀdimethylaminopyridine
(0.2 mmol) was added to the reaction mixture in one portion, and a solution of 2ꢀ
(chloroseleno)benzoyl chloride49 (5 mmol) in THF was added dropwise at ꢀ14°C. The
reaction mixture was stirred at room temperature and monitored using TLC (ethyl acetate as
eluent). When the reaction was complete (2–14 days), the solvent was removed in vacuo.
HClaq (0.5 M, 40 mL) and diethyl ether (10 mL) was added to the oily residue, followed by
stirring until the product solidified. The filtered product was washed (2 × 25 mL of water) and
crystallized (methanol/water, 3:2 or ethanol/water, 3:2).
General method C. Amino acid or amine (5 mmol) was suspended or dissolved in anhydrous
THF (30 mL), 4ꢀdimethylaminopyridine (0.1 mmol) was added to reaction mixture in one
portion and a solution of bis[(2ꢀchlorocarbonyl)phenyl] diselenide49 (0.5 g, 1.2 mmol) in
anhydrous THF was added dropwise at room temperature. The reaction mixture was
monitored using TLC. When the reaction was complete (1–3 days), the solution was
evaporated, and the residue was dissolved in ethyl acetate, washed with water, dried (MgSO4)
and evaporated. The product was recrystallized (methanol or ethanol/water).
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General method D. Amino acid methyl ester hydrochloride (4.4 mmol) was suspended in
anhydrous THF and cooled to –14°C, followed by the dropwise addition of a solution of Nꢀ
methylmorpholine (4.4 mmol) in anhydrous THF. The reaction mixture was stirred for 30 min
and subsequently, diselenide 3 (2.0 mmol), HOBt (4.4 mmol) and DCC (4.4 mmol) were
added. The reaction mixture was warmed to room temperature and incubated overnight.
Subsequently, 0.5 mL of water was added, the mixture was filtered, and the solution was
evaporated in vacuo. The residue was dissolved in ethyl acetate and washed with 5% citric
acid (three times), 10% NaHCO3aq, water and brine. The solution was evaporated, and the
residue was recrystallized (dichloromethane/hexanes).
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Compound 4b, method A, yield 89%, 1H NMR (MeOD): δ 4.16 (ABX, J = 11.7, 5.9, 4.9 Hz
2H), 5.74 (t, J = 5.3 Hz, 1H), 7.28 (dd, J = 7.3, 7.2 Hz, 1H), 7.34 (dd, J = 7.8, 7.4 Hz, 2H),
7.38 – 7.44 (m, 3H), 7.59 (dd, J = 7.7, 7.4 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.8
Hz, 1H). 13C NMR (MeOD): 61.33, 65.75, 126.83, 127.85, 129.58, 129.61, 129.81, 130.52,
133.82, 140.90, 143.01, 170.52. HRꢀESI MS (M + 1), expected 320.0190, found 320.0199.
Analytical HPLC, method A: tR = 3.30 min., purity >97%.
Compound 4g, method A, yield 70%, 1H NMR (MeOD): δ 2.73 (t, J = 6.6 Hz, 2H), 4.08 (t, J
= 6.6 Hz, 2H), 7.43 (dt, J = 7.5, 0.8 Hz, 1H), 7.61 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.89 – 7.94
(m, 2H). 13C NMR (MeOD): 36.25, 42.38, 127.05, 127.92, 129.49, 129.57, 133.92, 142.37,
170.28, 175.66. HRꢀESI MS (M – 1), expected 269.9670, found 269.9673. Analytical HPLC
method B: tR = 9.70 min., purity >96%.
Compound 4h, method A, yield 83%, 1H NMR (MeOD): δ 2.02 (p, J = 7.1 Hz, 2H), 2.39 (t, J
= 7.2 Hz, 2H), 3.88 (t, J = 7.2 Hz, 2H), 7.44 (dd, J = 7.6, 7.3 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H),
7.92 (d, J = 8.2 Hz, 1H). 13C NMR (MeOD): 27.55, 32.56, 45.55, 127.15, 128.02, 129.66,
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129.78, 133.89, 141.78, 170.23, 177.31. HRꢀESI MS (M – 1), expected 283.9826, found
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6
283.9836, Analytical HPLC method B: tR = 9.50 min, purity >96%.
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Compound 4k, method B, yield 78%, 1H NMR (MeOD): δ 0.90 (t, J = 7.4 Hz, 3H), 1.01 (d, J
= 6.7 Hz, 2H), 1.09 – 1.19 (m, 1H), 1.32 – 1.41 (m, 1H), 2.03 – 2.12 (m, 1H), 3.77 (s, 3H),
5.07 (d, J = 9.7 Hz, 1H), 7.44 (dd, J = 7.6, 7.4 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), (dd, J = 7.4,
7.3 Hz, 2H). 13C NMR (MeOD): 11.81, 16.64, 27.06, 40.52, 53.59, 62.88, 126.94, 127.99,
129.06, 129.82, 134.19, 142.86, 170.60, 173.55. HRꢀESI MS (M + 1), expected 328.0453,
found 328.0458. Analytical HPLC method A: tR = 7.17 min., purity >98%.
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Compound 4m, method B, yield 80%, H NMR (MeOD): δ 0.97 (d, J = 6.4 Hz, 6H), 1.49 –
1.59 (m, 1H), 1.80 – 1.92 (m, 2H), 3.74 (s, 3H), 5.49 (dd J = 7.9, 7.1 Hz, 1H), 7.44 (dd J =
13
7.4, 7.3 Hz, 1H), 7.63 (dd J = 7.6, 7.4 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H). C NMR (MeOD):
22.53, 24.12, 26.96, 43.64, 53.86, 56.64, 127.05, 128.01, 129.38, 129.78, 134.17, 142.57,
170.66, 174.00. HRꢀESI MS (M + 1), expected 328.0453, found 328.0452. Analytical HPLC
method A: tR = 7.16 min. purity >96%.
1
Compound 5b, method C, yield 87%, H NMR (MeOD): δ 1.04 (t, J = 7.4 Hz, 6H), 1.54 –
1.63 (m, 2H), 1.74 – 1.83 (m, 2H), 3.62 – 3.9 (m, 4H), 4.00 – 4.06 (m, 2H), 7.27 (dd, J = 7.5,
7.3 Hz, 2H), 7.30 (dd, J = 7.6, 7.4 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 7.8 Hz, 2H).
13C NMR (MeOD): 11.93, 25.90, 55.99, 65.66, 128.22, 129.53, 132.73, 133.34, 134.00,
136.46, 171.73. HRꢀESI MS (M + 1), expected 545.0463, found 545.0457. Analytical HPLC
method A: tR = 3.50 min., purity >96%.
1
Compound 5d, method C, yield 71%, H NMR (MeOD): δ 1.55 (d, J = 7.3 Hz, 6H), 4.64 (q,
J = 7.3 Hz, 2H), 7.28 (dd, J = 7.5, 7.3 Hz, 2H), 7.33 (dd, J = 7.4, 7.1 Hz, 2H), 7.75 (d, J = 7.5
Hz, 2H), 7.84 (d, J = 7.9 Hz, 2H). 13C NMR (MeOD): 18.45, 50.88, 128.20, 129.90, 132.84,
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133.65, 134.33, 135.45, 171.27, 179.94. HRꢀESI MS (M – 1), expected 542.9578, found
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542.9564. Analytical HPLC method A: tR = 2.50 min., purity >99%.
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Compound 5e, method C, yield 64%, 1H NMR (DMSO): δ 1.79 (p, J = 7.1 Hz, 4H), 2.32 (t, J
= 7.3 Hz, 4H), 3.31 (td, J = 7.3, 6.8, 6.0 Hz, 4H), 7.32 (ddd, J = 7.5, 7.0, 1.1 Hz, 2H), 7.38
(ddd, J = 7.8, 7.4, 1.3 Hz, 2H), 7.68 (dd, J = 8.1, 1.1 Hz, 2H), 7.78 (dd, J = 7.7, 1.1 Hz, 2H),
8.77 (t, J = 5.8 Hz, 2H). 13C NMR (DMSO): 25.41, 32.12, 127.15, 128.88, 130.84, 132.57,
132.84, 134.10, 168.39, 175.26. HRꢀESI MS (M + 1), expected 573.0048, found 573.0064.
Analytical HPLC method A: tR = 2.60 min., purity >99%.
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Compound 6a, method D, yield 91%, H NMR (MeOD): δ 3.78 (s, 6H), 4.16 (s, 4H), 7.30
(dd, J = 7.4, 7.3 2H), 7.34 (dd, J = 7.7, 7.6 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.85 (d, J = 8.0
Hz, 2H). 13C NMR (MeOD): 43.27, 53.60, 128.26, 129.78, 132.88, 133.85, 134.57, 134.93,
171.78, 172.67. HRꢀESI MS (M + 1), expected 544.9734, found 544.9737. Analytical HPLC
method A: tR= 3.60 min. HPLC purity >99%.
Compound 6b, method D, yield 88%, 1H NMR (MeOD): δ 1.96 (p, J = 7.1 Hz, 4H), 2.48 (t, J
= 7.4 Hz, 4H), 3.36 (t, J = 6.9 Hz, 4H), 3.67 (s, 6H), 7.28 (ddd, J = 7.6, 7.4, 1.2 Hz, 2H), 7.32
(ddd, J = 7.9, 7.4, 1.5 Hz, 2H), 7.63 (dd, J = 7.6, 1.5 Hz, 2H), 7.83 (dd, J = 8.1, 0.9 Hz, 2H).
13C NMR (MeOD): 26.65, 33.05, 41.20, 52.98, 128.28, 129.43, 132.83, 133.46, 134.11,
136.04, 171.62, 176.28. HRꢀESI MS (M + 1), expected 601.0361, found 601.0364. Analytical
HPLC method A: tR = 7.30 min. purity > 95%.
Enzymatic studies. Native urease from S. pasteurii CCM 2056TM and recombinant H. pylori
urease (expressed in E. coli strain Rosetta (DE3) + pGEM::ureOP) were purified using a
previously described method based on a fiveꢀstep chromatographic protocol (columns Q
Sepharose XK 50/20, Phenyl Sepharose XK 26/20, Sephacryl Sꢀ300 HR HiPrep 26/60,
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MonoQ TricornTM 5/50 GL and Superose 12 HR 10/30).57 The use of this multiꢀstep
chromatographic approach generated a homogenic urease preparation.
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The enzymatic hydrolysis of urea was performed in 3 mM phosphate buffer (pH 7.5) at 37°C.
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The kinetic parameters of the noninhibited urease reaction, ꢀꢆ and ꢇꢈꢉꢊ, were obtained after
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measuring the initial rates of the reactions conducted in mixtures containing 113.5 pM S.
pasteurii urease and 1–60 mM urea or 488 pM H. pylori urease and 0.1–2.5 mM urea using
the Berthelot colorimetric reaction as previously reported.58 One unit (U) of enzymatic
activity was defined as the amount of enzyme required to produce 1 µM ammonia per min
under specific conditions.
Inhibition studies. (i) S. pasteurii urease. The inhibitory activities of compounds 1-7 were
was analyzed using purified S. pasteurii urease. Reaction progress curves were performed in
standard assay mixtures at a single urea concentration (final concentration 25 mM) and at
different inhibitor concentrations according to their inhibitory strength. The inhibitors were
dissolved in water or DMF (the solvent proved nonꢀinhibitory up to a 20% concentration).
The reactions were stirred throughout the measurement period. The enzymatic hydrolysis of
urea was initiated by the addition of 500 µL of urease solution (final concentration 113.5 pM)
to the reaction mixture (final volume 5 mL) and monitored for 120 min by measuring the
ammonia concentration using a phenolꢀhypochlorite method in samples removed from the
reaction mixtures at set time intervals. For the unincubated reaction and when the inhibitor
ꢒ ꢓ
concentration was much higher than that of the enzyme (
ꢒ ꢓ
≫ ꢎ ), nonlinear reaction
ꢂ
ꢅ
ꢅ
progress curves were described using the integrated equation: ꢒꢔꢓ = ꢇꢋꢕ + (ꢇꢁ + ꢇꢋ)(1 −
ꢜ
ꢛ
ꢖꢗꢘ
)
(where P is the concentration of the reaction product formed at time t, which is
ꢙꢚꢚ
ꢘ
ꢙꢚꢚ
the reaction time, ꢇꢁ and ꢇꢋ are the reaction initial and steadyꢀstate rates, respectively, ꢌꢉꢍꢍ is
the apparent firstꢀorder rate constant for the interconversion between ꢇꢁ and ꢇꢋ). Control
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curves in the absence of inhibitor were linear. The equilibrium dissociation constants of the
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initial ꢎꢂ and final enzyme conformation ꢎꢂ∗ complex, known as inhibition constants (
ꢀ and
ꢁ
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ꢜ
ꢟ
ꢜ
ꢥ1 + ꢟꢠꢦ or
ꢠ
ꢀ∗ , respectively) were determined using equation:
=
ꢂ + ꢝ
ꢞ
ꢁ
ꢝ
ꢞ
ꢝ
ꢣ ꢟ
ꢤ
ꢣ
ꢤ
ꢡꢙꢢ
ꢡꢙꢢ
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ꢜ
ꢟ
ꢜ
ꢠ
=
∗ ꢂ + ꢝ
ꢥ1 + ꢟꢠꢦ (where ꢀꢆ and ꢇꢈꢉꢊ are the MichaelisꢀMenten constant and
ꢝ
ꢧ
ꢝ
ꢣ ꢟ
ꢤ
ꢣ
ꢤ
ꢡꢙꢢ
ꢡꢙꢢ
ꢞ
maximum rate of enzyme in a noninhibited reaction, S and I are substrate and inhibitor
concentration). The mechanism of inhibitor binding was determined using steadyꢀstate kinetic
measurements. The enzyme (final concentration 113.5 pM) was preincubated with increasing
concentrations of inhibitors in separate aliquots of reaction buffer prior to reaction initiation
through the incorporation of urea at a concentration ranging from 1 to 60 mM. The incubation
time was different for the assayed inhibitors, depending on when the equilibrium between
enzyme (ꢎ), inhibitor (ꢂ) and enzymeꢀinhibitor complexes (ꢎꢂ and ꢎꢂ∗
↔ ꢎꢂ ↔ ꢎꢂ∗) was achieved. The
: ꢎ + ꢂ
ꢀ
ꢁ
values were determined using LineweaverꢀBurk plots
after testing at least 5 inhibitor concentrations. (ii) H. pylori urease. Inhibition studies
(compounds 2, 5a, 5b, 6a, 6b) were conducted through the initiation of the enzymatic reaction
with the addition of the H. pylori urease (final concentration 488 pM) into assay mixtures
(200 µL total volume) containing increasing concentrations of inhibitors and 0.1–2.5 mM
urea. The
ꢀ values were determined using the LineweaverꢀBurk plots after testing at least 5
ꢁ
inhibitor concentrations in the range depending on their inhibitory strength.
Urease activity was determined using the Berthelot colorimetric reaction as previously
reported.58 The
ꢀ values for all analyzed compounds were run in triplicate.
ꢁ
Urease inhibition in expression strain E. coli Rosetta E. coli (pGEM::ureOP) was grown in
150 mL LB broth (Biocorp, Poland) containing 100 µg/mL of ampicillin (Polfa, Tarchomin,
Poland) at 37oC. When the culture reached the exponential growth phase (OD600~0.6), urease
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expression was induced with 0.75 mM IPTG and 0.75 mM Ni2+ for 24 hours at 22°C. Culture
samples (1 mL) were harvested through centrifugation (2 min, 20 376 x g, 37oC) and washed
with 10 mM PBS three times. The cell density in washed suspensions was adjusted using 0.5
McFarland standard. Urease assays were conducted in 96ꢀwell plates (Rotilabo, Uꢀprofile), in
300 µL total volume of 10 mM PBS containing 5 mM urea and various inhibitor
concentrations. The reactions were initiated through the incorporation of washed cells to a
final concentration of 1ꢃ109 cfu/mL. When necessary, the cells were preincubated for 2 h with
inhibitory compounds after which the reaction was initiated through addition of concentrated
urea solution, and 50ꢀµL aliquots of the reaction mixture were collected at different time
points and used in ammonia concentration determination using the Berthelot assay.59
Reactions containing no inhibitor were conducted as untreated reference. The influence of
DMF at concentrations present in several inhibitors stock solutions upon whole transformed
cells ureaꢀsplitting activity was also determined. All measurements were repeated three times.
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Urease inhibition in whole H. pylori J99 cells The strain was stored at ꢀ70°C in Tryptic Soy
Broth (Oxoid) with 15% glycerol. The cells were revived and grown on Columbia agar
(Difco) with 7% hemolyzed horse blood and selective Columbia agar containing 7%
hemolyzed horse blood plus Helicobacter Selective Supplement (Dent) from Oxoid. Cultures
were incubated for 3 days under microaerophilic conditions (5% O2, 10% CO2, and 85% N2)
at 37°C and subsequently passaged into new Columbia agar media and incubated for 3 more
days under the same conditions. H. pylori isolates were harvested with a sterile swab and
transferred to 10 mL Brucella broth (Oxoid) containing 5% fetal bovine serum (Sigma) and
1% IsoVitalex (from BBL). The cultures were grown at 37°C for 48 hours with vigorous
shaking, under microaerophilic conditions in anaerobic jars using Genbox microaer kits
(bioMerieux). The cultures were centrifuged once and resuspended in 10 mM PBS pH 7.2.
Additional washing was not performed to retain most of the extracellular urease on the cell
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surface. Urease assays containing approximately 1ꢃ107 cfu/mL of H. pylori cells were
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performed as described above. All measurements were repeated three times.
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Molecular modeling. The crystal structure of native S. pasteurii urease with 1.58ꢀÅ
resolution (PDB id 4CEU) was used as the starting point for all calculations.60 The Discovery
Studio v. 4.1 (BIOVIA) package was used. Hydrogen atoms were added automatically
assuming a pH of 7.0, and subsequently, the protonation of amino acid residues forming
active sites was checked and adjusted manually. The Cys322 residue was modified through
the attachment of an appropriate selenoorganic inhibitor molecule. At least two starting
conformations of inhibitor were prepared. The partial charges of all atoms were computed
using the MomanyꢀRone algorithm. Minimization was performed using the Smart Minimizer
algorithm and the CHARMm force field, up to an energy change of 0.0 or RMS gradient of
0.1. All residues that did not form an active site cleft were frozen. No implicit solvent model
was applied. The nonbond radius was set to 14 Å.
ASSOCIATED CONTENT
Supporting Information
The information concerning the IC50 initial and steady state inhibitory constants, example of
progress curves and LineweaverꢀBurk plot for inhibition of S. pasteurii urease by ebselen,
purity, analytical HPLC and HRꢀESI MS data of studied compounds is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding author
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* (Ł. B.), Phone: +48 71 320 3344, Fax: +48 71 320 2427, Eꢀmail:
lukasz.berlicki@pwr.edu.pl
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Notes
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The authors declare no competing financial interest.
Funding Sources
K. M. is grateful for support from the National Science Centre, Poland (grant no.
2015/17/N/NZ1/00027). A. G. acknowledges funding from the National Science Centre,
Poland (grant no. 2011/03/B/NZ6/04964).
ACKNOWLEDGMENTS
The Discovery Studio package was used under a Polish countrywide license. The software
resources (BIOVIA Discovery Studio program package) from the Wrocław Centre for
Networking and Supercomputing were used in the present study. The plasmid pGEM::ureOP
was a kind gift from Prof. Stefano Ciurli (University of Bolognia, Italy). This project was
financially supported through the Wroclaw Centre of the Biotechnology Program The
Leading National Research Centre (KNOW) for the years 2014–2018.
ABBREVIATIONS
AHA,
acetohydroxamic
acid;
DMAP,
4ꢀdimethylaminopyridine;
HOBt,
1ꢀ
hydroxybezotriazole; HPU, Helicobacter pylori urease; IPTG, isopropyl βꢀDꢀ1ꢀ
thiogalactopyranoside; NMM, Nꢀmethylmorpholine; SPU, Sporosarcina pasteurii urease;
TSB, tryptic soy broth.
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