Potent covalent inhibitors of bacterial urease identified by activity-reactivity profiling

Article abstract of DOI:10.1016/j.bmcl.2017.02.022

Covalent enzyme inhibitors constitute a highly important group of biologically active compounds, with numerous drugs available on the market. Although the discovery of inhibitors of urease, a urea hydrolyzing enzyme crucial for the survival of some human pathogens, is a field of medicinal chemistry that has grown in recent years, covalent urease inhibitors have been rarely investigated until now. Forty Michael acceptor-type compounds were screened for their inhibitory activities against bacterial urease, and several structures exhibited high potency in the nanomolar range. The correlation between chemical reactivity towards thiols and inhibitory potency indicated the most valuable compound — acetylenedicarboxylic acid, with K_i^?=42.5 nM and logk_GSH=-2.14. Molecular modelling studies revealed that acetylenedicarboxylic acid is the first example of highly effective mode of binding based on simultaneous bonding to a cysteine residue and interaction with nickel ions present in the active site. Activity-reactivity profiling of reversible covalent enzyme inhibitors is a general method for the identification of valuable drug candidates.

Full text of DOI:10.1016/j.bmcl.2017.02.022

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent covalent inhibitors of bacterial urease identified by
activity-reactivity profiling
a
b
a
c
c
Katarzyna Macegoniuk , Rafał Kowalczyk , Anna Rudzi n´ ska , Mateusz Psurski , Joanna Wietrzyk ,
Łukasz Berlicki
a
a,
⇑
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze z_ e Wyspia n´ skiego 27, 50-370 Wrocław, Poland
Department of Organic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze z_ e Wyspia n´ skiego 27, 50-370 Wrocław, Poland
Laboratory of Experimental Anticancer Therapy, Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of
b
c
Sciences, Weigla 12, 53-114 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Covalent enzyme inhibitors constitute a highly important group of biologically active compounds, with
numerous drugs available on the market. Although the discovery of inhibitors of urease, a urea hydrolyz-
ing enzyme crucial for the survival of some human pathogens, is a field of medicinal chemistry that has
grown in recent years, covalent urease inhibitors have been rarely investigated until now. Forty Michael
acceptor-type compounds were screened for their inhibitory activities against bacterial urease, and sev-
eral structures exhibited high potency in the nanomolar range. The correlation between chemical reac-
Received 5 January 2017
Revised 8 February 2017
Accepted 9 February 2017
Available online xxxx
Keywords:
Michael acceptors
Cysteine
Glutathione
Cytotoxicity
Thiols
tivity towards thiols and inhibitory potency indicated the most valuable compound
—
ꢀ
acetylenedicarboxylic acid, with K ¼ 42:5 nM and logkGSH ¼ ꢁ2:14. Molecular modelling studies
i
revealed that acetylenedicarboxylic acid is the first example of highly effective mode of binding based
on simultaneous bonding to a cysteine residue and interaction with nickel ions present in the active site.
Activity-reactivity profiling of reversible covalent enzyme inhibitors is a general method for the identifi-
cation of valuable drug candidates.
Ó 2017 Elsevier Ltd. All rights reserved.
Inhibitors binding covalently to the residues present in the
enzyme active site constitute a large group of biologically active
compounds of significant value.1 Numerous covalent drugs, rang-
ing from aspirin (discovered at the end of nineteenth century) to
ibrutinib (approved by the FDA in 2013 for Mantle Cell Lymphoma
treatment and in 2014–2016 for various leukemias), are available
most widely studied groups of covalent drug candidates. Although
cysteine proteases can be considered the primary target for this
,2
5
type of molecules, inactivation of non-catalytic cysteine residues
6
is also of interest. Recently, reversibly binding covalent inhibitors
have received attention because they are considered to be less
7
–9
prone to off-target modifications.
However, the main factor gov-
3
on the market. Although covalent enzyme inhibitors exhibit
erning the nonspecific binding and related toxicity and/or side
effects is the chemical reactivity of Michael acceptors towards
numerous crucial advantages resulting from their strong binding
to the target, they are often considered less attractive as drug can-
didates in comparison to non-covalent binding compounds
because of drawbacks in the later stages of drug studies; in partic-
ular, specificity and toxicity issues are of great concern. These
problems are often not predictable and are observed only for some
1
0–12
the –SH group.
Urease, an enzyme catalyzing the hydrolysis of urea,13 is an
interesting target for cysteine-binding compounds, although this
type of urease inhibitor has been rarely explored. Urease is related
to the development of infections caused by pathogenic bacteria,
such as Helicobacter pylori (gastroduodenal tract) and Proteus mir-
4
patients.
1
4,15
Michael acceptor-based enzyme inhibitors reacting with a cys-
teine residue in the active sites of proteins constitute one of the
abilis (urinary tract).
H. pylori produces large quantities of both
intra- and extracellular urease to increase the pH of its microenvi-
ronment by ammonia released during enzymatic urease hydroly-
1
6,17
sis.
Some urinary tract colonizing bacteria (e.g., Proteus
Abbreviations: AHA, acetohydroxamic acid; GHS, glutathione; PBMC, peripheral
blood mononuclear cells; PI, propidium iodide; SRB, sulforhodamine B; Tol, tollyl.
mirabilis) also produce urease, which causes rapid alkalinization
of urine and precipitation of inorganic salts, forming stones (stru-
⇑
Corresponding author.
E-mail address: lukasz.berlicki@pwr.edu.pl (Ł. Berlicki).
1
8–20
vites and/or apatites).
The goal of combatting the above-men-
http://dx.doi.org/10.1016/j.bmcl.2017.02.022
960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Macegoniuk K., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.022

2
K. Macegoniuk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
tioned pathogens stimulated research on urease inhibitors21–23 and
design assumption concerning the reaction with the cysteine resi-
due present at the entrance to the active site. Most probably, the
presence of neighboring histidine residue (His323), which could
act as a base, makes the covalent modifications of cysteine residue
reversible.
There are several interesting structure-activity relationships
visible in the study. First, the geometry of the central unsaturated
bond has a significant influence on the activity. E isomers of substi-
tuted double bond- and triple bond-containing compounds are
capable of urease inhibition, whereas Z isomers are completely
inactive against the target enzyme (e.g., 3a and 8a versus 4b or
3h and 8c versus 4a).
2
4–28
led to the discovery compounds such as phosphoramidates,
2
9–32
33–36
hydroxamates,
compounds.
phosphinates,
and
heterocyclic
3
7,38
Currently, only one urease inhibitor available on
39
the market, namely, acetohydroxamic acid, shows significant
side effects, including teratogenicity.40
The active site of urease contains two nickel ions coordinated by
four histidine residues (His137, His139, His249, His275), car-
bamoylated lysine (Kcx220) and aspartate (Asp363).41–43 The
active site is of relatively small volume (related to the size of urea)
and is covered by a movable flap (Fig. S8). This flap contains a cys-
teine residue that could be targeted by inhibitors.44 Currently, only
a limited number of urease inhibitors possess this mode of action,
including benzoquinone, ebselen derivatives, cyclohexanone and
Second, carboxylic acids showed similar, or in some cases even
higher, activity than analogous alkyl esters (e.g., 1a versus 1b, 3a
versus 3h, 8a versus 8c). This finding is of primary importance
because carboxylic acid-containing Michael acceptors are gener-
ally much less chemically reactive towards –SH groups than anal-
ogous esters. Therefore, the high activity of acids is related to their
specific interactions with the enzyme (see the following section).
Third, in most cases, the substituents of phenyl groups or
changes in ester functional groups have minor influence on the
activity (e.g., 1b versus 1d versus 1e or 3a-3f). This observation sug-
gests that these groups do not influence inhibitor binding.
Any compound showing high chemical reactivity with func-
tional groups available in biological systems, in particular –SH
groups, has to be avoided in medicinal chemistry due to unpre-
dictable influence on other biomolecules. Unspecific toxicity is cor-
4
5–50
cyclopentenone.
The product of reaction of urease and benzo-
51
quinone has been recently characterized structurally.
In this paper, we aimed to establish a general methodology to
evaluate the activity-reactivity profile of reversible Michael accep-
tor-type inhibitors based on studies of urease inhibitors. The vari-
ous types of Michael acceptors were extensively screened for their
inhibitory properties against bacterial urease.
The measured inhibitory activities were correlated with the
chemical reactivity (towards a model compound, glutathione) of
the studied compounds to find molecules that exhibit favorable
profiles related to specific interactions with the enzyme. Moreover,
the cytotoxicity (towards mouse fibroblasts BALB/3T3) of the cho-
sen compounds was analyzed. The most important findings were
also rationalized by modelling the structures of the inhibitor-
enzyme complexes.
A group of forty compounds of non-extended, Michael acceptor-
type compounds was selected for screening. These compounds
include molecules containing unsaturated functional groups of
various geometries: E and Z isomers of substituted double bonds
and linear triple bonds or allenes. All groups controlling the chem-
ical reactivity of double/triple bonds contained carbonyl groups
but showed significant differences in activating potency. Ketone
and ester functional groups are considered to activate strongly,
whereas carboxylates are much less reactive. Moreover, analogues
of known inhibitors, cyclopentenone (10a) and cyclohexanone
1
0
related with high reactivity of Michael acceptors. Therefore, the
preferred profile of the Michael acceptor-based active compound
is based on a combination of high inhibitory activity and low reac-
tivity towards thiols. The reactivity of representative compounds
was assayed using glutathione (GSH) as a model compound with
reactive cysteine residues. The modified Ellman method, where
the quantification of unreacted GSH was performed using a spec-
trophotometric-based
concentration-response
assay,
was
5
2
applied. The reactions of the extremely reactive Michael acceptor
compounds (3a, 8a) with GSH are completed within less than
15 min, whereas compound 1b demonstrates no reactivity, even
after 36 h of reaction (examples of progress curves and 1H NMR
spectra of reaction mixtures are shown in Figs. S3–S5, respec-
tively). The tested compounds spanned nearly six orders of magni-
tude of reactivity, with logkGSH values between ꢁ3.138 ± 0.077 and
2.588 ± 0.088 for ethyl cinnamonate (1a) and dimethyl
acetylenedicarboxylate (8a), respectively (Table S4). The depen-
(9a), were also included in the study for comparison.
All compounds were tested against model bacterial urease puri-
TM
fied from Sporosarcina pasteurii CCM 2056 with specific activity
of 2451 U/mg. The values of the kinetic parameters
ꢁ
1
(K
M
¼ 4:92 ꢂ 0:31 mM and vmax ¼ 4:224 ꢂ 0:060 mM s ) of highly
purified urease in an enzymatic reaction were determined by fit-
ting the initial reaction velocities measured over a range of urea
concentrations to the Michaelis-Menten equation by nonlinear
regression. The majority of the assayed compounds exhibited inhi-
bitory activity in the micromolar or nanomolar range (Table 1). The
nonlinear progress curves indicated that Michael acceptors pro-
duced time-dependent inhibition of urease activity, in which
dence between pK and logkGSH, shown graphically in Fig. 1, allows
i
for classification of the studied compounds into three groups. Com-
pounds from the first group show roughly linear dependence
between activity and reactivity (1a, 8a, 9a, 9c, 10a, 10b), which
suggests their nonspecific interaction with the enzyme. The second
group contains structures with high chemical reactivity and mod-
erate inhibitory activity (2a, 2b, 3a). In this case, the interactions of
the compound with the enzyme are specific but unfavorable due to
steric impairment. The third group is of the highest importance
because it combines compounds with a preferable profile: high
inhibitory potential and low –SH reactivity (1b, 3h, 8c). In these
cases, inhibition of the enzyme is driven not only by –SH reactivity
but also specific interactions between the ligand and protein. The
specificity of compounds 1b, 3h, 8c was additionally confirmed
by complete lack of inhibition of model cysteine-dependent
enzyme – papain. On the basis of the discussed graph, compounds
of interest are easily indicated; in this particular case, acetylenedi-
steady-state velocity (
) and steady-state velocity (
inhibitor concentration over the examined ranges. The linear
replots of 1= and 1= showed a slow binding mode of action
v
s
) was attained slowly with both the initial
(v
i
v
s
) decreasing with an increase in
v
i
v
s
according to mechanism B, in which the initial EI complex under-
ꢀ
goes conformational change into the final EI complex. The steady
À
Á
ꢀ
state inhibition constants
K
ranged from 0.00977 to 64.2 lM
for compounds 8a and 3e, respectively. Importantly, the potency
of some of the assayed compounds (5, 6a, 8a, 8b, 8c, 9a, 9b, 9c,
iLB
9
f, 10a and 10b) exhibited higher activities than the reference inhi-
ꢀ
bitor, acetohydroxamic acid (AHA). All inhibitors demonstrated
competitive and reversible type inhibition, as confirmed by Line-
weaver-Burk plots and fast dilution assay (data not shown). This
mode of action of the studied inhibitors is consistent with the
carboxylic acid (8c) with KiLB ¼ 42:5 nM and logkGSH ¼ ꢁ2:14 has
the most beneficial activity-reactivity profile.
The cytotoxicity towards normal mouse fibroblasts (BALB/3T3)
for
a group of selected Michael acceptor compounds was
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K. Macegoniuk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
Inhibitory activity of Michael acceptor-based compounds against S. pasteurii urease (detailed inhibitory characteristics are given in Table S3) and cytotoxicity (LC50) towards
mouse fibroblasts (BALB/3T3) of the selected compounds.
ꢀ
no
Compound
R
K
[lM]
LC50 [mM]
i LB
0
R
BALB/3T3
SRB assay
R
COOR'
1a
1b
1c
1d
1e
1f
Ph
Ph
Et
H
Et
H
Et
H
NI
100 mM >; 29.09%a
NT
21.5 ± 2.1
NI
34.2 ± 2.8
NI
p-Tol
p-Tol
p-NO
p-NO
O
2
-C
-C
6
H
H
4
-
-
2
6
4
11.9 ± 1.1
R
Ph
p-Tol
COOR'
COOR'
2
2
a
b
Et
H
3.956 ± 0.026
6.36 ± 0.45
24.5 ± 5.4
100 mM >; 31.28%a
ROOC
3
3
3
3
3
3
3
3
a
b
c
d
e
f
Me
Et
iPr
Bu
sBu
Et
Me
Et
21.3 ± 1.7
11.1 ± 1.0
42.4 ± 2.7
53.7 ± 3.7
64.2 ± 3.5
22.1 ± 1.8
52.4 ± 5.0
36.5 ± 3.2
39.7 ± 7.0
iPr
Bu
sBu
tBu
H
g
h
Et
H
H
NT
ROOC
COOR'
4
4
a
b
H
Me
H
Me
NI
NI
O
0.0928 ± 0.0076
5
EtOOC
COOEt
COR
.
OEt
NHBn
R
6
6
a
b
2.52 ± 0.20
NI
COOR'
7
7
7
7
7
7
a
b
c
d
e
f
H
H
Me
Me
Ph
Me
H
Et
H
Et
H
46.0 ± 4.1
44.4 ± 3.5
126 ± 12
109.3 ± 8.7
52.3 ± 4.5
37.1 ± 3.4
Ph
ROOC
Me
Et
COOR'
8
8
8
a
b
c
Me
Et
H
0.00977 ± 0.00084
0.01020 ± 0.00095
0.0425 ± 0.0036
29.8 ± 1.7
NT
H
O
R
9
9
9
9
9
9
a
b
c
d
e
f
H
0.766 ± 0.063
0.784 ± 0.071
0.694 ± 0.064
17.8 ± 1.6
21.2 ± 2.0
0.592 ± 0.045
100 mM >; 29.76%a
CH = CHCN
CH = CHCOOMe
CH = CHCOOH
CH = CHC(O)Me
CH = CHC(O)Ph
O
R
1
1
0a
0b
H
0.291 ± 0.016
0.285 ± 0.026
86.5 ± 5.4
CH = CHCOOMe
100 mM >; 35.23%a
COOR
ROOC
COOR
1
1
AHA
1a
1b
Me
H
5.21 ± 0.37
6.13 ± 0.48
3.30 ± 0.40
cis-platin
1.92 ± 0.63
NI – no inhibitory activity, NT – not toxic.
a
Percent of proliferation inhibition at 100 mM.
determined as the concentration required to inhibit 50% prolifera-
expected, compounds with low reactivity towards glutathione dis-
tion of the cell population (LC50) and is reported in Table 1. As
played little (1a, 9c, 10b) to no or almost no (1b, 3h, 8c) cytotoxic
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4
K. Macegoniuk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
9
8
7
6
5
4
3
2
1
0
Taking into account the result of cytotoxicity assay (Table 1)
and whole-cell inhibition of native P. mirabilis urease for com-
pound 3h and 8c (Table 2), it seems that this inhibitors may be
clinically useful for the treatment of the human urinary tract infec-
tions. Moreover, our results also emphasize the importance of
experiments analyzing the influence of the tested compounds on
living cells. This type of assay is indispensable step in the screening
of chemical compounds enabling to determine the side effects of
the tested inhibitors as well as their actual biological activities.
Analysis of the mode of binding for the most active inhibitor of
bacterial urease — compound 8c — was performed using a molec-
ular modelling approach. The detailed analysis of the active site of
urease revealed that the cysteine residue (Cys322) located at the
movable flap is accompanied by a histidine residue (His323). This
residue is expected to catalyze the reaction of the Michael acceptor
addition to Cys322. We assume that the mechanism of inhibition is
composed of two steps: reaction with Cys322 and rearrangement
to an energetically favorable conformation. This mechanism is in
agreement with the experimental results indicating slow binding
mechanism type B. In the case of compound 8c, which has a triple
bond, the reaction can lead to two potential configurations of the
product (E and Z). However, the enzyme is expected to act stereos-
electively, and modelling studies indicate the formation of the pro-
duct with E stereochemistry (both carboxylic acid groups placed at
the same side, Fig. 2). This arrangement is supported by two pieces
of evidence: (a) His323 (acting as a base) and Cys322 are located at
the same side of the inhibitor molecule; therefore, the sulfur and
hydrogen atoms should be added to the reacting molecule at the
same side (putative reaction mechanism given in Fig. S7); (b) the
final conformation of the adduct is thermodynamically more stable
in the case of E stereochemistry. Similar mode of binding was
observed in crystal structure of citrate-urease complex (PDB id
8a
8
c
1
0b 10a
9c
9a
2
3a
a
2b
1b
3h
1
a
-
5
-4
-3
-2
-1
0
1
2
3
logkGSH
Fig. 1. The inhibitory activity – chemical reactivity profile of selected Michael
acceptors (for detailed data, see Table S4).
effects (against BALB/3T3) compared to cis-platin as the positive
control. However, compounds with the highest chemical reactivity
2a, 8a) demonstrate significant toxicity both in the antiprolifera-
tive studies against BALB/3T3. These results are in agreement with
(
previous results on the relationships between various types of tox-
icity and –SH reactivity.1
0,53,54
and confirm that the applied
methodology of the selection of compounds of interest by the anal-
ysis of their activity-reactivity profile is appropriate.
The biological relevance of the inhibitors was studied in vitro
against Proteus mirabilis strain, which is a urease-positive pathogen
of the human urinary tract.55 P. mirabils urease is shows high
sequence similarity to S. pasteurii enzyme (Fig. S6). The whole-cell
urease inhibition studies were performed under conditions corre-
sponding to the acidity of physiological urine (pH 5.5). The effec-
tiveness of urease inhibition against intact cells was evaluated
for compounds 1b, 3h, 8c and 1a (negative control), and compared
with well-established acetohydroxamic acid — AHA (Table 2). In
5
6
4
AC7). The molecule of citric acid also chelates two nickel ions
by one carboxylate and forms hydrogen bonds between the second
carboxylate and Arg339 (see Fig. S9 for superimposition of these
two structures).
Compound 8c is the first example of an interesting mode of
binding, which combines the formation of a covalent bond with a
cysteine residue and interactions with two nickel ions. This inter-
action pattern is promising because inhibitors acting this way have
high activity and selectivity towards bacterial urease.
8
0
5
the presence of 5 mM urea using 1 ꢃ 10 cfu/mL, the IC values
0
in non-preincubated assays were in range from 169 to 492 mM
for AHA and compound 1b, respectively). The IC50 parameter after
h preincubation of cells with tested inhibitors was lower for all
(
2
compounds, what means that the degree of inhibition increased
with time. The highest activity enhancement was observed for
Covalent inhibitors of urease are a virtually unexplored field,
and the presented results considerably increased the number of
2
0
h
2h
50
3
h (IC5 ¼ 65:5 ꢂ 7:2 mM) and 8c (IC ¼ 88:6 ꢂ 9:3 mM), this classi-
fied them as more potent urease inhibitors than AHA
2
0
h
(
IC5 ¼ 91:4 ꢂ 8:8 mM). The non-preincubated system likely
requires time to allow the interaction between the enzyme and
the inhibitor. This is related to the fact that the studied compounds
constitute a time-dependent class of urease inhibitors. Secondly,
the P. mirabilis urease is located only in the cell cytoplasm, there-
fore the inhibitory efficiency is strongly dependent on the com-
pound diffusivity. The obtained result show that the newly
developed small structures are able to cross bacterial cell mem-
branes and inhibit urease in intact bacterial cells.
Table 2
Whole-cell inhibition (IC50) of native P. mirabilis urease.
Compound
0
2 h
IC [mM]
IC [mM]
5
0
50
1
1
3
8
a
b
h
c
NI
NI
492 ± 54
182 ± 17
326 ± 38
169 ± 15
125 ± 14
65.5 ± 7.2
88.6 ± 9.3
91.4 ± 8.8
Fig. 2. Modelled structure of the product of addition of compound 8c to S. pasteurii
urease. The movable flap is shown as a solid ribbon, residues of the active site are
presented as sticks, and metal ions are shown as blue spheres. Non-covalent
interactions of the inhibitor are shown as solid green lines. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
this article.)
AHA
0
5
IC – parameter assayed without preincubation of cells with inhibitory com-
0
2
5
h
0
pounds; IC – parameter assayed using cells subjected to 2 h of preincubation with
inhibitors in PBS; NI – no inhibitory activity.
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K. Macegoniuk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
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2
011/03/D/ST5/05766. The Discovery Studio package was used
under a Polish country-wide license. The use of software resources
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Please cite this article in press as: Macegoniuk K., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.022