N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases

Article abstract of DOI:10.1021/acs.jmedchem.0c00584

In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-Aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.

Full text of DOI:10.1021/acs.jmedchem.0c00584

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Article
N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting
Pseudomonas aeruginosa Elastase and Clostridium Collagenases
Jelena Konstantinovi#, Samir Yahiaoui, Alaa Alhayek, Jörg Haupenthal, Esther
Schönauer, Anastasia Andreas, Andreas M Kany, Rolf Mueller, Jesko Köhnke, Fabian
Berger, Markus Bischoff, Rolf W. Hartmann, Hans Brandstetter, and Anna K.H Hirsch
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2020
Downloaded from pubs.acs.org on May 30, 2020
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N-Aryl-3-mercaptosuccinimides as Antivirulence
Agents Targeting Pseudomonas aeruginosa Elastase
and Clostridium Collagenases
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Jelena Konstantinović , Samir Yahiaoui , Alaa Alhayek
, Jörg Haupenthal , Esther
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Schönauer , Anastasia Andreas , Andreas M. Kany , Rolf Müller , Jesko Köhnke ,
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Fabian K. Berger , Markus Bischoff , Rolf W. Hartmann , Hans Brandstetter , Anna K.H.
Hirsch1,2,*
1Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI), Campus
Building E8.1, 66123 Saarbrücken, Germany
2Department of Pharmacy, Saarland University, Campus Building E8.1, 66123
Saarbrücken, Germany
3Department of Biosciences, University of Salzburg, Billrothstr. 11, 5020 Salzburg, Austria
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4Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI), Campus
Building E8.1, 66123 Saarbrücken, Germany
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5Workgroup Structural Biology of Biosynthetic Enzymes, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Campus Building E8.1, 66123, Saarbrücken,
Germany
6Institute of Medical Microbiology and Hygiene, Saarland University, 66421
Homburg/Saar, Germany
Key words: virulence factor, LasB, ColH, metalloprotease inhibitor, succinimide, thiol,
Hathewaya histolytica
ABSTRACT: In light of the global antimicrobial-resistance crisis, there is an urgent need
for novel bacterial targets and antibiotics with novel modes of action. It has been shown
that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya
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histolytica) collagenase (ColH) play a significant role in the infection process and thereby
represent promising antivirulence targets. Here, we report novel N-aryl-3-
mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent
activities in vitro and high selectivity for the bacterial over human metalloproteases.
Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human
cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH
inhibitor shows a significant reduction of collagen degradation in an ex vivo pig skin
model.
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▪ INTRODUCTION
The growing number of antibiotic-resistant bacteria represents one of the biggest risks
to public health, leading to an enhancement of infections that are difficult to treat. Bacterial
resistance to antibiotics is natural, yet overuse and misuse of antibiotics accelerate
resistance development, bringing the world to the verge of the so-called “post-antibiotic
era”. Of special importance are infections caused by multi-drug resistant bacteria on the
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WHO priority-pathogen list, such as carbapenem-resistant variants of the Gram-negative
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pathogen Pseudomonas aeruginosa, which are responsible for many nosocomial, eye
and burn infections,4,5 as well as fatal lung infections in cystic-fibrosis and bronchiectasis
patients.6 P. aeruginosa also affects injured tissue such as skin via surgical or wound
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infections. The versatile pathogen is known to produce numerous virulence factors. One
of them is elastase, the metalloenzyme that shows hydrolytic activity towards connective
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tissue, which significantly contributes to the effectiveness of this bacteria. Clostridiaceae
represent a family of Gram-positive bacteria that are known as causative agents of
numerous fatal diseases with high mortality rates worldwide, such as botulism
(
Clostridium botulinum), soft-tissue infections like gas gangrene and wound infections
(Clostridium perfringens, Clostridium histolyticum) and tetanus (Clostridium tetani).1
1,12
Bacillus cereus is another Gram-positive bacterium responsible for foodborne illnesses
and traumatic wound infections in humans.1
3,14
The high lethality of these bacteria is
closely related to the production of collagenases, extracellular enzymes that enable the
bacteria to colonize specific niches in the host, to evade the host immune response and
to obtain nutrition from infected cells. Moreover, collagenases cause tissue destruction
via collagen degradation, which plays a significant role in the infection process by allowing
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the bacteria to reach anaerobic sites in host tissue and spread the infection.1
5,16
This
especially affects the wound-infection prognosis and results in delayed healing
process.1
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Recently, particular emphasis has been put on targeting bacterial virulence as an
alternative approach for fighting microbial infections. The pursued ‘pathoblockers’
preserve the commensal microbiome and are expected to be less susceptible to the
development of resistance than conventional antibiotics. In our work, we focus on two
zinc metalloproteases that are secreted virulence factors: elastase (LasB) from P.
aeruginosa and collagenase H (ColH) from C. histolyticum (recently renamed as
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Hathewaya histolytica ). Both enzymes have a major impact on the infectivity of P.
aeruginosa and C. histolyticum.11,20 Moreover, their extracellular localization makes these
enzymes particularly attractive targets, considering the difficulties associated with
crossing the Gram-negative bacterial cell walls in the former species.
Most of LasB and ColH inhibitors found in the literature contain different metal-chelating
warheads.20,21,22,23,24 Among them, hydroxamates represent the most common structural
motif.21,25,26,27 The main problem with such hydroxamate-containing protease inhibitors is
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their lack of stability under physiological conditions and their lack of selectivity over human
matrix metalloproteases (MMPs), which makes them unsuitable candidates for
antibacterial treatment in vivo26,28 and rationalizes why there is still no drug on the market
that could effectively inhibit these virulence factors. A selection of LasB and ColH
inhibitors described in the literature is shown in Figure 1.
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NH
S
O
O
H
HS
N
N
NH2
HO
N
H
O
1
2
OH
SH
IC50 (LasB) = 2.73 ± 0.03 M
Ki (LasB) = 40.5 nM
O N
O
2
H
N
S
HO
O
3
N
H
O
NH
K (ColH) = 5 nM
i
HN
O
N
N
N
F
SO2
F
NH
O S
2
F
4
K (ColH) = 0.2 M
i
F
F
F
Figure 1. Structural motifs of some LasB and ColH inhibitors described in the literature
(
Zn-binding groups are highlighted in blue).21,22,24,26
In our previous work, we discovered N-aryl mercaptoacetamide inhibitors with low
micromolar and nanomolar affinities toward LasB and ColH, respectively.29,30 To constrain
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the flexibility and freeze the active conformation of our previously published thiols, we
designed a novel succinimide class (Figure 2). The succinimide core has been reported
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for inhibitors of various enzyme targets, such as serine proteases, human leukocyte
32
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elastase, cathepsin G and proteinase 3, tumor necrosis factor and phosphodiesterase.
It can also be found in several drugs, such as ethosuximide, phensuximide, methsuximide
and lurasidone, used to treat absence seizures, schizophrenia and bipolar disorder.34
O
H
N
SH
SH
N
R
O
R
O
N-aryl mercaptoacetamides
3-mercapto-N-arylsuccinimides
5: R= 3,4-di-Cl (IC50 (LasB) = 6.6 ± 0.3 µM)
6: R= 4-Ac (IC50 (ColH) = 0.017 ± 0.002 µM)
Figure 2. Our previous results and the design of new inhibitors.29,30
Here, we report new N-aryl-3-mercaptosuccinimides, showing low micromolar
potencies against P. aeruginosa elastase and nanomolar potencies against Clostridium
collagenases. The most active compounds were investigated for their cytotoxicity and
selectivity for the bacterial over human metalloproteases. To validate collagenases as
targets, we have established an ex vivo pig skin model and demonstrated the impact of
our most potent inhibitor on this human skin mimic.
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▪ RESULTS AND DISCUSSION
Design of new compounds. We designed the initial succinimide core based on our
previously published N-aryl mercaptoacetamide inhibitors.29,30 To expand the structure–
activity relationships (SAR) further and provide more detailed information on the aromatic
moiety’s influence on the activity, we designed a series of compounds bearing polar,
lipophilic, electron-withdrawing or electron-donating functional groups. In order to prevent
disulfide formation, we protected the free thiol group in the form of thioacetate. Finally,
we explored the possibility of growing the structure further by introducing an additional
carbon spacer between the succinimide and the free thiol.
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Synthesis of new compounds. Reaction of anilines with mercaptosuccinic acid at 120–
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60 °C afforded 21 new free thiol-containing succinimides 7–27 with yields of 6–68%. Due
to the presence of an ortho-substituent, compounds 16–18 and 21 were obtained as
mixtures of atropisomers. Acetic anhydride in presence of pyridine and DMAP at room
temperature led to protection of the free thiol group to obtain derivatives 28–30 with
moderate yields (65–78%). The general synthetic route is presented in Scheme 1.
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: R = H (51%)
8: R = 4-Me (47%)
9: R = 3,4-di-Me (34%)
1
1
0: R = 2-Cl (66%)
1: R = 3-Cl (55%)
SH
12: R = 4-Cl (68%)
3: R = 3,4-di-Cl (56%)
O
S
1
NH2
14: R = 2,6-di-Cl (30%)
15: R = 3,4-di-F (50%)
16: R = 2-Me-5-Cl (35%)
17: R = 2,6-di-Me-3-Cl (54%)
18: R = 2-OMe (42%)
O
O
N
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a
b
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9: R = 3-OMe (30%)
0: R = 4-OMe (11%)
1: R = 2,4-di-OMe (25%)
2: R = 3,4-di-OMe (20%)
3: R = 3-Cl-4-OCF (58%)
R
2
8: R = 4-OMe (65%)
29: R = 3,4-di-Cl (78%)
0: R = 3-Cl-4-OCF (73%)
3
3
3
4: R = 3-NO (6%)
2
25: R = 4-Ac (32%)
26: R = 3-Ph (52%)
27: R = 4-Ph (40%)
o
Reagents and conditions: (a) mercaptosuccinic acid, 120160 C, Ar, neat, overnight; (b) Ac O, Pyr, DMAP, DCM, r.t., overnight
2
Scheme 1. Synthesis of novel free thiol succinimides and thioacetate derivatives
Synthesis of N-aryl-3-mercaptomethylsuccinimides started from itaconic anhydride and
corresponding anilines (Scheme 2). Cyclization of intermediate α-itaconamic acids 31,
35
3
2, 34 and 35 in presence of acetic anhydride and sodium acetate at 100 °C, afforded
itaconimides 36–39 with relatively low yields (8–39%). Michael addition of thioacetic acid
on obtained itaconimides in presence of triethylamine in dimethoxyethane at room
temperature, led to the final compounds 40–43 with high yields (61–99%). α-Itaconamic
acids 32–34 under the same reaction conditions in THF as a solvent, provided
compounds 44–46 with quantitative yields. Hydrolysis of thioacetate using sodium
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hydroxide in methanol at room temperature afforded free thiol-containing carboxylic acids
47–49 with moderate to quantitative yields (61–99%). A final neat cyclization step
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furnished target compounds 50–52 with low yields (14–36%).
O
S
OH
NH2
b
O
O
c
O
O
a
O
N
N
O
NH
R
R
R
R
31: R=H
36: R=H (33%)
40: R=H (94%)
3
7: R=3,4-diCl (8%)
8: R=4-Ac (10%)
41: R=3,4-diCl (61%)
42: R=4-Ac (99%)
43: R=4-OMe (91%)
3
3
3
3
2: R=3,4-diCl
3: R=3,4-diF
4: R=4-Ac
3
39: R=4-OMe (39%)
5: R=4-OMe
c
O
SH
OH
O
S
SH
O
OH
O
d
O
NH
e
O
N
O
NH
R
R
R
44: R=3,4-diCl (99%)
45: R=3,4-diF (99%)
46: R=4-Ac (99%)
47: R=3,4-diCl (99%)
48: R=3,4-diF (61%)
49: R=4-Ac (98%)
50: R=3,4-diCl (25%)
51: R=3,4-diF (36%)
52: R=4-Ac (14%)
Reagents and conditions: (a) itaconic anhydride, CHCl , 2 h, r.t.; (b) Ac O, NaOAc, 12 h, 100 °C; (c) AcSH, Et N, DME or THF,
3
2
3
r.t.; (d) 2 M NaOH, MeOH, 2 h, r.t.; (e) 120 °C, Ar, neat, overnight
Scheme 2. Synthesis of N-aryl-3-mercaptomethylsuccinimides and their acetylthio
analogues
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Activity against antivirulence targets. We evaluated all compounds synthesized in this
work for their inhibitory activity against both LasB and ColH. IC values and percentage
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of inhibition results were calculated from at least two independent experiments performed
in duplicates.
SAR of novel succinimides on LasB. To expand the SAR, we designed and synthesized
3
1 succinimide-based derivatives and investigated their inhibitory activity against LasB
3
6
using a functional FRET-based in vitro assay (Table 1). Among the first group of
compounds 7–27, electronegative substituents such as chlorine or fluorine were found to
be favorable for the activity. In particular, compounds 13 and 15, both with a 3,4-dihalo
pattern, displayed more potent inhibitory activities when compared to the ortho-, meta- or
para-monosubstituted analogues. Furthermore, several examples indicate that polar
groups, particularly electron-donating substituents are detrimental for the activity. In fact,
all mono and dimethoxy derivatives were less potent than their chlorine analogues, with
3,4-dimethoxy derivative 22 showing the most dramatic loss in activity (47-fold compared
to 13). This is further supported by the five-fold difference in activity between 3,4-dimethyl
(9) and 3,4-dimethoxy analogues (22). Among both chlorine and methoxy isomers, ortho-
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5
5
6
(
10 and 18) and meta-derivatives (11 and 19) proved to be more potent than the para-
derivatives (12 and 20), most probably due to electronic effects or disruptions of the planar
structure caused by the vicinity of the substituents to the succinimide core. Compound 25
with its polar electron-withdrawing para-acetyl substituent was 1.7 times more active than
its methoxy-analogue, but still much less potent than compounds bearing lipophilic
chlorine substituents. The observation that polar, electron-withdrawing substituents are
more tolerated than electron-donating ones, is illustrated through the example of nitro-
compound 24, being more active than its 3-methoxy analogue 19. Compounds with an
additional phenyl ring in positions 3 (26) and 4 (27) showed comparable activity to the
compound with a naked core, with 3-phenyl derivative even being 1.6–fold more active
than compound 7. Although these derivatives were less potent than compound 13, the
fact that they show inhibition of LasB paves the way for further optimization of this part of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
the structure. In addition, the fact that compounds 8 (R =4-Me) and 27 (R =4-Ph) show
no significant difference in the activity suggests that there are no steric limitations in para-
position. An additional carbon spacer next to the free thiol in compounds 50 and 51 did
not improve the activities of the most potent derivatives 13 and 15, respectively. However,
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the IC50 values determined for 50 and 51, being in the range of 5–10 μM, open the
possibility to further grow the structure in the direction of the free thiol group. All
thioacetate derivatives, with no exception, proved to be inactive, with <50% of inhibition
at 200 μM, which confirms that the free thiol is crucial for the activity against LasB.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Structures and LasB inhibition of a series of novel succinimide derivatives: 3,4-
di-halo pattern in 13, 15 and 50 proved to be beneficial for the activity.a
O
SR2
()n
N
O
R1
Cpd.
7
R1
R2
n
IC50 (μM)
Cpd.
23
R1
R2
n
IC50 (μM)
3-Cl-4-
H
H
0
44.2±2.3
H
0
55.2±4.4
OCF
3
8
9
4-Me
H
H
H
H
H
H
0
0
0
0
0
0
50.6±1.6
29.4±0.9
8.5±0.4
8.1±0.5
16.5±0.8
3.4±0.2
24
25
26
27
28
29
3-NO
4-Ac
3-Ph
4-Ph
2
H
0
0
0
0
0
0
22.2±0.4
64.0±7.5
27.6±4.0
44.6±1.1
>200
3,4-di-Me
2-Cl
H
10
H
11
3-Cl
H
1
2
3
4-Cl
4-OMe
Ac
Ac
1
3,4-di-Cl
3,4-di-Cl
>200
3
-Cl-4-
14
2,6-di-Cl
H
0
16.0±3.7
30
Ac
0
>200
OCF
3
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5
5
6
1
5
6
3,4-di-F
H
H
H
H
H
H
H
H
0
0
0
0
0
0
0
0
3.5±0.2
15.0±0.6
30.5±5.4
28.9±1.6
40.2±1.4
40
41
42
43
50
H
Ac
Ac
Ac
Ac
H
1
1
1
1
1
1
1
>200
>200
1
2-Me-5-Cl
2,6-di-Me-3-Cl
2-OMe
3,4-di-Cl
4-Ac
17
>200
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
9
4-OMe
3,4-di-Cl
3,4-di-F
4-Ac
>200
1
3-OMe
5.4±0.7
10.1±1.4
>200
20
4-OMe
111.8±8.9 51
H
2
1
2
2,4-di-OMe
3,4-di-OMe
45.0±0.8
52
H
2
160.2±10.1
aMeans and SD of at least two independent experiments.
SAR of novel succinimides on ColH. In our previous work, we have shown that there is
a structural similarity between the inhibitors of LasB and ColH.29,30 It was therefore of
interest to investigate the activity of all new compounds against ColH and to compare the
SAR with that observed for LasB. Figure 3 represents the inhibition of the peptidase
domain of ColH (ColH-PD) in the presence of 1 μM of the selected compounds.
Previously, we reported that polar substituents in para-position have the most beneficial
30
effect on the activity of N-aryl mercaptoacetamides. Here, we observed the same trend
with the new succinimide class, with compound 25, bearing a para-acetyl substituent,
being the most active one with 95% of ColH-PD inhibition. The para-methoxy derivative
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2
0 was slightly less active with 74% of inhibition, but still following the trend of polar
substituents being more favorable for the activity than non-polar, lipophilic substituents,
such as chlorine. The protection of the free thiol functional group proved to be detrimental
for the activity, as in the case of LasB. The two compounds showing >50% of inhibition of
ColH-PD were further tested in a dose-response manner in the presence of the reducing
agent TCEP. This experiment revealed nanomolar IC values for 25 (0.06 ± 0.01 μM)
1
1
1
1
1
1
1
1
1
1
2
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2
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2
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4
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5
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0
1
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
and 20 (0.32 ± 0.05 μM). Overall, since the inhibitors presented in this work have similar
structure and SAR we described for N-aryl mercaptoacetamides, we expect the
interactions with LasB and ColH to be similar as those in our previously published co-
crystal structures.29,30
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0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Inhibition of the peptidolytic activity of ColH-PD in the presence of 1 μM of the
respective compound in a FRET-based assay: para-acetyl substituent in compound 25
proved to be the most beneficial substitution for the activity. Data are presented as means
and SD of three independent experiments.
We consider the difference observed in SARs between LasB and ColH inhibitors
beneficial in terms of their selectivity for each particular target. Each hit can be further
developed for the treatment of single infections caused by either P. aeruginosa or C.
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histolyticum. However, wound infections, due to their nature, are likely to be colonized
with other bacteria.37,38 Therefore, having a common structural motif that inhibits both
targets is particularly interesting and if the inhibitors were to be used for Clostridia and P.
aeruginosa co-infections purpose, their structure could be further optimized and adapted
as dual inhibitors of ColH and LasB.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Broad-spectrum inhibition of other bacterial collagenases. In addition to ColH from C.
histolyticum, other Clostridium and Bacillus species also secrete collagenases that play
pivotal roles in the pathogenesis of these bacteria by destroying the connective tissue
components in the infected host.16 We therefore tested the two most active ColH-PD
inhibitors (20 and 25) on three additional collagenases, using the collagenase unit of ColG
(ColG-CU) from C. histolyticum, the peptidase domain of ColT (ColT-PD) from C. tetani,
and the collagenase unit of ColQ1 (ColQ1-CU) from B. cereus strain Q1. As anticipated,
the succinimide-based scaffold retained the broad-spectrum inhibitory properties of the
mercaptoacetamide-based inhibitors (Table 2).30
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Inhibition of ColH-PD, ColT-PD, ColG-CU and ColQ1-CU in presence of 100 μM
of compounds 20 and 25a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
%
inhibition @ 100 μM
Cpd.
20
ColH-PD
95 ± 1
ColG-CU
85 ± 3
ColT-PD
89 ± 4
ColQ1-CU
99 ± 1
25
96 ± 2
100 ± 2
102 ± 3
99 ± 2
aMeans and SD of at least two independent experiments.
Selectivity against MMPs, HDACs and TACE as human off-targets. Previously, we
described N-aryl mercaptoacetamides with high selectivity for the bacterial over a broad
range of human MMPs.29,30 MMPs are calcium-dependent zinc metalloproteases that play
a pivotal role in numerous biochemical processes in humans.39,40 Based on the depth of
their S1’ binding pocket, MMPs can be divided into three classes: deep (e.g., MMP-3 and
-14), intermediate (e.g., MMP-2 and -8) and shallow (e.g., MMP-1 and -7). With the aim
to explore the interactions of our inhibitors with all three pocket types, which could help
us to assess potential effects on other, not-tested representatives, we chose a panel of
six MMPs, comprising two members of each class. In addition, HDAC-3, HDAC-8 and
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TACE (ADAM-17) – enzymes involved in gene expression and the processing of
TNF-α41,42 respectively – were selected as important additional human off-targets. Our
results showed that the most potent inhibitors of LasB and ColH (13 and 25, respectively)
fortunately possess a high selectivity over most of the off-targets tested. While both
compounds did not inhibit MMPs -1, -3 and -7 as well as both HDAC enzymes, we
observed certain inhibition of MMP-2, -8 and -14 at 100 µM. Inhibition of TACE, which
was observed for both compounds, will be considered as high priority in the future
optimization of the structures (Tables 3 and 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Inhibition of six MMPs in presence of 100 μM of compounds 13 and 25a
%
inhibition @ 100 μM
Cpd.
13
MMP-1
n.i.
MMP-2
39 ± 32
14 ± 4
MMP-3
MMP-7
n.i.
MMP-8
84 ± 8
94 ± 1
MMP-14
n.i.
n.i.
n.i.
25
n.i.
n.i.
84 ± 8
aMeans and SD of at least two independent experiments. n.i. = <10% inhibition.
Table 4. Activity of compounds 13 and 25 against HDAC-3, HDAC-8 and TACEa
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5
5
6
IC50 (μM)
Cpd.
HDAC-3
>100
HDAC-8
>100
TACE
1
3
5
5.2 ± 0.6
3.4 ± 1.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
>100
>100
aMeans and SD of at least two independent
experiments.
Cytotoxicity assays. Broad-spectrum inhibition of bacterial collagenases and selectivity
against a panel of off-targets supported the further evaluation of the compounds’ toxicity
in vitro. In this context, we investigated 13 and 25, the two most active compounds against
both targets, for their cytotoxicity against the three human cell lines – HepG2
(
hepatocellular carcinoma), HEK293 (embryonal kidney) and A549 (lung carcinoma).
Neither compounds showed cytotoxic effects, with IC values > 100 μM (Table 5), making
50
them suitable for further investigation in in vivo model systems. Compared to our previous
hits, 5 and 6, they displayed similar or even lower toxicities in most of the cell lines tested.
Particularly, compound 25 proved to be even less toxic than 6, which showed an IC of
50
1
00 μM in HEK293 cells.
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Table 5. Cytotoxicity of compounds 13, 25, 5 and 6 against HepG2, HEK293 and A549
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cell linesa
IC50 (μM)
Cpd.
HepG2
>100
>100
>100
>100
HEK293
>100
>100
>100
100
A549
>100
>100
>100
>100
1
2
5
6
3
5
aMeans and SD of at least two independent
experiments
In vivo toxicity in zebrafish embryo model. Due to the promising in vitro activities against
antivirulence targets LasB and ColH, and the lack of cytotoxicity against three human cell
lines, we subjected compounds 15 and 25 to a toxicity study based on zebrafish embryos.
An advantage of this non-mammalian in vivo model is the high genetic homology to
humans and that it provides follow-up information on the type of toxicity encountered (e.g.,
hepatic, cardio-vascular etc.). In addition, this model can also predict mammalian
teratogenicity by evaluation of lethality and malformation during the development of
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1
1
1
2
2
2
2
2
2
2
2
2
2
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5
5
5
5
5
6
embryonic zebrafish.4
3,44
Both compounds tested showed a maximum tolerated
concentration (MTC) of ≥ 30 µM, which is higher than for the corresponding
mercaptoacetamide-based LasB inhibitor 5 we published previously (MTC = 10 µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table S1).29
(
Ex vivo pig skin model. We established an ex vivo model based on pig skin to address
the impact of our inhibitors on living mammalian tissue and on the contained collagen as
the natural substrate of collagenase. We challenged the skin, prepared from the ear of
freshly slaughtered pigs, with pure ColQ1 from B. cereus to degrade collagen. We
assessed the activity of ColQ1 by quantifying the formation of hydroxyproline as an
indicator for collagen turnover (Figure 4). Optimization of the assay conditions for the
model consisted of examining different buffer conditions and different protein
concentrations (Figure S1 and Figure S2). To evaluate the potential effect of 25 on
collagen turnover, we incubated the skin with ColQ1 in absence and presence of defined
concentrations of this compound. The subsequent quantification of hydroxyproline
revealed that 25 inhibited the collagenolytic activity of ColQ1, as demonstrated by the
significantly reduced amount of hydroxyproline released compared to control (Figure 5).
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These results support our previous finding that 25 is an inhibitor of a broad range of
bacterial collagenases. The good performance of our inhibitors in this model is a sound
starting point for their subsequent testing under in vivo conditions. We expect that our
inhibitors will accelerate the in vivo healing process, by preventing the distribution of
infection. The immune system will clear the bacteria, promoting remodeling of collagen
and skin regeneration.
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Figure 4. Representation of the pig skin model: the composition of the skin, the dermal
layer and the amino acids of collagen are illustrated. The concept of the hydroxyproline
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quantification assay is explained by mixing chloramine-T and Ehrlich’s reagent to obtain
a product that can be detected with a spectrophotometer.
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Figure 5. The amount of hydroxyproline at different concentrations of 25. Data shown
represent the means ± SD from three independent measurements. One-way ANOVA
followed by Tukey’s HSD test (** = p < 0.01, **** = p < 0.0001).
Antibacterial activity. The aim of this study was to create ‘pathoblockers’ that target
bacterial virulence factors without directly affecting bacterial viability. These have the
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ability to disarm, rather than to kill pathogens in order to make them less pathogenic. In
this context, it was of interest to test the antibacterial activity of the new derivatives against
P. aeruginosa strain PA14 and C. histolyticum strain DSM 1126 to exclude growth
inhibition by selected compounds. We therefore selected the four most potent compounds
against LasB (13, 15, 50 and 51) and the most active succinimide against ColH (25). As
shown in Table 6, it is clear that compounds 13, 15, 50 and 51 did not affect the growth
of PA14 and 25 did not affect the growth of DSM 1126, with a minimal inhibitory
concentration (MIC) > 100 μM. This assures that the activity of these compounds is only
through targeting the secreted bacterial virulence factors.
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Table 6. Antibacterial activity of selected compounds against P. aeruginosa and C.
histolyticum
MIC (μM)
Cpd.
P.
C. histolyticum
aeruginosa
1
3
>100
>100
>100
n.d.
n.d.
n.d.
15
50
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▪ CONCLUSIONS
In this study, we aimed to constrain the conformation of previously published N-aryl
mercaptoacetamide inhibitors,29,30 which led to a series of novel succinimide inhibitors of
the antivirulence targets LasB and ColH. Among these compounds, we identified 13 and
2
5 to show the best potency against LasB and ColH, respectively. Compound 13
improved the activity against LasB compared to our previous hit 5 by two times, while
compound 25 was slightly less potent than 6 against ColH; however, it maintained the
activity in the nanomolar range. Both compounds showed sufficient selectivity for the
bacterial metalloproteases over human MMPs and three other off-targets. The two most
active compounds against ColH, 25 and 20, showed in addition broad-spectrum inhibition
of homologous bacterial collagenases. These most potent LasB and ColH inhibitors
showed no signs of cytotoxicity in three human cell lines. Interestingly, compounds 15
and 25 showed a MTC of ≥ 30 μM in our zebrafish model. This is, for 15, a threefold lower
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toxicity compared to our published LasB hit 5. Moreover, we have established a pig skin
model to further characterize the most promising collagenase inhibitor. In this ex vivo
assay, compound 25 showed a promising effect in preventing collagen degradation,
which paves the way for this compound’s further evaluation under in vivo conditions. To
investigate the compounds in such in vivo models, additional work should be carried out
to improve their pharmacokinetic profiles. However, we strongly believe that the new
succinimide inhibitors presented in this work have a great potential to be developed
further and to be used as therapeutics.
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▪ EXPERIMENTAL SECTION
Chemistry. All reagents were used from commercial suppliers without further
purification. Procedures were not optimized regarding yield. NMR spectra were recorded
on a Bruker AV 500 (500 MHz) spectrometer. Chemical shifts are given in parts per million
1
13
(
ppm) and referenced against the residual proton, H, or carbon, C, resonances of the
>
99% deuterated solvents as internal reference. Coupling constants (J) are given in
Hertz. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t
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=
triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, br =
broad and combinations of these) coupling constants, and integration. Liquid
chromatography-mass spectrometry was performed on a LC-MS system, consisting of a
Dionex UltiMate 3000 pump, autosampler, column compartment and detector (Thermo
Fisher Scientific, Dreieich, Germany) and ESI quadrupole MS (MSQ Plus or ISQ EC,
Thermo Fisher Scientific, Dreieich, Germany). High resolution mass was determined by
LC-MS/MS using Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS system. Purity
of the final compounds was determined by LC-MS using the area percentage method on
the UV trace recorded at a wavelength of 254 nm and found to be >95%. Melting points
were determined by using a Stuart melting point SMP30 device.
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General procedure A: Synthesis of succinimides 7–27 and 50–52
Mercaptosuccinic acid (1.0 eq) and corresponding aniline (1.0 eq) were mixed in a crimp
vial under Ar atmosphere and heated at 120–160 °C from 3.5 h to overnight. The crude
product was purified using column chromatography. In case of N-aryl-3-
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mercaptomethylsuccinimides, 4-(aminoaryl)-2-(mercaptomethyl)-4-oxobutanoic acid was
heated at 120 °C overnight.
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General procedure B: Synthesis of thioacetates 28–30 by acetylation of free thiol
Succinimide (1.0 eq) was dissolved in DCM and the solution was cooled in an ice bath.
Pyridine (2.0 eq) and DMAP (0.1 eq) were added, followed by dropwise addition of Ac O
2
(2.0 eq). After 30 minutes at 0 °C, the reaction mixture was allowed to warm up to r.t. and
stirred overnight. Volatiles were evaporated under reduced pressure and crude product
was purified using column chromatography.
General procedure C: Synthesis of a-itaconamic acids 31–35
α-Itaconamic acids were synthesized following the procedure described in the
35
literature. Itaconic anhydride (1.0 eq) was dissolved in CHCl . The corresponding aniline
3
(1.0 eq) was added to the vigorously stirring solution. After 2 h, the product was collected
by filtration and washed with small amount of chloroform. The product was used in the
next step without further purification.
General procedure D: Synthesis of itaconimides 36–39
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