Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acsinfecdis.8b00010

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

Full text of DOI:10.1021/acsinfecdis.8b00010

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Article
Binding Mode Characterization and Early in vivo
Evaluation of Fragment-like Thiols as Inhibitors of the
Virulence Factor LasB from Pseudomonas aeruginosa
Andreas Martin Kany, Asfandyar Sikandar, Jörg Haupenthal, Samir Yahiaoui,
Christine K. Maurer, Ewgenij Proschak, Jesko Koehnke, and Rolf W. Hartmann
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00010 • Publication Date (Web): 27 Feb 2018
Downloaded from http://pubs.acs.org on March 2, 2018
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Binding Mode Characterization and Early in vivo Evaluation of Fragmentꢀ
like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas
aeruginosa
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Andreas M. Kany,†,⊥ Asfandyar Sikandar,‡,⊥ Jörg Haupenthal,† Samir Yahiaoui,† Christine K.
Maurer,† Ewgenij Proschak,ǁ Jesko Köhnke,‡ Rolf W. Hartmann*,†,§
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†
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Design and
Optimization, Campus E8.1, 66123, Saarbrücken, Germany
‡ Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Workgroup Structural Biology of
Biosynthetic Enzymes, Campus E8.1, 66123, Saarbrücken, Germany
§
Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus
E8.1, 66123 Saarbrücken, Germany
ǁ
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, MaxꢀvonꢀLaueꢀStraße 9,
60438 Frankfurt, Germany
* Rolf.Hartmann@helmholtzꢀhzi.de
The increasing emergence of antibiotic resistance necessitates the development of antiꢀinfectives with
novel modes of action. Targeting bacterial virulence is considered a promising approach to develop
novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a
pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive
antiꢀvirulence target. Mercaptoacetamideꢀbased thiols have been reported to inhibit LasB as well as
collagenases from clostridia and bacillus species. The present work provides an insight into the
StructureꢀActivityꢀRelationship (SAR) of these fragmentꢀlike LasB inhibitors, demonstrating an
inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An Xꢀray
coꢀcrystal structure is presented, revealing distinct binding of two compounds to the active site of
LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in
a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human
matrixꢀmetalloproteinases (MMPs).
Keywords: antibiotic resistance, antiꢀvirulence agent, elastase, LasB, binding mode, selectivity,
Galleria mellonella
The increasing emergence of resistant bacteria poses a threat to public health, especially in case of
Gramꢀnegative species.1,2 Pseudomonas aeruginosa is one of the three most problematic pathogens on
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the WHO priority list.1 It is the reason for many hospitalꢀacquired infections as well as fatal lung
infections in cystic fibrosis and bronchiectasis patients.3,4 To combat the rise of antibioticꢀresistant
Pseudomonas aeruginosa infections, novel treatment options are urgently needed.5,6
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A promising new approach to reduce selection pressure is to target bacterial virulence in order to
disarm pathogens rather than to kill them.7–10 Pseudomonas aeruginosa produces numerous virulence
factors contributing to disease progression, which provide attractive antiꢀinfective targets.11–14 The
secreted enzyme LasB is a major virulence factor, playing a crucial role for the pathogenicity of P.
aeruginosa.15 The enzyme is a zincꢀmetalloprotease with high structural similarity to thermolysin.16,17
One of its main functions is the cleavage of components of the connective tissue like elastin15 or
collagen18 to allow the bacteria to colonize a niche in the host. Tissue damage is further caused by
disruption of cellꢀtoꢀcell junctions.19,20 Additionally, LasB enables P. aeruginosa to evade the human
immune response by cleaving i.a. IgG21, cytokines22, surfactant proteins A and D23, complement factor
C324 or pulmonary defense receptor PAR2.25 Consequently, this protease represents an attractive antiꢀ
infective target and its extracellular localization facilitates drug discovery as permeation of the Gramꢀ
negative cell wall is not needed.9,26
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LasB belongs to the thermolysin (M4) family of enzymes.27 For this class it has been reported that the
rather open active site cleft of the protease adopts a more closed conformation upon inhibitor
binding.28 To date, several zincꢀchelating inhibitors of LasB have been described,29–32 including
compounds bearing a mercaptoacetamide motif attached either to small peptides33 or to aniline.34
Crystallographic data shedding light on the binding mode of these thiols to the protease has not been
available yet. We have recently described Nꢀaryl mercaptoacetamideꢀbased compounds as very potent
clostridial collagenase inhibitors with high selectivity toward human matrix metalloproteinases
(MMPs).35 The compounds contain a prodrugꢀlike thiocarbamateꢀmotif, which liberates free thiols as
active form after hydrolysis in buffer. The best ColH inhibitor 1 (Figure 1) was also found to inhibit
LasB.
In this work, we report a functional screening followed by LCꢀMS validation for LasB inhibition of
the focused TimTec ActiTargꢀP library previously used to discover the ColH inhibitors. The only
inhibitor resulting from the screening was an Nꢀaryl mercaptoacetamide. Encouraged by the promising
inhibitory activity and selectivity, we wanted to gain further insight into LasB inhibition by this
compound class. We describe the activity and selectivity profiles of a broad range of Nꢀaryl
mercaptoacetamides. An Xꢀray crystal structure of a mercaptoacetamideꢀbased inhibitor in complex
with LasB is presented revealing an unprecedented open conformation of the active site, which harbors
two inhibitor molecules. To the best of our knowledge, this is the first description of inhibitor binding
to LasB which does not lead to a closure of the active site cleft.
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Figure 1. Structure of most potent ColH inhibitor 1.
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RESULTS AND DISCUSSION
Screening for Novel LasB Inhibitors.
In order to expand the chemical space of LasB inhibitors, we performed a functional screening based
on the FRETꢀbased in vitro assay developed by Nishino and Powers.36 We used a protease inhibitorꢀ
enriched library which, after removing structures known as PAINS37, comprised 1192 low molecular
weight compounds. In addition, we included 330 fragments (Maybridge Fragment Library) into the
screening. The only compound showing more than 50% inhibition when tested at 100 µM was
mercaptoacetamide 2 (Figure 2). Several falseꢀpositive hits had to be excluded because of quenching
of substrate fluorescence.
Figure 2. Structure of screening hit 2.
Development of an LCꢀMSꢀbased Readout for the FRET Assay.
In the FRETꢀbased proteolytic assay, active LasB cleaves a quenched substrate, which results in an
increase of fluorescence.36 Enzyme inhibition leads to reduced substrate cleavage and consequently to
reduced fluorescence. However, compounds interfering with the fluorophore by quenching effects38
can pretend enzyme inhibition, resulting in falseꢀpositive hits.39,40 Several quenching compounds were
found, especially among fragments. To clarify if reduced fluorescence was caused by protease
inhibition or to quenching effects, we developed an LCꢀMSꢀbased readout for the FRET Assay.
Elastase cleaves the synthetic substrate AbzꢀAlaꢀGlyꢀLeuꢀAlaꢀNba (3) at the GlyꢀLeu bond36,41
,
forming cleavage products 4 and 5 (Figure 3, A) which could be separated chromatographically. Using
the published LasB inhibitor phosphoramidon42 as a positive control, the FRETꢀassay results were
excellently reproduced using an LCꢀMSꢀbased readout which was based on the mass peak of cleavage
product 5 (Figure S1). Applying this technology, we could detect several false positives whose
apparent inhibition in the FRET assay was only due to quenching. Two examples are shown in
Figure 3, B.
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Figure 3. (A) Cleavage of FRET substrate AbzꢀAlaꢀGlyꢀLeuꢀAlaꢀNba (3) into products
Abz = 2ꢀAminobenzoyl; Nba = 4ꢀnitrobenzylamide. (B) Structure and comparison of FRET (blue, v/v0) vs. LCꢀMS results
(red, A/A0) for fragments 6 and 7.
4 and 5 by LasB.
SAR of NꢀAryl Mercaptoacetamides.
To elucidate the SAR of Nꢀaryl mercaptoacetamides, 35 derivatives were purchased or synthesized
(for further information see SI), and tested for LasB inhibition applying the FRETꢀbased in vitro assay
(Table 1 and Table S1). Nonpolar aromatic substituents, especially halogens, turned out to be
favorable for activity while polar hydrogenꢀbond accepting moieties led to significantly reduced
inhibition. For the o-, m- and p-Cl derivatives 9, 12 and 17 as well as m- and p-CH3 derivatives 13 and
21 no substantial effect of the substitution position on activity was found. The same holds true for the
methoxy (11, 15, 23) and o-,p-phenyl (10, 18) analogues. Introduction of a second substituent
improved the IC50 at least twofold. Addition of methyl or chlorine substituents to 17 resulted in the
best compounds 26, 27 and 28, displaying IC50 values in the oneꢀdigit micromolar range. An exception
is dimethoxy derivative 32, which showed an activity similar to the monoꢀmethoxy analogues 11, 15
and 23. In comparison to the initial screening hit 2, the activity could be improved by more than 2.5ꢀ
fold. Two examples showed that the introduction of a third substituent was not beneficial for
inhibition: Introduction of a methyl group to compound 26 led to a twofold drop in activity (34), while
no difference was observed between diꢀ and triꢀmethyl derivatives 29 and 33.
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Table 1: Chemical structures and LasB inhibition of a series of Nꢀaryl mercaptoacetamides.
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Cp.
8
R
IC50 (µM)
11.4 ± 0.2
14.1 ± 0.5
25.0 ± 0.8
51.7 ± 4.0
19.4 ± 0.5
47.5 ± 1.4
59.2 ± 0.9
61.7 ± 2.1
Cp.
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1
R
4ꢀCl
IC50 (µM)
15.7 ± 0.4
19.8 ± 1.4
22.8 ± 1.1
32.9 ± 1.3
36.1 ± 0.7
47.6 ± 1.1
47.7 ± 1.0
73.1 ± 2.5
81.5 ± 2.5
85.6 ± 2.3
Cp.
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27
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30
2
R
IC50 (µM)
5.9 ± 0.3
2ꢀBr
2ꢀCH3ꢀ3ꢀCl
3,4ꢀdiꢀCl
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2ꢀCl
4ꢀPh
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2ꢀPh
4ꢀBr
2ꢀCH3ꢀ5ꢀCl
2,4ꢀdiꢀCH3
2,3ꢀdiꢀCH3
3,4ꢀdiꢀCH3
3ꢀClꢀ4ꢀCH3
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2,4,6ꢀtriꢀCH3
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2ꢀOCH3
3ꢀCl
4ꢀI
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4ꢀCH3
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4ꢀOCH3
3ꢀCH3
3ꢀF
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3ꢀOCH3
4ꢀCOCH3
3ꢀNHSO2CH3 127.2 ± 3.5
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4ꢀCOOCH3
4ꢀ(CH2ꢀ1,2,4ꢀ
triazole)
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34a 3ꢀClꢀ2,6ꢀdiꢀCH3 11.9 ± 0.4
aCompounds 33 and 34 were synthesized as free thiols.
Comparison to the SAR for ColH inhibition
Interestingly, the here reported SAR shows an inverse activity profile of the Nꢀaryl
mercaptoacetamides compared to ColH. We previously reported polar, hydrogen bondꢀaccepting
substituents, especially in para-position to the aniline function, to be most potent for ColH
inhibition.35 Contrary to that, oxygenꢀcontaining compounds like e.g. the methoxyꢀderivatives 11, 15,
23 and 32 showed considerably weaker LasB inhibition compared to their halogenꢀsubstituted analogs.
Strikingly, our best ColH inhibitor 1 is one of the weakest inhibitors described in this study. The
position of the substituent substantially affects inhibition of ColH but not of LasB.
Confirmation of Thiols as Active Compounds.
As previously described35, thiocarbamates are rapidly cleaved to the corresponding free thiols in
aqueous buffers (Figure 4, A). Consequently, to confirm that the observed weaker activity on LasB
compared to clostridial collagenases was not due to slower hydrolysis in LasB assay buffer, we
performed the recently reported LCꢀMSꢀbased stability test. Fast hydrolysis of 17 in 50 mM Tris pH
7.2 was observed with a thiocarbamate halfꢀlife of 3.7 ± 0.1 min (Figure 4, B). A third component was
formed which is most likely the disulfide oxidation product. Compound hydrolysis proceeded faster
than described for ColH assay at 22.5 °C35, indicating temperature dependence of hydrolysis.
Additionally, we found the same in vitro activity of selected thiols compared to their prodrugs,
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confirming the results from the LCꢀMS assay that compound activation occurs within the preꢀ
incubation time of our assay (Figure 4, C and Figure 5). Unlike thiols, which can be oxidized to
disulfides43, these thiocarbamate prodrugs have the advantage of being stable toward oxidation. As
expected, the inactivity of dithiocarbamates 75ꢀ82 (Table S1) could be explained by their stability
toward hydrolysis (Figure S2).
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Figure 4. (A) Conversion of thiocarbamate 17 into corresponding free thiol 35. (B) LCꢀMS stability assay showing fast
hydrolysis of 17 into 35 in 50 mM Tris pH 7.2 (10% methanol) at 37 °C. (C) In vitro results for 17 and 35 are in accordance
with the LCꢀMS results.
Figure 5. (A) Structure of thiol 36. (B) FRET assay results for 36 and prodrug 27. Nonꢀlinear regression was performed with
the Hill Slope constrained to 1.
Binding Mode of Compound 36 to LasB.
To elucidate the binding mode of the Nꢀaryl mercaptoacetamides to LasB we coꢀcrystallized
compound 27, with LasB purified from Pseudomonas aeruginosa PA14 culture supernatant. The
putative LasBꢀ27 complex crystallized in space group P21 and crystals diffracted to 1.3 Å resolution
(Figure 6). The structure was solved by molecular replacement using the published LasB apo structure
(PDB ID 1EZM) as a search model. Full details of the data collection and refinement statistics can be
found in Table S2.
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Hydrolysis of 27 to 36 was expected from the stability tests and from the in vitro assay using the
corresponding thiol 36 and indeed there was no electron density for the thiocarbamate group observed.
These observations confirm that the prodrug moiety does not contribute to the inhibitory activity. To
our surprise, instead of one molecule, the active site of LasB contained unambiguous electron density
for two molecules of 36, arranged in an antiparallel fashion (hereafter referred to as 36A and 36B). In
the LasB apo structure, the activeꢀsite zinc ion is coordinated by the sideꢀchains of His140, His144 and
Glu164 as well as one water molecule. The free thiol of 36A displaces the water molecule to complete
the tetrahedral coordination sphere (sulfur – zinc distance 2.3 Å). The carbonyl oxygen of 36A forms a
bidentate hydrogen bond with Arg198 in the S1′ binding site (2.2 Å / 2.3 Å), while the sideꢀchain of
His223 forms a hydrogen bond with the thiol and amide nitrogen (2.4 Å / 3.5 Å). The amide nitrogen
of the second molecule hydrogen bonds with Asn112 in the edge strand (2.0 Å), while its aromatic
core lies in the lipophilic S2′ binding pocket. The tolerance of substitution at different positions
without crucial changes in activity can be explained by the relatively large size of the binding site in
comparison to the rather narrow binding pocket of ColH. Calculations performed with Molecular
Operating Environment (MOE) software44 enabled us to determine differences in the acidity of the
thiol groups: Zinc coordination significantly increased thiol acidity, reducing the pka value from 9.0 in
solution to 4.0. These results indicate full ionization of this sulfur atom when bound to the active site.
By contrast, no significant change in the acidity of the nonꢀzinc bound thiol could be determined,
indicating a protonated state of this sulfur atom. It appears probable that binding of 36A is required for
36B to bind to the protein by providing a hydrophobic surface anchored to the activeꢀsite zinc ion 36B
can interact with. Regarding the Hill Slope of 1 in the in vitro assay for 36 (Figure 5, B), it seems that
only one binding event is necessary for full inhibition. This indicates that a second molecule might not
be required for the activity of the compound and its absence in solution cannot be excluded.
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Elastase belongs to the M4 family of peptidases, showing high similarity to thermolysin from Bacillus
thermoproteolyticus.27 Its LasB Cꢀterminal domain is composed mostly of αꢀhelices, while the
Nꢀterminal domain is formed predominantly by antiparallel βꢀsheets. The active site cleft is positioned
within the hingeꢀregion in between the two domains.17 Interestingly, binding of 36A and 36B does not
lead to a closure of the active site cleft as it has been reported after binding of phosphoramidon45 and
another peptidic inhibitor.46 In fact, the open conformation observed in the apo structure is virtually
unperturbed (Cα rmsd of just 0.24 over 290 residues, Figure S3). It seems likely that the antiparallel
binding of two molecules of 36 prevents the active site from closing and thus allows addressing the
enzyme in an open conformation. This offers a completely new avenue for the design of LasB
inhibitors.
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Figure 6. Structure of LasB in complex with 36. Cartoon representations of LasB (cyan) in complex with 36 (black, grey).
The difference electron density (FoꢀFc) contoured to 3σ with phases calculated from a model that was refined in the absence
of 36 is shown as a yellow isomesh. The activeꢀsite zinc ion is shown as a grey, calcium ion as a green sphere. Residues
involved in binding of 36A and 36B are shown as sticks.
In the work of Zhu et al., a primed binding mode of Nꢀaryl mercaptoacetamides to the LasB active site
was proposed based on docking studies.34 We experimentally confirmed the inhibitor to be placed in
the primed binding pocket. However, there are substantial differences in the orientation of the
inhibitor, mainly owing to the presence of two molecules. The reported docking studies suggested
additional chelation of the zinc atom by the carbonyl group of the inhibitor, which we demonstrated to
hydrogen bond with Arg198. The proposed hydrogen bond between Asn112 and the amide nitrogen of
the inhibitor is indeed present, yet it is formed by 36B and not by the zinc chelating 36A.
Structureꢀbased Optimization
Binding of a single molecule to the binding site instead of two might have an entropic benefit resulting
in improved inhibition. Hence, we tried to replace the second, nonꢀzinc chelating molecule and to
grow the zincꢀbinding molecule deeper into the binding pocket, using the crystal structure of the LasBꢀ
36 complex. Modeling approaches showed a benzyl group to be appropriate to fill the part of the
binding pocket occupied by the aromatic core of the nonꢀzinc binding molecule (Figure 7).
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Figure 7. (A) Structure of 37, the Nꢀbenzyl derivative of active thiol 36. (B) Surface representation of LasB bound to two
molecules of 27. (C) Modeling of 37, the Nꢀbenzyl derivative of the active thiol 36 based on the Xꢀray crystal structure.
A straightforward approach was used to synthesize Nꢀbenzyl derivatives of compound 27 (Scheme 1):
Nꢀbenzyl substituted anilines 47ꢀ54 were obtained via reductive amination, using aniline 38 and a
variety of benzaldehydes (39ꢀ46). Amide coupling with chloroacetyl chloride led to intermediates
55ꢀ62. Replacement of the αꢀchlorine by a thiocyanate group was followed by hydrolysis to the
respective thiocarbamates 63ꢀ70 using a mixture of concentrated sulfuric acid and acetic acid.47
Scheme 1. Synthesis of Nꢀbenzyl mercaptoacetamides. Reagents and conditions: (a) sodium triacetoxyborohydride, DCM,
RT, 20 h (47ꢀ52, 54) or 3ꢀmethoxybenzylbromide, K2CO3, DMF, 120°C, 20 h (53); (b) chloroacetyl chloride, acetone, 0°C –
r.t., 1.5 h; (c) ammonium thiocyanate, ethanol, 80°C, 2 h; (d) sulfuric acid, acetic acid, 0°C, 30 min
Introduction of a benzylic group at the position of the amide nitrogen of 27 led to an active compound
with an IC50 of 20.4 ± 0.9 µM (63, Table 2). The activity was approximately threefold lower compared
to the nonꢀbenzylated compound (Table 1). We introduced halogens at several positions of the benzyl
group (64ꢀ67) to improve hydrophobic interactions with the lipophilic part of the binding pocket. The
activity was slightly increased compared to 63, however, with IC50s in the twoꢀdigit micromolar range
our compounds were still less active than 27. The polar phenol and methoxyphenol derivatives 68 and
69 inhibited LasB in the same range as the halogenated compounds 64ꢀ67, indicating that the
lipophilic part of the binding pocket might not be reached. Combination of the 4′ꢀfluorine of 65 and
the 3′ꢀmethoxy group of 69 did not lead to an additive effect (70).
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Table 2. Structure and LasB inhibition of Nꢀsubstituted derivatives 63ꢀ74.
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Cp.
63
64
65
66
67
68
69
70
71
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73
74
R
Ph
IC50 (µM)
20.4 ± 0.9
12.6 ± 0.4
15.3 ± 0.6
17.3 ± 0.8
15.9 ± 0.9
17.6 ± 0.6
14.3 ± 0.6
18.3 ± 0.9
15.9 ± 0.7
15.0 ± 0.5
54.5 ± 2.3
27.6 ± 1.4
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3′ꢀFꢀPh
4′ꢀFꢀPh
3′ꢀClꢀPh
2′,3′,5′ꢀtriꢀClꢀPh
3′ꢀOHꢀPh
3′ꢀOCH3ꢀPh
3′ꢀOCH3ꢀ4´ꢀFꢀPh
H
nꢀC4H9
tꢀC4H9
C6H11
Given that the activity could not be improved by introducing rigid aromatic functions, we investigated
conformationally more flexible alkyl substituents. Synthesis was achieved following the same
procedure (Scheme 1) using the respective alkyl aldehydes, giving compounds 71ꢀ74 (Table 2).
Similar to the benzyl compounds 63ꢀ70, introduction of alkyl substituents at the amide nitrogen led to
a loss of activity, especially in case of the rather bulky neopentyl and cyclohexylmethyl substituents
(73 and 74). Considering that replacement of the amide hydrogen by methyl group already resulted in
a more than twofold loss of activity (71) we concluded that the hydrogen bond formed between
Asn112 and the second molecule in the binding pocket plays an important role for compound binding.
To assess the impact of Nꢀsubstitution on thiocarbamate halfꢀlife, the stability of 68 and 74 was
analyzed. Again, we proved that thiocarbamates were cleaved rapidly, thus the reduced activity was
not caused by slower compound activation in assay buffer (Figure S4).
Selectivity Toward Further Proteases.
MMPs are ubiquitously present in the human body, playing pivotal roles in the progress of various
diseases, but also exerting beneficial effects on human health. The unselective inhibition of various
MMPs was the reason for the failure of several protease inhibitors, while selectively inhibiting specific
MMPs remains a challenging task in protease drug discovery.48–53 Several bacterial protease inhibitors
lacking MMP selectivity have been reported.54–56 Thiolꢀbased LasB inhibitors have been tested toward
selected MMPs.33,34 In this context, the inhibition of range of six MMPs with structural variation in
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their S1
′
binding pocket57 (deep: MMPꢀ3 and ꢀ14; intermediate: MMPꢀ2 and ꢀ8; shallow: MMPꢀ1
and ꢀ7) was analyzed for 27. As we previously demonstrated for other Nꢀaryl mercaptoacetamides35,
27 did not inhibit this broad range of MMPs at concentrations up to 100 µM either (Figure S5).
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In contrast to these antitargets, bacterial metalloꢀβꢀlactamases represent attractive additional targets,
since cleavage of βꢀlactam antibiotics by these proteases is a crucial mechanism of antibiotic
resistance.58 Inhibition of metalloꢀβꢀlactamases by thiolꢀbased inhibitors has been reported to restore
the activity of βꢀlactam antibiotics.59–62 To this end, we tested IMPꢀ7, a metalloꢀβꢀlactamase present in
clinical isolates of P. aeruginosa for in vitro inhibition by prodrug 27 and thiol 36. Interestingly, the
enzyme was found to be inhibited by 27 (1.16 ± 0.07 µM) as well as 36 (0.86 ± 0.06 µM).
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Cytotoxicity Assays.
For a potential therapeutic application in humans, we analyzed the cytotoxicity of thiocarbamate 17 as
well as thiols 35 and 36 toward two different human cell lines (Table 3). As described previously35, the
mercaptoacetamides had only a low cytotoxic effect on HEP G2 cells at a concentration as high as 100
µM. Likewise, the cytotoxic effect on HEK298 cells was also low at this high concentration.
Table 3. Cytotoxicity of 17, 35 and 36 in HEP G2 and HEK298 cells.
Reduction of viability [%]
Cp.
Concn [µM]
HEP G2
26 ± 16
28 ± 12a
25 ± 3
HEK298
51 ± 12
51 ± 4
22 ± 5
49 ± 7
19 ± 3
2 ± 2
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35
100
100
100
1
36
Doxorubicin
Rifampicin
Batimastat
50 ± 5a
29 ± 5a
13 ± 7a
100
100
aValues taken from Schönauer, Kany et al.35
Galleria mellonella Infection Model
Stimulated by the promising in vitro results, the in vivo efficacy of our compounds was investigated.
Infection models with Galleria mellonella larvae have previously been employed by us to assess novel
treatment options for Pseudomonas aeruginosaꢀinduced infections63,64 and were reported to highly
correlate with mouse models.65 We analyzed the effect of prodrug 27 and active thiol 36 by coꢀ
injecting them with PA14. The larvae were challenged with PA14 concentrations corresponding to two
times the LD50.65 Compared to PA14ꢀinfected larvae receiving no treatment, 2.5 nmol of 36
significantly increased the survival of G. mellonella from 43% to 72% after 65 hours (Figure 8).
Notably, the survival rate was increased and not only the survival time, as reported for LasB inhibitors
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in a C. elegans model.34 However, administering the same concentration of thiocarbamate prodrug did
not lead to a significant effect, indicating an insufficient release of the thiol in G. mellonella
hemolymph. These findings prove the potential of LasB inhibition to effectively reduce P. aeruginosa
pathogenicity.
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Figure 8. Survival curves for PA14ꢀchallenged Galleria mellonella larvae receiving treatment with 2.5 nmol 27 or 36 in
comparison to larvae receiving treatment with PBS only. Curves represent results from at least three independent
measurements. The survival rate was significantly higher for larvae treated with 36 (p = 0.0003, logꢀrank test) but not with
27. The survival rate for larvae treated with the compounds in PBS was 100%.
CONCLUSION
In this study, a functional screening of a proteaseꢀinhibitor enriched library and a fragment library was
performed using a wellꢀestablished FRET assay, with the aim to expand the chemical space of LasB
inhibitors. An LCꢀMSꢀbased counterscreen was developed, allowing identification of falseꢀpositives.
Only one real screening hit was identified, an Nꢀaryl mercaptoacetamide. Compounds of this class are
known to be inhibitors of LasB as well as of ColH from C. histolyticum. We analyzed the inhibition of
further 35 derivatives of this class of fragmentꢀlike molecules. Interestingly, the SAR was inverse to
the activity profile we discovered for ColH, allowing rational optimization of the compound class to
selectively inhibit either LasB or ColH. The Xꢀray crystal structure of a LasBꢀinhibitor complex was
solved, revealing a primed binding mode of two thiol molecules to the active site of the protease.
Importantly, our results show that inhibitor binding does not necessarily lead to a closure of the
binding pocket, as it has been described for thermolysinꢀlike proteases. These findings pave the way
for the development of novel LasB inhibitors targeting the open conformation of the enzyme,
providing an important starting point for lead optimization. The structural information obtained by Xꢀ
ray crystallography was used to grow the fragments by introducing benzyl or alkyl groups. These
compounds indeed inhibited LasB, yet the activity was not improved. Supposably, the conformation of
the Nꢀsubstituted compounds is not ideal to replace the second inhibitor molecule in the binding
pocket. With the βꢀlactamase IMPꢀ7 we discovered an additional target of the Nꢀaryl
mercaptoacetamides, which is attractive for the development of novel antiꢀinfectives. Furthermore, in
vivo efficacy was demonstrated using a Galleria mellonella infection model. The survival rate of
PA14ꢀinfected larvae was increased significantly when treated with our best thiol. Considering the
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fragmentꢀlike character of our inhibitors, and the fact that they represent antiꢀvirulence agents instead
of traditional antibiotics, this is a remarkable effect. These results underline the potential of reducing
bacterial pathogenicity to develop novel antiꢀbacterial drugs, which are urgently needed to combat
antibiotic resistance. Nꢀaryl mercaptoacetamides prove particularly interesting for the development of
such drugs, since they display high selectivity toward human MMPs.
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EXPERIMENTAL SECTION
Expression and Purification of LasB. Pseudomonas aeruginosa PA14 were grown in lysogeny broth
medium for 72 h at 37 °C. Cells were removed by centrifugation (5000 rpm, 4 °C, 60 min) and the
supernatant was filtered through a bottleꢀtopꢀfilter (0.20 µm). Purification was performed according to
the method described by Morihara et al.66 with few modifications. The precipitate formed by acetone
treatment was dissolved in water and dialyzed against buffer A (20 mM Tris, pH 8.0). The dialyzed
sample was then loaded onto a Hitrap Q HP column (GE Healthcare, Little Chalfont, UK) and washed
back to baseline in buffer A, before being eluted with a gradient from buffer A into buffer A2 (20 mM
Tris, pH 8.0, 1 M NaCl). The fractions with the strongest activity were pooled together (in vitro assay
described below) and loaded onto a Superdex 200 gel filtration column (GE Healthcare) that was preꢀ
equilibrates with buffer B (20 mM Tris, pH 8.0, 2 mM CaCl2). Protein purity and activity was assessed
by SDSꢀPAGE and in vitro inhibition assay.
In vitro Inhibition Assay. For the initial screening, Elastase was purchased from Elastin Products
Company (Owensville, MO, USA) or Merck (Darmstadt, Germany). Later, purified elastase prepared
according to the procedure described above was used. The fluorogenic substrate 2ꢀAminobenzoylꢀAlaꢀ
GlyꢀLeuꢀAlaꢀ4ꢀNitrobenzylamide36 was purchased from Peptides International (Louisville, KY, USA).
Fluorescence intensity was measured for 60 min at 37°C in black 384ꢀwell microtiter plates (Greiner
BioOne, Kremsmünster, Austria) using CLARIOstar microplate reader (BMG Labtech, Ortenberg,
Germany) with an excitation wavelength of 340 ± 15 nm and an emission wavelength of 415 ± 20 nm.
The assay was performed in a final volume of 50 µl assay buffer (50 mM Tris pH 7.2, 2.5 mM CaCl2,
0.075% Pluronic Fꢀ127, 5% DMSO) containing LasB at a final concentration of 10 nM (commercial
batch) or 0.3 nM (purified batch) and the substrate at 150 µM. Before substrate addition, compounds
were preꢀincubated with the enzyme for 15 min at 37°C. Experiments were performed in duplicates
and repeated for at least two times. Blank controls without enzyme were performed. After blank
subtraction, the slope of samples containing inhibitors (v) was divided by the slope of a
simultaneously started uninhibited enzymatic reaction (v0). IC50 values were determined with nonꢀ
linear regression using GraphPad Prism 5 (Graph Pad Software, San Diego, CA, USA) and are given
as mean values ± standard deviation (SD). The slope factor was constrained to 1.
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LCꢀMSꢀbased Readout for the FRET Assay. The FRETꢀbased fluorescence assay was performed
according to the procedure described above using 50 mM Tris pH 7.2. At the end of the measurement,
the enzymatic reaction was stopped by adding formic acid at a final concentration of 2%.
Simultaneously, amitryptiline were added as internal standard. The resulting mixture was diluted 1:10
in a mixture of 10% acetonitrile in MilliQ water containing 2% formic acid, resulting in a 10 µM
amitryptiline concentration. The analyses were performed using a TF UltiMate 3000 binary RSLC
UHPLC (Thermo Fisher, Dreieich, Germany) equipped with a degasser, a binary pump, an
autosampler and a thermostated column compartment and a MWD, coupled to a TF TSQ Quantum
Access Max mass spectrometer with heated electrospray ionization source (HESIꢀII). For gradient
elution, an Accucore RPꢀMS column (150 × 2.1 mm, 2.6 µm, Thermo Fisher, Dreieich, Germany) was
used with a mobile phase consisting of acetonitrile containing 1‰ formic acid (FA; v/v; eluent A) and
water containing 1‰ FA (v/v; eluent B) at a flow rate of 400 µL/min under the following conditions: 0
– 0.9 min 10% A, 0.9 – 2.5 min 10 ꢀ 50% A, 2.5 – 5.5 min 50% ꢀ 70 % A, 5.5 – 6 min hold, 6 – 6.5
min 10% A, giving a total run time of 6.5 min. The injection volume was 10 µL. The divert valve was
set to 1.6 min. The autosampler temperature was set to 6°C. The following MS conditions were used:
electrospray ionization (ESI), positive mode, sheath gas, nitrogen at a flow rate of 35 arbitrary units;
auxiliary gas, nitrogen at flow rate of 10 arbitrary units; vaporizer temperature, 50 °C; ion transfer
capillary temperature, 270 °C; capillary offset, 15 V; spray voltage, 3000 V. The mass spectrometer
was operated in the SIM mode with the following masses: 4 m/z 266.1 (tube lens offset 120 V),
amitryptiline m/z 278.1 (tube lens offset 90 V), 5 m/z 337.0 (tube lens offset 120 V), 3 m/z 584.1 (tube
lens offset 120 V) with a scan width of m/z 2.0 and a scan time of 0.1 s, respectively. Measurements
were performed in duplicates and repeated for at least two times. Observed retention times were as
follows: 4 2.00 min, 5 3.52 min, amitryptiline 4.02 min, 3 4.37 min. MSꢀpeak areas were determined
using TF Xcalibur Software. Peak areas were normalized by ISTD peak area (giving A) and divided
by the peak area of the respective ISTD normalized sample without inhibitor (A0). IC50 values were
determined using the method described above (GraphPad Prism 5 software).
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LCꢀMSꢀbased Stability Assay. The assay was performed as described previously35 using 50 mM Tris
pH 7.2 and a temperature of 37°C.
Screening Library. The protease inhibitor enriched screening library (ActiTargꢀP) was purchased
from TimTec (Newark, DE, USA). Fragments were obtained from the Maybridge Fragment Library
(Maybridge, Altrincham, UK). Compounds were stored as DMSO stock solutions.
Chemistry. All reagents were used from commercial suppliers without further purification.
Procedures were not optimized regarding yield. NMR spectra were recorded on a Bruker Fourier 300
(300 MHz) spectrometer. Chemical shifts are given in parts per million (ppm) and referenced against
1
the residual proton, H, or carbon, 13C, resonances of the >99% deuterated solvents as internal
reference. Coupling constants (J) are given in Hertz. Data are reported as follows: chemical shift,
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multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad and combinations of these)
coupling constants and integration. Mass spectrometry was performed on a SpectraSystemsꢀMSQ
LCMS system (Thermo Fisher, Dreieich, Germany). Flash chromatography was performed using the
automated flash chromatography system CombiFlash Rf+ (Teledyne Isco, Lincoln, NE, USA)
equipped with RediSepRf silica columns (Axel Semrau, Sprockhövel Germany) or Chromabond Flash
C18 columns (MachereyꢀNagel, Düren, Germany). Purity of compounds synthesized by us was
determined by LCMS using the area percentage method on the UV trace recorded at a wavelength of
254 nm and found to be >95%. Thiols and thiocarbamates were synthesized according to the
procedures described previously.35 Nꢀbenzyl and Nꢀaryl mercaptoacetamides were synthesized
according to Scheme 1 and Scheme S2 using General Procedures 1ꢀ3 as described in more detail in the
Supporting Information.
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Human MMP Inhibition Assay. The catalytic domains of MMPꢀ1, ꢀ2, ꢀ3, ꢀ7, ꢀ8 and ꢀ14 along with
the SensoLyte 520 Generic MMP Activity Kit were purchased from AnaSpec (Fremont, CA, USA).
The assay was performed as described previously using Batimastat as a positive control35, according to
the guidelines of the manufacturer.
βꢀlactamase Inhibition Assay. Activity assays for IMPꢀ7 were carried out, as described by Klingler et
al.60 Final protein concentrations of 0.1 nM in a 50 mM HEPES buffer (pH 7.5, 0.01 % Triton Xꢀ100).
Substrate (Fluorocillin™ (Invitrogen, Darmstadt, Germany) was dissolved in assay buffer to a final
concentration of 888 nM. Test compounds were dissolved and preꢀdiluted in DMSO (final
concentration: 1 %). In a black polystyrol 96ꢀwell plate (Corning) an amount of 1 µL of the respective
inhibitor solution at different concentrations was incubated with 89 µL of IMPꢀ7 containing buffer for
30 minutes at room temperature. 10 µL of substrate solution was added. The readout of the emitted
fluorescence was started immediately (45 s for 30 cycles) using a Tecan Infinite F200Pro (Tecan
Group Ltd; excitation at 495 nm and emission at 525 nm). Blank controls were performed without
enzyme. Positive controls were performed with enzyme but without inhibitor. The inhibitory activity
of each test compound was measured in three independent experiments. For calculation of IC50 values,
data obtained from measurements with eight different inhibitor concentrations were used. For the
evaluation of the sigmoidal dose response equation (variable slope with four parameters) GraphPad
Prism 5 (GraphPad Software, La Jolla, CA, USA) was used.
Cytotoxicity Assays. Hep G2 or HEK298 cells (2 x 105 cells per well) were seeded in 24ꢀwell, flatꢀ
bottomed plates. Culturing of cells, incubations and OD measurements were performed as described
previously67 with small modifications. 24 h after seeding the cells the incubation was started by the
addition of compounds in a final DMSO concentration of 1 %. The living cell mass was determined
after 48 h. At least two independent measurements were performed for each compound.
Galleria mellonella Virulence Assay. The virulence assay was performed as described by Lu, Maurer
et al63 with some modifications: Galleria mellonella larvae (TruLarv™) were purchased from
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BioSystems Technology (Exeter, United Kingdom). Injections were performed using a LA120 syringe
pump (Landgraf Laborsysteme, Langenhagen, Germany) equipped with 1 ml InjektꢀF tuberculin
syringes (B. Braun, Melsungen, Germany) and Sterican 0.30 x 12 mm, 30G x 1½ needles (B. Braun).
The following treatment conditions were applied: (a) sterile PBS solution, (b) PA14 suspension, (c)
2.5 nmol 27 in “b”, (d) 2.5 nmol 36 in “b”, (e) 2.5 nmol 27 in “a”, (f) 1.25 nmol 36 in “a”. Samples aꢀe
contained 1% DMSO and f 0.5%. For each treatment, data from at least three independent
measurements were combined.
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Xꢀray Crystallography. LasB was concentrated to 10 ꢀ12 mg/mL and mixed with inhibitor 27 at a
final concentration of 1 mM. Complex crystals were grown by the sitting drop method using a
reservoir solution containing 0.2 M Magnesium Chloride and 30% (w/v) PEG 3350. Crystals were
cryoprotected in glycerol and diffraction data was collected from single crystals at 100 K at beamline
ID23ꢀ2 (ESRF) at a wavelength of 0.873 Å. Data was processed using Xia268 and the structure solved
using PHASER69 molecular replacement with Pseudomonas aeruginosa elastase (PAE, PDB ID
1EZM) as a search model. The solution was manually rebuilt with COOT70 and refined using
PHENIX71 and Refmac572. The final refined structure of LasB in complex with compound 27 was
deposited in the Protein Data Bank (PDB) as entry 6F8B.
Molecular Modeling. Molecular modeling was performed with Molecular Operating Environment
2015.10 (MOE) software (Chemical Computing Group, Montreal, Canada) using standard parameters.
In the coꢀcrystal structure of LasB and 36, inhibitor molecule 36B was removed and 36A was grown
using the Builder function. The final structure was energyꢀminimized using the QuickPrep function.
AMBER10:EHT was used as a force field.
ASSOCIATED CONTENT
Supporting Information. Supporting Table T1ꢀT2 and Figures S1ꢀS4, giving structures and stability
data of inactive compounds, Xꢀray data collection and refinement statistics, FRET and LCꢀMS results
for phosphoramidon, stability data for 68 and 74, MMP inhibition assay, experimental procedures and
spectral data for synthetic compounds
AUTHOR CONTRIBUTIONS
⊥A.M.K. and A.S. contributed equally.
ACKNOWLEDGEMENTS
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We thank Jeannine Jung, Simone Amann, Tabea Schramm and Lilia Weizel for technical support, Dr.
Jens Eberhard and Dr. Giuseppe Allegretta for help with the establishment of the LCꢀMS assay, Dr.
Teresa Röhrig for support with the G. mellonella assay and Dr. Martin Empting for help with
molecular modeling. E.P. thanks German Research Foundation (DFG, HeisenbergꢀProfessur
PR1405/4ꢀ1) for financial support.
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