Tackling Pseudomonas aeruginosa Virulence by a Hydroxamic Acid-Based LasB Inhibitor

Article abstract of DOI:10.1021/acschembio.8b00257

In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial proteases represent promising targets for pathoblocker development. A common motif for protease inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described. The present inhibitor was developed based on a recently reported, highly selective thiol scaffold. Using X-ray crystallography, the lack of inhibition of a range of human matrix metalloproteases could be attributed to a distinct binding mode sparing the S1′ pocket. The inhibitor was shown to restore the effect of the antimicrobial peptide LL-37, decrease the formation of P. aeruginosa biofilm and, for the first time for a LasB inhibitor, reduce the release of extracellular DNA. Hence, it is capable of disrupting several important bacterial resistance mechanisms. These results highlight the potential of protease inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even with a strong zinc anchor.

Full text of DOI:10.1021/acschembio.8b00257

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Article
Tackling Pseudomonas aeruginosa Virulence
by a Hydroxamic Acid-Based LasB Inhibitor
Andreas M. Kany, asfandyar sikandar, Samir Yahiaoui, Jörg Haupenthal,
Isabell Walter, Martin Empting, Jesko Koehnke, and Rolf W. Hartmann
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00257 • Publication Date (Web): 08 Aug 2018
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Tackling Pseudomonas aeruginosa Virulence by a Hydroxamic Acid-Based
LasB Inhibitor
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Andreas M. Kany†, Asfandyar Sikandar‡, Samir Yahiaoui†, Jörg Haupenthal†, Isabell Walter†, Martin
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Empting†, 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
* Rolf.Hartmann@helmholtz-hzi.de
ABSTRACT
In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial
proteases represent promising targets for pathoblocker development. A common motif for protease
inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific
inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc
metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described.
The present inhibitor was developed based on a recently reported, highly selective thiol scaffold.
Using X-ray crystallography, the lack of inhibition of a range of human matrix-metalloproteases could
be attributed to a distinct binding mode sparing the S1´ pocket. The inhibitor was shown to restore the
effect of the antimicrobial peptide LL-37, to reduce the formation of P. aeruginosa biofilm and, for
the first time for a LasB inhibitor, the release of extracellular DNA. Hence, it is capable of disrupting
several important bacterial resistance mechanisms. These results highlight the potential of protease
inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even
with a strong zinc anchor.
Proteases have proven to be attractive targets for the treatment of various diseases, including
infections.1 While antiviral protease inhibitors are in clinical use for the treatment of e.g. HIV or HCV,
no bacterial protease inhibitors have been approved as anti-infective drugs yet.2,3 However, in order to
combat the spread of antibiotic resistance, new antibacterial agents with novel modes of action are
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urgently needed.4–6 This applies especially for Gram-negative pathogens which are challenging to treat
as their cell wall is difficult to permeate.7 The development of pathoblockers which target bacterial
virulence rather than killing bacteria is of growing interest in anti-infective drug discovery, due to the
reduced selection pressure such a strategy is supposed to have.8–12 In this context, bacterial proteases
represent attractive targets.2,3,13 Notably, the only FDA-approved anti-virulence drugs are
immunoglobulins that target secreted virulence factors, highlighting the potential of extracellular
targets to circumvent cell wall permeation problems.8 The highly problematic Gram-negative pathogen
Pseudomonas aeruginosa has been assigned critical priority by the WHO14 and urgently requires novel
treatment options because of increasing resistance.15,16 P. aeruginosa is i.a. responsible for fatal lung
infections in cystic fibrosis patients.17 Among its numerous virulence factors representing potential
drug targets,12,16,18–20 the zinc-metalloprotease elastase (LasB) is of specific interest, given its
extracellular location.21 LasB substantially contributes to disease progression in P. aeruginosa infected
individuals by facilitating host invasion and immune evasion.22 It was for example found to degrade
and thereby inactivate the endogenous antimicrobial peptide (AMP) LL-37.23 Furthermore, LasB was
reported to be involved in the formation of P. aeruginosa biofilms either by periplasmic activation of
nucleoside diphosphate kinase (NDK) required for alginate synthesis24, or by upregulation of
rhamnolipids.25 The aggregation of bacteria in the biofilm matrix seriously impedes successful
antibiotic treatment and blocks host defense mechanisms.26,27
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Several thiol-based inhibitors with promising activity on LasB have been described.28–32 A class of
N-aryl mercaptoacetamides turned out to be particularly attractive since these thiols display high
selectivity against a range of human matrix metalloproteinases (MMPs).32,33 Thiol-containing
compounds are in clinical use for the treatment of various diseases.34–36 However, a disadvantage of
this class compared to other zinc-chelating inhibitors is the possible oxidation to the respective
disulfides, resulting in inactivation of the compounds.37
In this study, we describe the synthetic replacement of the sulfhydryl function of LasB inhibitor 132
(IC50: 6.6
0.3 µM) by zinc-binding groups insensitive to oxidation (Figure 1). Among the
compounds tested, a hydroxamic acid derivative was found to inhibit LasB in the low micromolar
range as well. Hydroxamates have been reported as LasB inhibitors, but the described compounds lack
selectivity against human MMPs.38,39 In contrast, the inhibitor described in this work maintained the
remarkable selectivity of thiol 1 against human MMPs, despite a binding mode equivalent to described
hydroxamate-MMP complexes. The new compound was further able to reduce biofilm formation and
eDNA release by P. aeruginosa and to restore the antimicrobial effect of LL-37.
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Figure 1. Structures of lead LasB inhibitor 1 and compounds 3–8 synthesized in this article.
RESULTS AND DISCUSSION
Synthesis of novel compounds 3–8.
Among the variety of chemically diverse zinc binding groups in literature,40 we focused on hydroxyl,
carboxyl and hydroxamate functions. By introducing these relatively small zinc chelating groups,
drastic changes in the size of the thiol function of 1 were avoided in order to allow the inhibitor
backbone to preserve the previously observed binding mode.32 Carboxylic acid derivatives 3, 4 and 6
were obtained either by reacting aniline 2 with succinic/maleic anhydride or via hydrolysis of
methylester intermediate 5. Similarly, 7 was synthesized by reacting 5 with hydroxylamine (Scheme 1,
A-B). As an isosteric modification we further synthesized the alcohol derivative of 1, compound 8,
using glycolic acid in a neat reaction (Scheme 1, C).
Scheme 1. Reagents and conditions: (a) succinic anhydride, dioxane, 70 °C, 6 h, (b) maleic anhydride, dioxane, 70 °C, 6 h;
(c) methyl malonyl chloride, Et3N, DCM, RT, 4 h; (d) NaOH, THF, RT, 24 h; (e) H2NOH, DIPEA, MeOH, 8 h reflux, 16 h
RT; (f) glycolic acid, 130°C, 24 h
Identification of compound 7 as a promising LasB inhibitor
Using a FRET-based inhibition assay41 it turned out that replacement of the thiol function of 1 by a
hydroxy group led to a complete loss of activity when tested at 600 µM (8). Compounds bearing a
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carboxylic acid in γ- (3,4) or in β-position (6) to the carbonyl group were also inactive. Contrary to
that, the activity was maintained for the β-hydroxamic acid derivative 7, displaying an IC50 of 17.4
0.8 µM and a Ki,app of 12.3 0.6 µM. This is slightly less active compared to the free thiol analogue 1,
however, the oxidation issue was resolved by replacing the thiol by a hydroxamic acid function.
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Binding Mode of 7 to LasB.
In order to rationalize whether the minor difference in activity compared to 1 was due to a different
binding mode, the X-ray co-crystal structure of the LasB-7 complex was solved. The complex
crystallized in space group P212121 and crystals diffracted to 2.1 Å resolution (Figure 2, A). The
structure was solved by molecular replacement using the published LasB structure (PDB ID 1EZM) as
a search model. Full details of the data collection and refinement statistics can be found in
Supplementary Table 1. Hydroxamate 7 was found to be orientated toward the primed binding site of
the protease. As expected, the active site zinc atom is coordinated by both the carbonyl oxygen and the
hydroxamide oxygen of 7, leading to a distorted trigonal-bipyramidal geometry. The carbonyl oxygen
further undergoes a weaker interaction with His223 (3.5 Å). Additionally, the hydroxamide oxygen
forms a hydrogen bond with the adjacent Glu141 (2.5 Å), while the amide nitrogen interacts with the
carbonyl group of Ala113 (3.0 Å). These observations are in excellent accordance with the reported
binding of hydroxamate functions to MMP-7,42 MMP-343 or to thermolysin.44 Inhibitor binding to
thermolysin-like proteases like LasB was described to lead to a closure of the binding pocket due to
hinge-bending motion.45 Intriguingly, thiol 1 has recently been discovered by us to keep the active site
cleft in an open conformation due to the unexpected binding of two molecules to the primed binding
site (Supplementary Figure S1).32 In contrast, only one molecule of the hydroxamate binds to the
protease, which undergoes the characteristic hinge-bending. Unlike the zinc-chelating thiol, the
hydroxamate directly interacts with the edge strand via a hydrogen bond with the main chain oxygen
of Ala113. This interaction presumably promotes closure of the active site cleft, which is hampered in
case of thiol 1 by the second molecule interacting with Asn112. Due to the twisted orientation of the
aromatic core of 7 compared to 1, a previously observed bidentate hydrogen bond with Arg198 in the
S1′ binding site is not possible. This observation could explain the slightly weaker activity of the
hydroxamate compared to the thiol.
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Figure 2. (A) Structure of LasB in complex with 7. Cartoon representations of LasB (cyan) in complex with 7 (black). The
difference electron density (Fo-Fc) contoured to 3σ with phases calculated from a model that was refined in the absence of 7
is shown as a blue isomesh. The active-site zinc ion is shown as a grey, calcium ion as a green sphere. Residues involved in
binding of 7 are shown as sticks, their interactions depicted as yellow dashed lines. Zinc-liganding distances are 2.1 Å (OH)
and 2.4 Å (NH). (B) Overlay of LasB (cyan) in complex with 7 (grey) and MMP-3/-7 (green) occupied by hydroxamate
inhibitors 9 and 10 (brown, PDB codes 4G9L/1MMQ). Zinc ligands are highlighted.
Selectivity against six MMPs and ADAM-17.
Strong zinc chelating groups like hydroxamic acids can be the reason for poor selectivity against
further metalloproteases, when compound binding is driven more by the chelating moiety than by the
rest of the molecule.46 In fact, the lack of selectivity against MMP anti-targets has been one reason for
the failure of various hydroxamate-based MMP inhibitors in clinical trials.47,48 Considering the high
similarity in zinc chelation by 7 and the hydroxamates in MMP-3 or MMP-7, it was investigated
whether the previously demonstrated selectivity of N-aryl mercaptoacetamides toward six human
MMPs32,33 could be maintained. Fortunately, 7 did not inhibit these MMPs comprising members with
differing depth of the S1′ binding site49 including MMP-3 and -7 (Table 1). By contrast, the
unselective inhibitor Batimastat50 (Supplementary Figure S2) inhibited all tested enzymes in the low
nanomolar range.
Table 1. Residual activity of six MMPs and ADAM-17 in presence of 100 µM 7 and IC50 values of Batimastat33; Means and
SD of at least two independent measurements are displayed. *: test at 25 µM. n.d.: not determined.
7
Batimastat
IC50 [nM]
Residual activity at
100 µM [%]
MMP-1
MMP-2
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1.8 0.1
5.6 0.9
7.0 0.2
0.7 0.2
2.8 0.2
n.d.
MMP-3
MMP-7
MMP-8
MMP-14
ADAM-17
ADAM-17
38 12
61 23*
n.d.
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This of course prompted the question why, despite comparable interactions of the hydroxamate
function, LasB was inhibited but not MMP–7. Logically, the selectivity might be related to differences
in the positioning of the inhibitor backbone in the pocket. In order to investigate this, published X-ray
structures of MMPs in complex with hydroxamates were overlaid with the LasB-7 complex. The
shallow S1´ pocket of MMP-7 is occupied by an isobutyl moiety of inhibitor 942 and the deep S1´
pocket of MMP-3 by an aromatic core of inhibitor 1043 (for inhibitor structures see Supplementary
Figure S3). In contrast, the core of 7 does not bind to the respective pocket of LasB (Figure 2, B). The
ability to bind the S1´ pocket of the respective MMP is a common feature of reported hydroxamate-
based MMP inhibitors, which can also be the cause for a lack of selectivity.46,51 Consequently, the high
selectivity against various MMPs with differing depth of the S1´ binding pocket might be explained by
an inability of 7 to bind to this site of the protease. ADAMs (A Disintegrin and Metalloproteinase), a
class of zinc-dependent proteases playing essential roles in muscle development, cell migration or
shedding, display additional antitargets for the presented LasB inhibitor. In this context, an in vitro
inhibition assay toward ADAM-17 revealed that 7 exerted moderate inhibitory effects (Table 1).
Consequently, further optimization regarding selectivity toward this antitarget is needed. Still, the
promising selectivity toward a range of MMPs makes 7 an attractive starting point for LasB inhibitor
development.
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Cytotoxicity Assays.
In addition to selectivity, it was also investigated whether the cytotoxicity of 7 toward human cell lines
was as low as described for thiol 1.32 This was of specific interest since cytotoxic properties are a
known drawback of hydroxamates.52 Notably, 7 had only low effects on the viability of HEP G2 cells,
comparable to thiol 1 and rifampicin (Figure 3). The effect on HEK293 cells was more pronounced at
the tested concentrations, which might be related to the inhibition of antitargets such as ADAM-17.
For comparison, residual enzyme activities in the LasB in vitro assay are listed for each inhibitor
concentration in Supplementary Table S2.
Figure 3. Cytotoxicity of 1, 7 and two reference compounds toward HEP G2 and HEK293 cells (Values for 1 are taken
from32)
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Selectivity against HDACs.
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Considering the cytotoxic effects of 7 against HEP G2 cells, it was investigated whether this
observation could furthermore be related to inhibition of histone deacetylases (HDACs).53 These zinc-
dependent enzymes are involved in the epigenetic regulation of cell proliferation and differentiation.54
Given that HDACs are known to be inhibited by hydroxamic acids like vorinostat or trichostatin A,55
assessing a potential inhibitory effect of hydroxamate 7 was of special interest. Figure 4 shows that 7
does not exert any inhibitory effect on HDAC3 and HDAC8, while these enzymes are efficiently
inhibited by the positive control trichostatin A (Supplementary Figure S2). The observed selectivity
against these additional antitargets could be explained by the absence of a long linker separating the
zinc chelating function from the aromatic core, as it is typical for hydroxamate-based HDAC
inhibitors.52,56
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Figure 4. Residual activity of selected HDAC enzymes in presence of 100 µM 7.
Restoration of LL-37 antibacterial activity against P. aeruginosa PA14.
LL-37 is an α-helical human cathelicidin peptide, which shows increased prevalence in cystic fibrosis
patients.57 Its effectiveness against P. aeruginosa is considerably reduced as it is susceptible toward
cleavage by LasB.23 In order to assess a potential restoration effect of 7 on the activity of LL-37, a
bacterial growth assay with PA14 and LL-37 in presence/absence of 7 was performed (Figure 5). The
antibacterial effect of LL-37 alone was only minor at 25 µg/mL (p = 0.0211). However, when
combined with the LasB inhibitor, it recovered its ability to reduce bacterial growth in a dose-
dependent way. Significant reduction of the OD600 was observed starting from a hydroxamate
concentration of 62.5 µM (p = 0.0047). At high concentrations, 7 itself slightly inhibited PA14
growth, yet to a much lower extent than in combination with LL-37 (p = 0.0255 at 250 µM 7). These
findings highlight the potential of LasB inhibitor 7 to restore a host defense mechanism which is
otherwise hampered by LasB.
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Figure 5. Growth of PA14 cultures incubated with 7 in absence (blue) and presence (red) of 25 µg/mL LL-37. Means and SD
of three independent measurements are depicted. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001
Biofilm volume and eDNA reduction
The biofilm matrix, a key element of P. aeruginosa resistance,26 is composed of extracellular
polysaccharides, lipids, proteins and, importantly, extracellular DNA (eDNA).58,59 Inhibition of LasB
was shown to inhibit biofilm formation.28 Therefore, it was of great interest to investigate whether
treatment of PA14 cultures with 7 would result in reduced biofilm formation as well. Indeed, the
hydroxamate caused a concentration-dependent reduction of eDNA release (Figure 6, A) and of the
overall biofilm volume (Figure 6, B). Since significant inhibition was observed at concentrations
lower than 250 µM, these effects can be attributed to the on-target activity of the compound and are
not due to a reduction of bacterial growth or lysis of cells. Hence, LasB inhibitor 7 has demonstrated
important pathoblocker activity. It is able to interfere with crucial factors leading to bacterial
resistance of P. aeruginosa toward antibiotics and host defense molecules.27,60 To the best of our
knowledge, no direct correlation between LasB inhibition and a reduction of eDNA release has been
reported to date. Regarding the two mechanisms discussed in literature, the reduced biofilm formation
is either due to a reduced NDK-mediated alginate synthesis,24 or rhamolipid-mediated.25 Which of
these mechanisms inhibitor 7 interferes with cannot be concluded. Yet, these findings could hint at the
capacity of 7 to permeate at least the outer membrane of the Gram-negative cell wall, as far as an
inhibition of periplasmic NDK24 is concerned.
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Figure 6. Reduction of eDNA release by treatment of PA14 cultures with 7. (A) Reduction of overall PA14 biofilm volume
by 7. (B) Columns represent mean and SD of at least four independent measurements. ** = p < 0.01, **** = p < 0.0001
CONCLUSION
Based on our recent findings that N-aryl mercaptoacetamides are promising highly selective inhibitors
of the virulence factor LasB from P. aeruginosa, the lead inhibitor was modified by changing the zinc-
chelating moiety to a hydroxamate. Using X-ray crystallography, it was shown that similar to the thiol
analogue 1, hydroxamate 7 occupied the primed binding site. Contrary to our recent observations for
1, only one inhibitor molecule was bound to the protease, which could undergo the characteristic
hinge-bending motion resulting in a closed conformation of the enzyme. This was attributed to the
ability of the inhibitor to interact with catalytic zinc and the edge strand alike. Despite the high
similarity of zinc binding to hydroxamates inhibiting MMP-7, inhibitor 7 was unexpectedly able to
maintain the remarkable selectivity of 1 toward a range of MMPs. This could be rationalized by the
fact that the S1´ pocket was not occupied by 7, unlike observed for MMP inhibitors. These findings
show that despite the selectivity issues related to hydroxamic acids, selectivity can indeed be achieved
by sparing the S1´ pocket. Since cytotoxic effects toward mammalian cell lines were observed, the
activity of 7 was investigated regarding other antitargets revealing a moderate inhibition of ADAM-17
and high selectivity against HDAC3 and 8. Consequently, there will be a special focus on selectivity
and cytotoxicity during future compound optimization. Below an antibacterial concentration of 250
µM, LasB was efficiently inhibited in vitro. This inhibition could be translated into more complex,
cellular assays, highlighting this compound as a promising anti-virulence agent. After reduction of
their pathogenicity by such an agent, bacteria are supposed be cleared by host defense mechanisms or
with the help of conventional antibiotics.10,11 In this context, our results highlight a direct restoration
effect of the antibacterial activity of the host defense peptide AMP LL-37 in vitro. Indirectly, the
activity of host defense mechanisms might be further improved by the inhibition of biofilm formation
and eDNA release also observed for 7. The same holds true for the effectiveness of conventional
antibiotics, which is seriously hampered by bacterial biofilms. Overall, these findings show
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hydroxamate 7 to be an interesting lead, which could pave the way for the rational development of
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selective protease inhibitors as potential new antibiotics.
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METHODS.
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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,
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 SpectraSystems-MSQ
LCMS system (Thermo Fisher). Flash chromatography was performed on silica gel 60 M, 0.04–0.063
mm (Machery-Nagel) or using the automated flash chromatography system CombiFlash Rf+
(Teledyne Isco) equipped with RediSepRf silica columns (Axel Semrau) or Chromabond Flash C18
columns (Macherey-Nagel). 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%.
Expression and Purification of LasB. LasB was expressed and purified as described previously.32
In vitro Inhibition Assays. The LasB in vitro inhibition assay and the MMP assay were performed as
described previously.32 The LasB concentration that was used in the assay was 0.3 nM. The Km value
for LasB was determined to be 363.0 µM.
ADAM Inhibition Assay. ADAM-17 (TACE) Inhibitor Screening Assay Kit was purchased from
Sigma-Aldrich. The assay was performed according to the guidelines of the manufacturer.
Fluorescence signals were measured in a CLARIOstar plate reader (BMG Labtech).
HDAC Inhibition Assay. HDAC3 and HDAC8 inhibitor screening kits were purchased from Sigma-
Aldrich. The assay was performed according to the guidelines of the manufacturer. Fluorescence
signals were measured in a CLARIOstar plate reader (BMG Labtech).
Cytotoxicity assays. Hep G2 or HEK293 cells (2 x 105 cells per well) were seeded in 24-well, flat-
bottomed plates. Culturing of cells, incubations and OD measurements were performed as described
previously61 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 three independent measurements were performed for each compound.
X-ray Crystallography and image preparation. LasB was concentrated to 10–12 mg mL–1 and
mixed with inhibitor 7 at a final concentration of 1 mM. Complex crystals were grown by the sitting
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drop method using a reservoir solution containing 1.8 M AMSO4 and 0.1 M Tris-Cl, pH 8.8. Crystals
were cryoprotected in glycerol and diffraction data was collected from single crystals at 100 K at
beamline ID29 (ESRF) at a wavelength of 1.738 Å. Data was processed using Xia262 and the structure
solved using PHASER863 molecular replacement with Pseudomonas aeruginosa LasB (PAE, PDB ID
1EZM) as a search model. The solution was manually rebuilt with COOT64 and refined using
PHENIX65 and Refmac566. The final refined structure of LasB in complex with compound 7 was
deposited in the Protein Data Bank (PDB) as entry 6FZX. Structural superimposition of complex
structures of human MMPs (1MMQ and 4G9L) and LasB (6FZX) was achieved through alignment of
residues 201–205 of 4G9L (corresponds to residues 140–144 of 1MMQ) using the align atoms
algorithm of YASARA structure (YASARA Biosciences GmbH).67 Image was rendered using PovRay
(http://www.povray.org/).
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Bacterial growth assay. The assay was performed in 96 well plates (Greiner) with a final volume of
200 µL. LL-37 was purchased from AnaSpec (Fremont) and diluted to a final concentration of 25 µg
mL–1 from 125 µg mL–1 stocks in 18Mꢀ H2O. Prior to culture addition 7 was serially diluted in
DMSO. A pre-culture of PA14 was adjusted to the final start OD600 0.02 in lysogeny broth medium.
All samples contained 1% DMSO and 40% of 18Mꢀ H2O. OD600 was measured using a FLUOstar
Omega (BMG Labtech) after inoculation and after incubation for 16.5 h at 37°C with 200 rpm. Given
OD600 values were obtained after subtraction of the respective start OD600 and represent three
independent measurements with at least two replicates each. One-way ANOVA was performed using
GraphPad Prism 6 software.
Biofilm and eDNA assays. The assays were performed as described previously.68
ASSOCIATED CONTENT
Supporting Information Available: This information is available free of charge via the Internet.
Supplementary Table 1, giving X-ray data collection and refinement statistics, Supplementary Table
T2, showing residual activities of LasB in the in vitro assay, Supplementary Figure S 1–3, showing a
comparison of the LasB-1 complex to LasB-7 and structures of hydroxamate-based MMP inhibitors 9,
10, Batimastat and HDAC inhibitor Trichostatin A, synthetic procedures and spectral data for
compounds 3–8
The image of LL-37 used in the TOC graphic was created using PDB entry 2K6O.
ACKNOWLEDGEMENTS
J. Köhnke acknowledges the DFG for an Emmy-Noether Fellowship (KO 4116/3–1). We thank J.
Jung, S. Amann and D. Jener for technical support and T. Röhrig for help with statistical analysis.
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