Synthetic quinolone signal analogues inhibiting the virulence factor elastase of Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c6cc06295d

We explore the chemical space of Pseudomonas quinolone signal analogs as privileged structures and report the discovery of a thioquinolone as a potent inhibitor of the important virulence factor elastase of the human pathogen Pseudomonas aeruginosa. We provide evidence that the derivative binds to the active site zinc of elastase and additionally acts as a fluorescent zinc sensor.

Full text of DOI:10.1039/c6cc06295d

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DOI: 10.1039/C6CC06295D
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Synthetic quinolone signal analogues inhibiting the virulence
factor elastase of Pseudomonas aeruginosa
D. Szamosvári,a V. F. Reichle,a M. Jureschi,a and T. Böttcher*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We explore the chemical space of Pseudomonas quinolone signal precursor 2-heptyl-4-quinolone (HHQ) that may both have
analogs as privileged structures and report the discovery of a distinct roles in cell-to-cell communication.14, 15 PQS and HHQ
thioquinolone as potent inhibitor of the important virulence have been demonstrated to regulate virulence factor
factor elastase of the human pathogen Pseudomonas aeruginosa. expression of P. aeruginosa and it has thus been suggested
We provide evidence that the derivative binds to the active site that targeting the pqs system may be a promising anti-
17
zinc of elastase and additionally acts as fluorescent zinc sensor.
virulence strategy.16,
Furthermore, the HHQ and PQS
quinolone scaffolds represent chemically privileged structures
and we hence reasoned that heteroatom substituted
derivatives may lead to functional diversity that could be
applied to screen for potential virulence inhibitors. We thus
synthesized a library of non-natural quinolone derivatives and
report here the discovery of a potent inhibitor of the virulence
factor elastase of pathogenic P. aeruginosa.
Pseudomonas aeruginosa is an important opportunistic human
pathogen responsible for severe diseases ranging from urinary
tract infections via life-threatening sepsis, endocarditis, and
meningitis to chronic respiratory infections in cystic fibrosis
patients.1, 2 The increasing emergence of multi-drug resistant
strains of P. aeruginosa poses major threats to public health
and P. aeruginosa is one of the leading causes of hospital-
acquired infections worldwide.3 The pathogenicity of P.
aeruginosa is mediated by its enormous arsenal of virulence
factors including toxins, extracellular enzymes, siderophores,
and secretion systems that directly inject virulence factors into
the eukaryotic host cell.4 A major virulence factor hereby is the
enzyme elastase (LasB) that supports the infection and
colonization process by damaging tissue and degrading
immune proteins.5 The fine-tuned production of virulence
factors that is responsible for the pathogen’s broad spectrum
of infective life-styles is orchestrated by the interactions of
several intertwined quorum sensing systems.6-8 Inhibition of
quorum sensing and its downstream circuits has attracted
much attention as potential strategy to disarm pathogens for
the future treatment of infectious diseases.9-12 One of the
quorum sensing systems of P. aeruginosa, the pseudomonas
quinolone signalling (pqs) system, uses a series of 2-alkyl-4-
quinolones (AQs) as signalling molecules.13 While various
different AQs are known, the most abundant and well-studied
are 3-hydroxy-2-heptyl-4-quinolone (PQS) and its biosynthetic
While previous studies have aimed to chemically inhibit or
deregulate the pqs quorum sensing system using derivatives
with modifications on the 2-alkyl-4-quinolone scaffold,18-20 we
focussed on the synthetically more demanding approach of
systematically changing the core scaffold by substituting its
functional groups and replacing its heteroatoms. We thereby
aimed to explore the chemical space of the privileged
structures of HHQ and PQS-like non-quinolone compounds and
investigate their biological activity as potential virulence
inhibitors of P. aeruginosa. We first developed and evaluated
various synthetic strategies towards the quinolone scaffold
whereby we obtained PQS and HHQ as control compounds.
HHQ (1) was prepared as reported previously by the synthesis
of 3-oxodecanoic acid methyl ester, condensation with aniline
and subsequent Conrad-Limpach cyclization (Figure 1A).21
Although, HHQ was often used as starting point for the
synthesis of 2-heptyl-3-hydroxyquinolin-4-one (PQS) by Duff-
formylation and Dakin-oxidation as described by Pesci et al.,22
both reactions appeared to be problematic.21 Formylation of
HHQ was only obtained when the HHQ was previously
transferred into its quinoline tautomer. The following
oxidation gave PQS in only 23% yield. PQS was therefore
synthesized after the method of Hradil et al. which turned out
to be a much more reliable and up scalable approach to
prepare PQS (Figure 1B).23, 24
a.Department of Chemistry, Konstanz Research School Chemical Biology, University
of Konstanz, 78457 Konstanz, Germany.
E-mail: Thomas.Boettcher@uni-konstanz.de
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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To generate a structurally diverse library of HHQ and PQS potential biological activity we performed a series of virulence
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DOI: 10.1039/C6CC06295D
derivatives, heteroatom substitutions were intended at assays with live cells of the highly virulent P. aeruginosa strain
positions 1, 3, and 4 of the 2-alkyl-4-quinolone scaffold. 4- PA14.30 We screened the library for inhibition of three
Thioketo-analoges
the appropriate 4-keto-compounds HHQ (
7
and
8
were synthesized by thionation of important extracellular virulence factors, pyocyanine,
) and PQS ( ) using rhamnolipid, and elastase. Cultures of P. aeruginosa PA14
1
2
P4S10 in pyridine under reflux conditions (Figure 1A and 1B).25 were grown in liquid medium supplemented with 500 µᴍ of
This reaction was found to be extremely reliable giving the each compound and after incubation for 24 h the production
desired products in good yields without interfering with or activities of the corresponding virulence factors were
hydroxyl- or amine functionalities at the same time whereas quantified in spent culture supernatants.
the Lawessons-reagent did not result successful thionation of
the ketones.
Figure 2. Active compound screening and investigations on the
mechanism of 8. A) Screening of the compound library for the
effect on elastin-congo red degradation with DMSO as control.
1
B) In vitro inhibition of elastase activity by
of
fluorescence increase of
concentration. Inset: fluorescence of
8
. C) H NMR shifts
8
in dependence of equivalents of added zinc (II). D) Relative
in dependence of the zinc (II)
under excitation by UV
8
8
light at 365 nm. ***Independent two-sample t-test p<0.00001.
While most compounds did not result in significant changes or
Fig. 1. Synthetic library of HHQ and PQS derivatives. A) slightly increased elastolytic activity (
Synthesis scheme for HHQ ( ) and the derived thioketone ) almost completely inhibited elastin degradation at a
and B) for PQS ( ) and its analog . C) Synthesis of compounds concentration of 500 µᴍ (Figure 2A, Supporting Figures S1 and
3-6. D) Structures of the compound library tested in the S2). The active compound did not inhibit growth of P.
2 and 6), one compound
1
7
,
(8
2
8
bioassays.
aeruginosa at the maximum tested concentration of 1 mᴍ
indicating that the effect on was not an artefact of cell toxicity
were or reduced growth (Supporting Figure S3). Elastolytic activity in
Chromen-4-one
(3
)
and thiochromen-4-one
(4)
synthesized from their corresponding 1-3-diketones 3b and 4c cultures of P. aeruginosa is mainly caused by the extracellular
which were prepared by Baker–Venkataraman rearrangement virulence factor LasB (elastase), a zinc metalloprotease that
of the octanoyloxy esters of 2-hydroxyacetophenone and 2- contributes as major virulence factor to the infectious lifestyle
mercaptoacetophenone 4a respectively.26, 27 Synthesis of the of P. aeruginosa.31 As no other virulence factors tested were
1-O and 1-S-PQS-derivatives
5 and 6 started from 2-hydroxy- impacted, we speculated that coincidently 8 may inhibit
and 2-mercaptoacetophenone, respectively via the directly the activity of the enzyme elastase rather than its
corresponding chroman-4-one 5a and thiochroman-4-one 6a production via the pqs quorum sensing system. To test this
by pyrrolidine catalyzed Knoevenagel-reaction with octanal hypothesis we used purified elastase and employed an activity
(Figure 1C).28, 29 Oxidation of the α-keto position turned out to assay with a fluorogenic peptide substrate. This in vitro assay
be difficult since an oxidation of the sulfide group to a resulted in a very potent inhibition by
8 with an IC50 of 4 µᴍ,
sulfoxide or sulfone had to be avoided. The product was confirming the direct mode of inhibition of elastolytic activity
obtained by nitrosation with isoamyl nitrite and subsequent on enzyme level (Figure 2B).
oximation and oxime hydrolysis.29 The combination of these It is known that analogous structures of
8 bind to zinc and are
diverse synthetic strategies resulted in a small library of 10 metalloproteinase inhibitors and PQS is known as iron
compounds (Figure 1D). In order to investigate our library for chelator.32-35 We thus speculated that the mechanism of action
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of
8
may involve binding to the zinc ion in the active site of sensor allowed to detect fluorescence quenchingVaietwinArctirceleaOsnilninge
DOI: 10.1039/C6CC06295D
elastase whereby its activity is inhibited. To investigate if
directly binds to zinc(II), we applied a combination of competed with
spectroscopic and NMR-based methods. NMR-titration of
8
concentrations of
9
and 10, indicating that these compounds
8
for zinc binding (Supporting Figure S8).
8
Consequently, the inhibition of elastase involved most likely
with zinc chloride in DMSO resulted in significant shifts and for all three compounds the binding of a hydroxyl thioketone
1
broadening of H and 13C signals in dependence of the zinc(II) or its corresponding thioenol-form to the active site zinc. For
concentration (Figure 2C, Supporting Figures S4 and S5). the in vitro studies the higher activity of the electron deficient
Surprisingly, when zinc(II) was added to a solution of
8 in aromatic systems of compounds 9 and 10 first appeared to be
ethanol, a strong fluorescence was observed and a titration puzzling. However, analysis of the crystal structure of elastase
experiment revealed that zinc concentrations down to 2.5 µᴍ revealed a carboxyl group (Glu141) in proximity to the active
could still be detected by fluorescence intensities two-fold site which might stabilize the positive charge in the thioenol-
over baseline making the compound also a formidable zinc form (Figure 4B). Hydrophobic pockets in proximity to the
sensor (Figure 2D). In contrast, no fluorescence was observed active site appear to be ideal for accommodating the lipophilic
for other biologically relevant divalent cations, confirming its heptyl chain of our compounds and also may explain the
high selectivity for zinc. These results suggest that
8 inhibits preference for hydrophobic side groups in natural substrates
elastase by binding to the zinc ion in active site of this and previously reported elastase inhibitors.34, 37, 38
metalloenzyme.
In order to further elucidate the influence of heteroatoms in
position 1 for elastase inhibition, we synthesized a second
generation of analogues of
nitrogen in position 1 of the 3-hydroxy-4-thioquinonolone
scaffold replaced by oxygen and sulphur 10. Additionally we
generated a 3-hydroxy-4-oxime derivative as an alternative
metal chelator (11). The 4-thioketones and 10 were
synthesized by thionation with P4S10 in pyridine as described
for and (Figure 3A). Interestingly, the oxime 11 could not
8 including derivatives with the
9
9
7
8
be obtained by base catalyzed reaction of PQS with
hydroxylamine hydrochloride which is probably because the
keto-form can be also understood as vinylogous amide.36
Instead, 3-hydroxyl TBDMS protected PQS was transferred into
its enol-tautomer and 4-hydroxyl benzylated to allow the
oxime formation at position 4 (Figure 3B).
Figure 4. Inhibition of Pseudomonas elastase activity. A)
Activity of the second generation of compounds derived from
8
with elastase in vitro. B) Active site of LasB with His140,
His144, and Glu164 coordinating the zinc ion and proposed
mechanism of inhibition. C) In situ inhibition of elastolytic
activity with compound 8 in culture of P. aerugionsa PA14.
To quantify the in situ efficacy, we measured the
concentration dependent inhibition of elastolytic activity by
Figure 3. Synthesis of a second generation of compounds
compounds
Hereby, only compound
8
-
11 with live cultures of P. aeruginosa PA14.
was an efficient inhibitor of
based on the active elastase inhibitor
8
. A) Scheme for the
and 10, and B) for
8
synthesis of the thioquinolone derivatives
the oxime 11.
9
elastolytic activity in situ resulting in sigmoidal inhibition
behaviour with an IC50 of 40 µᴍ (Figure 4C). Compound 10
resulted in only low efficacy with an in situ IC50 of 351 µᴍ and
Using this set of compounds, we first investigated their ability
to inhibit elastase activity in vitro. Hereby, compounds and
10 were even slightly more active than , each with an IC50 of 2
compounds
Figure S9). The discrepancy between in vitro and in situ activity
of and 10 may be explained by their similarity to flavones
9 and 11 were inactive up to 500 µᴍ (Supporting
9
8
9
that are known to be degraded by Pseudomonads.39 We thus
suspect that the compounds may be fed into bacterial
metabolism reducing their half-life and thus their efficacy
whereby the magnitude of the drop in activity from in vitro to
in situ experiments correlates with the increasing similarity of
µᴍ (Figure 4A, Supporting Figure S6). In contrast, compound
11 did not inhibit elastase activity in vitro at concentrations up
to 50 µᴍ indicating that oxygen in position 4 is required for
activity of the compound and cannot be simply replaced by
other chelating groups (Supporting Figure S7). While
compounds
9 and 10 did not exhibit fluorescence in presence
the compounds with the flavone scaffold. With
8 being the
of zinc, a competitive spectroscopic experiment with
8 as zinc
most active compound in situ we have discovered a potent
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