Flexible Fragment Growing Boosts Potency of Quorum-Sensing Inhibitors against Pseudomonas aeruginosa Virulence

Article abstract of DOI:10.1002/cmdc.201900621

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.

Full text of DOI:10.1002/cmdc.201900621

Accepted Article
Title: Flexible Fragment Growing Boosts Potency of Quorum Sensing
Inhibitors against Pseudomonas aeruginosa Virulence
Authors: Michael Zender, Florian Witzgall, Alexander Felix Kiefer,
Benjamin Kirsch, Christine K. Maurer, Andreas M. Kany,
Ningna Xu, Stefan Schmelz, Carsten Börger, Wulf
Blankenfeldt, and Martin Empting
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900621
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900621
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10.1002/cmdc.201900621
ChemMedChem
RESEARCH ARTICLE
Flexible Fragment Growing Boosts Potency of Quorum Sensing
Inhibitors against Pseudomonas aeruginosa Virulence
Michael Zender,[a] Florian Witzgall,[c] Alexander Kiefer,[a] Benjamin Kirsch,[a] Christine K. Maurer,[a]
Andreas M. Kany,[a] Ningna Xu,[e] Stefan Schmelz,[c] Carsten Börger,[f] Wulf Blankenfeldt,[c,d] Martin
Empting*[a,b]
Dedicated to Professor Rolf W. Hartmann
Abstract: Hit-to-lead optimization is a critical phase in drug discovery.
Herein, we report on the fragment-based discovery and optimization
of 2-amino pyridine derivatives as a novel lead-like structure for the
treatment of the dangerous opportunistic pathogen Pseudomonas
aeruginosa. We pursue an innovative treatment strategy by interfering
with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing
(QS) system leading to an abolishment of bacterial pathogenicity. Our
compounds act on the PQS receptor (PqsR), a key transcription factor
controlling the expression of various pathogenicity determinants. In
this target-driven approach, we made use of biophysical screening via
surface plasmon resonance (SPR) followed by isothermal titration
calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit
optimization then involved growth vector identification and exploitation.
Astonishingly, the latter was successfully achieved by introducing
flexible linkers rather than rigid motifs leading to a boost in activity on
the target receptor and anti-virulence potency.
Alarmingly, the occurrence of multi-resistant and pan-resistant
strains renders currently available antibiotics ineffective and leads
to an urgent need for novel treatment options.[2] P. aeruginosa
employs an arsenal of virulence-associated factors that allow this
pathogen to be effective in various host organisms and
environments.[3] The release of many virulence factors is
controlled and synchronized by a process called quorum sensing
(QS).[4] QS allows bacteria to collectively regulate gene
expression depending on their population density. Small diffusible
molecules (auto-inducers) are secreted from the cells and once a
certain threshold concentration has been achieved,
transcriptional regulators are activated. This leads to
a
population-wide alteration of gene expression, resulting in
concerted phenotypic actions.[5] This ability is essential during the
course of acute and chronic infections[6] as well as for lowered
antibiotic susceptibility.[7]
Introduction
Pseudomonas aeruginosa is an opportunistic Gram-negative
pathogen. It provokes different acute and chronic infections
especially in immune-compromised and hospitalized patients.[1]
[a]
Dr. M. Empting, Dr. M. Zender, Dr. A. Kiefer, Dr. B. Kirsch, Dr. C. K.
Maurer, Dr. A. M. Kany
Design and Optimization (DDOP)
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)-
Helmholtz Centre for Infection Research (HZI)
Campus E8.1, 66123 Saarbrücken, Germany
E-mail: martin.empting@helmholtz-hzi.de
Dr. M. Empting
[b]
[c]
[d]
[e]
[f]
Figure 1. Schematic representation of the pqs quorum sensing system in
Pseudomonas aeruginosa highlighting PqsR as an attractive point-of-
intervention. Activation of PqsR by native agonists PQS (and to a lower extend
HHQ) leads to autoinduction of the biosynthetic enzyme cascade PqsA-E as
well as regulation of bacterial virulence like e.g. production of pyocyanin.
Department of Pharmacy
Saarland University
Campus E8.1, 66123 Saarbrücken, Germany
Dr. F. Witzgall, Dr. Stefan Schmelz, Prof. Dr. W. Blankenfeldt
Structure and Function of Proteins (SFPR)
Helmholtz Centre for Infection Research (HZI)
Inhoffenstr. 7, 38124 Braunschweig, Germany
Prof. Dr. W. Blankenfeldt
Biotechnology and Bioinformatics, Institute for Biochemistry
Technische Universität Braunschweig
Spielmannstr. 7, 38106 Braunschweig, Germany
Dr. N. Xu
Lehrstuhl für Biochemie
Universität Bayreuth
Universitätsstr. 30, 95447 Bayreuth, Germany
Dr. C. Börger
PharmBioTec GmbH
Respective cell-to-cell communication in P. aeruginosa is mainly
based on four distinct QS circuitries. The las[8] and the rhl[9] QS
systems use different N-acylated homoserine lactones (AHLs) as
signaling molecules, while recently discovered iqs relies on 2-(2-
hydroxyphenyl)-thiazole-4-carbaldehyde (Integrated Quorum
Sensing Signal, IQS).[10] AHL-based communication is most
widespread ‘language’ found in Gram-negative bacteria.[11] On the
contrary, the forth system called pqs[12] employs alkylquinolones
(AQs)[13,14] and occurs only in Pseudomonas and Burkholderia
species (Figure 1).[15] The signaling molecules PQS
(Pseudomonas Quinolone Signal; 2-heptyl-3-hydroxy-4(1H)-
quinolone) and its precursor HHQ (2-heptyl-4(1H)-quinolone)
Science Park 1, 66123 Saarbrücken, Germany
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cmdc.201900621
ChemMedChem
RESEARCH ARTICLE
activate the receptor PqsR (also referred to as MvfR).[13,14] PqsR
is a LysR-type transcriptional regulator that controls a subset of
genes that is responsible for the production of various virulence
factors like pyocyanin, elastase, and lectins.[16] Moreover, it drives
the expression of the pqsABCDE operon.[13,17] This operon
encodes the enzymes PqsABCDE required for the biosynthesis
of HHQ,[18] which is converted by the monooxygenase PqsH to
the more potent agonist PQS. PqsR-deficient strains showed
reduced pathogenicity in several in vivo infection models[16,19]
demonstrating its central role during the infection process.
Therefore, PqsR is a potential drug target to attenuate P.
aeruginosa virulence without affecting bacterial viability. This
approach promises only a low selection pressure towards
resistance development.[20]
This enabled us to devise an elegant structure-guided
optimization strategy considering the binding pose of the natural
ligand HHQ. The introduction of a flexible linker finally led to
compounds with nanomolar inverse-agonistic activities in an E.
coli reporter gene assay and complete pyocyanin inhibition in P.
aeruginosa.
Results and Discussion
Selection of an optimal starting point. Selecting the best
starting point is a pivotal decision in the early stage of a drug
discovery project. In previous projects, ligand efficiency[30] (LE)
was used as major criterion for hit selection.[27] The LE values
represent the mean binding contribution per heavy atom
(LE= -∆G/N) allowing to compare hits of different sizes.[30] Taken
into account that the ligand binding site of PqsR is highly
lipophilic,[25] the engineering of well-placed hydrogen bonds will
be a difficult task. Hence, an ideal screening hit should establish
most effective non-covalent interactions. Enthalpic contribution
(∆H) is a convenient indicator for the establishment and the
breaking of specific interactions during the formation of the
protein-ligand complex.[29] In a simple model, ∆H describes the
sum of bond breaks between solvent and ligand and on the other
hand bond formation between ligand and protein.[29] Hence,
enthalpic efficiency (EE) was used as guideline to derive an
alternative starting point. EE normalizes the enthalpic contribution
for the heavy atom count (EE= -∆H/N). Hence, this concept allows
comparing ligands of different sizes.
Our group has previously obtained the first PqsR-targeting
quorum sensing inhibitor (QSI) by chemical modification of the
natural ligand HHQ.[21] These compounds were further improved
regarding their efficacy in P. aeruginosa.[22,23] We revealed that
most potent QSI of this class act as inverse agonists rather than
antagonists on the target receptor.[24] In a similar approach, QSI
containing a quinazolinone scaffold was discovered and helped
resolving the crystal structure of the ligand binding domain of
PqsR.[25] Furthermore, an HTS campaign led to benzamide-
benzimidazole compounds showing efficacy in different mouse
models, which emphasizes the in vivo relevance of targeting
PqsR in infectious diseases.[26] To overcome the poor physico-
chemical profiles of the HHQ-derived QSI, we initiated two
fragment screenings using surface plasmon resonance (SPR)
technology.[27,28] These approaches led to the hydroxamic acid 1
and the 2-amino-oxadiazole 2 (Table 1). First attempts to enlarge
these structures have not been successful so far or led to
compounds lacking activity in P. aeruginosa (data not shown).
During two previously reported fragment-optimization approaches,
compounds 1[27] and 2[28] (Table 1) were discovered. The
thermodynamic profiles of these optimized fragments were
compared to the best screening hits derived from previously
reported SPR screenings.[27,28] Initially, compound 3 was ranked
lower due to its lower affinity.[28] A re-evaluation of these three
fragment scaffolds making use of the above-mentioned ranking
metrics showed that 3 possesses strikingly better EE and LE
values (Table 1).
Here, we applied enthalpic efficiency[29,30] as a metric to select
screening hit 3 as an alternative starting point. During initial
fragment–growing efforts, we were successful in stepwise
enlarging the fragment structure. Notably, we determined the
crystal structure of a ligand-receptor complex (PDB ID 6Q7V).
Table 1. Thermodynamic profiling of fragment-sized PqsR ligands guiding the selection of the optimal starting point.
Compound
KD (ITC) [µM]
4.1 ± 0.6
1.3±0.3
10.0±1.3
> 50[c]
ΔG [kcal mol-1]
ΔH [kcal mol-1]
-8.9±0.2
-8.5±0.5
-9.3±0.4
-
-5.8±0.1
-
-11.5±0.9
-TΔS [kcal mol-1]
1.5±0.3
0.4±0.6
2.5±0.4
-
-0.6±0.2
-
3.7±1
EE[a] [kcal mol-1]
0.63
0.53
1.17
-
0.72
-
1.05
LE[b] [kcal mol-1]
1
2
3
4
5
6
7
-7.4±0.1
-8.0±0.1
-6.8±0.1
-
-6.4±0.1
-
0.53
0.50
0.85
-
0.80
-
21.3±3.4
> 50[c]
3.1±0.5
-7.5±0.1
0.70
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10.1002/cmdc.201900621
ChemMedChem
RESEARCH ARTICLE
ITC titrations were performed at 298 K. Data represent mean ± SD from at least two independent experiments; [a] EE = -ΔH/(heavy
atom count); [b] LE = –ΔG/(heavy atom count). [c] no heat release detectable at solubility maximum
the formation of specific interactions were hindered due to the
attached moiety.
In the light of these considerations, fragment 3 was evaluated as
a promising alternative starting point.
Table 2. Activity of compounds 7-22 on the target receptor PqsR.
Fragment growing. Ideally,
a fragment-based screening
campaign is supported by structure-based drug design strategies
as this improves the chances to develop highly active lead
molecules tremendously.[31] However, for 3 no crystal structure in
complex with PqsR could be obtained. In order to derive a first
SAR impression and to find a potential vector for growing the
fragment, close commercial analogues (4-8) were investigated for
their affinity to PqsR by ITC analysis (Table 1). This revealed that
the position of the bromine substituent was fundamental for
affinity. A bromo substituent in 3- and 5-position (4 and 6)
abolished the affinity, whereas in 4-position (5) the affinity was
slightly decreased, which promoted the 4-position as potential
growth vector. The exchange of the bromine for a sterically more
demanding trifluoromethyl group in 6-position (7) led to a threefold
increase of affinity caused by a gain in the enthalpic contribution.
Candidate compounds were tested in an E. coli lacZ reporter gene
system for their ability to antagonize/inverse agonize PqsR.[32]
This heterologous system provides higher sensitivity and a clear-
cut readout due to the absence of the entire pqs system present
in P. aeruginosa. Our previous studies indicated that the system
employing an E.coli laboratory strain poses a less restrictive
biological barrier to small molecules for reaching the intracellular
target.[22] Hence, it facilitates a straightforward evaluation and
comparison of PqsR-targeting QSI regarding their on-target
activities. Noteworthy, this assay system provides a more reliable
estimation of cellular effectivity than cell-free affinity
measurements. For example, the reported affinity of the native
agonist PQS towards the ligand binding domain is rather low (KD
= 1.2 ± 0.3 µM).[33] In contrast, the PQS-mediated effect in E.coli-
based assays and P. aeruginosa occurs at drastically lower
[b]
Compound
7
Structure
IC50 [µM][a]
clogD7.4
33.6±8.2
1.7
H
8
9
n.i.
n.i.
1.6
3.1
10
11
29 % @ 100 µM
2.6±0.8
3.7
12
13
5.1±0.5
5.9±0.9
3.5
3.1
14
15
16
4.9±0.9
3.6
3.9
3.6
18.5±7.3
concentrations (EC50(
= 6.3 nM,[22] EC50(
= 24 nM).
P.aeruginosa
)
E.coli)
This discrepancy might be explained by the fact that functional
PqsR as a LysR-type transcriptional regulator exists as a
homotetramer capable of binding to DNA and presumably able to
adopt different conformational states.[34] This higher-ordered
architecture of the bacterial target is not resembled in cell-free
assays employing only a monomeric N-terminally truncated
protein.
In contrast to the congeners (3-6; data not shown), compound 7
showed a moderate antagonistic activity (Table 2). The acetylated
analog 8 was tested in order to assess the amine function as a
possible handle to grow the fragment. However, the attached
acetyl moiety abolished the affinity and antagonistic activity
completely. Taken together, these findings led to the decision to
keep scaffold 7 constant and to introduce further substituents into
the 4-position (Figure 2). At first, rigid cyclopropyl-ethynyl (9) and
4-fluorophenyl (10) moieties were attached as molecular probes.
Both modifications abolished the antagonistic activity almost
completely.
35±5 % @ 100
µM
17
18
42.8±19.5
n.i.
3.3
4.2
19
3.4±0.2
20
21
22
0.14±0.04
3.6±1.1
4.3
3.4
3.8
A closer look at the thermodynamic profile of 9 disclosed a
dramatic loss in the enthalpic contribution (SI Table S1: 7: ∆H = -
0.49±0.17
11.5 kcal/mol vs. 9: ∆H
= -3.7 kcal/mol) that is partially
compensated by a gain in entropy. These findings suggest that
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10.1002/cmdc.201900621
ChemMedChem
RESEARCH ARTICLE
Figure 2. Schematic illustration of the optimization pathway starting from SPR
hit 3 leading to optimized fragment 7 and identification of a growth vector (5)
followed by fragment growing. Enlarged fragment 11 finally enabled further
structure-guided optimization through flexible fragment growing.
[a] Inverse agonistic/antagonistic activity was determined in
the presence of 50 nM PQS; n.i. = no inhibition (activity ≤ 15%).
IC50 represents the concentration of the half maximal activity;
otherwise activity at
a given concentration is provided.
bCalculated using ACD/Percepta 2015.
Hence, we introduced a more flexible linker into the 4-position with
the aim to regain specific interactions of the amino-pyridine
headgroup. For this purpose, several compounds enlarged with
different benzylamine moieties (11-13) were synthesized. Indeed,
these compounds (11-12) displayed up to 12-fold increase in the
on-target activities. Overall, the 4-fluoro substituted compound 11
evolved as the most potent QSI of this series so far. Notably, also
the affinity measured by ITC was improved up to 5-fold. (SI, Table
S1). Taken together, the following general concept was derived
from these previous findings (Figure 2): the amino-pyridine
headgroup should be connected via a flexible linker part to
another aromatic moiety. We were able to solve a co-crystal
structure of 11 in complex with the ligand binding domain of PqsR
(Figure 3a), clearly showing that the linker establishes an angled
connection between the 2-amino-pyridine headgroup, which
occupies the space of the quinolone core of the natural ligands
while the 4-fluorophenyl ring which points into the alkyl-chain
pocket. Both aromatic systems of 11 are flanked by the alkyl side
chains of isoleucine residues enabling CH-π interactions. The 2-
amino moiety can form an H-bond interaction with the backbone
carbonyl of Leu207 and the NH of the benzylamine linker
interacted with the hydroxyl moiety of Thr265. However, a
comparison of the thermodynamic signatures of 11 binding to
PqsRwt and PqsRT265A, respectively (SI, Figure S3) displayed no
significant difference in the enthalpic contribution, which argues
against a beneficial H-bond interaction. Notably, we detected an
additional putative H-bond between the pyridine nitrogen lone pair
and a water molecule that had been observed in ligand-receptor-
complexes of the congener 22 (described below) and PDB entry
4JVC.[25]
Figure 3. a) Crystal structure of 11 (light blue carbon) in complex with PqsR91-319
(ribbon: black carbon; surface: white carbon; PDB ID 6Q7V). b) Crystal structure
of HHQ (grey carbon) in complex with PqsR91-319 (ribbon: black carbon; surface:
white carbon; PDB ID 6Q7U). c) Overlay of a) and b). The yellow arrow indicates
the applied rational for linker modification toward 20. Fluorine: green, nitrogen:
blue, oxygen: red, sulfur: yellow. Hydrogen omitted for clarity.
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ChemMedChem
RESEARCH ARTICLE
After obtaining this structural knowledge it became clear, why
linear compounds 9 and 10 showed low or even no activity
although they were enlarged along the correct growth vector. We
concluded that a certain flexibility in the linker part is essential for
the compound to adopt an angled binding conformation. Hence,
in the further proceeding, modifications of this region were
prioritized (Figure 2) in order to optimize compound 11.
Following this strategy, we evaluated the role of the NH within the
linker through synthesis of N-methylated derivative 14 and benzyl
alcohol 15. Compound 14 showed a slightly decreased on-target
activity corroborating the notion that the H-bond observed in the
X-ray structure of 11 does not translate into a significant gain in
binding affinity. Interstingly, the exchange of NH to O (compare
11 and 15) led to a more significant drop in potency.
Usually, unfavorable entropic penalties resulting from ligand
flexibility are addressed by rigidification of compounds through
reduction of rotatable bonds.[35] Hence, we made several efforts
to introduce less flexible and cyclic structures into the linker region
(16-18). However, none of these modifications led to improved
potency. For example, the rigidified amide linker (16) is not able
to adapt the angled conformation of 11. As a consequence, the
antagonistic activity was almost abolished. The same trend was
observed for cyclized derivatives 17 and 18. Especially in the case
of the indane derivative 17, this loss of activity was surprising.
Based on the crystal structure of 11 we assumed that neither the
angled conformation nor the observed interactions would be
affected by introduction of this 5-membered ring (SI, Figure S2).
These findings underline that the optimal geometric arrangement
of the pyrimidine headgroup and the second aromatic system is
difficult to be mimicked by rigid ligand structures. In this regard,
we used the crystal structure in complex with the native ligand
HHQ (Figure 3b).
A superimposition with HHQ and 11 (Figure 3c) raised the idea to
introduce a prolonged, even more flexible linker to mimic the alkyl
sidechain of these compounds. Therefore, compounds 19-22
were synthesized. The activity of 19 against PqsR was
unchanged compared to 11. Interestingly, introduction of four-
atom linkers resulted in remarkable potencies in the heterologous
reporter gene assay (20-22). The N-propyl amine linker (20)
showed a 20-fold boost in potency (Table 2; 11 IC50 = 2.6±0.8 µM
vs. 20 IC50 = 0.14±0.04 µM). A co-crystal structure of 20 in
complex with PqsR91-319 showed that the extended linker pointed
deeper into the pocket occupied by the alkyl chain of the natural
ligand, enabling additional CH-π interactions with Tyr258 while
the interactions of the aminopyridine head group were retained
(Figure 4).
Figure 4. a) Overlay of crystal structures of 20 (orange carbon) and HHQ (grey
carbon) in complex with PqsR91-319 (ribbon: black carbon; surface: white
carbon; PDB ID 6Q7W). b) Minimal energy structure of 20 derived from a
constrained molecular dynamics simulation based on solved X-ray structure.
The main interactions of 20 with PqsR observed within 300 ps of simulation are
shown (hydrogen bonds: light blue, CH-π interactions: yellow). Fluorine: green,
nitrogen: blue, oxygen: red, sulfur: yellow. Hydrogen omitted for clarity.
Effects in P. aeruginosa. Pyocyanin is one of the most
prominent and characteristic virulence factors released by P.
aeruginosa during acute and chronic infections.[36] The redox-
active blue pigment has cytotoxic and immunomodulating
properties.[36] Moreover, it has been clearly demonstrated that
pyocyanin promotes the development of
a
pulmonary
Lastly, we exchanged the alkyl chain (20) by an ethanolamine (21)
and glycine (22) linker moiety to lower the clogD values.
Compound 22 showed only a slightly reduced antagonistic activity,
whereas 21 showed a 20-fold drop. Overall, 20 emerged as the
frontrunner compound from this fragment-growing approach,
pathophysiology in mice similar to the lung of infected cystic
fibrosis patients.[37] Hence, it was of major interest to demonstrate
that 2-amino-pyridines were able to translate the PqsR-directed
activity measured in E.coli into antivirulence activity in P.
aeruginosa. Compounds 7, 11 and 20-22 were evaluated for the
inhibition of pyocyanin in the highly virulent clinical isolate PA14
(Table 3). In this regard, it is important to point out that P.
aeruginosa shows low intrinsic outer-membrane permeability for
xenobiotics and applies a multitude of different efflux pumps.[36]
Astonishingly, fragment 7 already showed an inhibitory significant
effect on pyocyanin, albeit at high concentration.
showing
a
superior combination of nanomolar inverse
agonistic/antagonistic activity, enthalpy-driven binding (EE =
0.50 kcal/mol, SI, Table S1) and reasonable physico-chemical
properties (clogD=3.8). Remarkably, in contrast to the usual drug
optimization process relying on reducing the amount of rotatable
bonds, we gained potency through allowing ligand flexibility.
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ChemMedChem
RESEARCH ARTICLE
The activation of PqsR drives the expression of the HHQ
biosynthesis operon pqsA-E, leading to enhanced alkyl quinolone
(AQ) levels and finally to an auto-inductive loop. P. aeruginosa
produces more than 50 different AQ congeners.[39] Among others
HHQ and HQNO are most prevalent.[39] HQNO showed Gram-
positive antibacterial activity that promotes a growth advantage
Table 3. Effects on pyocyanin in P. aeruginosa
for
P
aeruginosa in mixed microbial communities.[40]
Subsequently, the most interesting compounds were also
evaluated for their effect on HHQ and HQNO levels in a
PA14pqsH strain. QSI 11 was able to affect HHQ (48±4%
inhibition at 200 µM) and HQNO (36±3% inhibition at 200 µM).
The enlarged compound 20 showed an improved efficacy (56±4%
HHQ inhibition at 100 µM and 48±1 HQNO inhibition at 100 µM).
These results were in line with already reported data showing that
the expression of virulence factors and the levels of AQs are
differently sensitive to the antagonization/inverse agonisation of
PqsR.[22,26]
Pyocyanin[a]
IC50 [µM]
PEI[b]
MW
R1
R2
7
0.12
0.0165
420.4
331.4
162.1
285.2
313.3
315.3
328.3
2.0
0.0172
Conclusion
The rapid development and spreading of resistances against
conventional antibiotics has raised the awareness of a broader
public in recent years. This ability of pathogens demonstrates the
power of evolutionary adaption processes.[41] The development of
PqsR-targeting QSI pursues the strategy to limit the virulence of
P. aeruginosa without affecting bacterial fitness. Hence, these
antivirulence compounds might be more robust to adaptive
evolution because they avoid exerting high selective pressure.[42]
Herein, we report on the discovery and optimization of 2-amino-
pyridines as promising PqsR-targeting QSI. Starting from an SPR
fragment screening[28] ligand 3 was selected based on its unique
EE, although it did not show significant antagonistic activity.
Successfully growing and improving a fragment-screening hit
43% @ 250 µM
102
-
11
20
21
22
0.0140
5.9
0.0168
58% @ 100 µM
37% @ 100 µM
-
-
[a] Photometric quantification of pyocyanin from PA14
without structural information is
a challenging approach.
cultures; given values represent the mean of at least two
independent experiments; SD<25%. [b] PEI Pyocyanin Efficiency
Index PEI=pIC50/MW.
Therefore, close commercial analogs were investigated, leading
to compound 7, which showed improved affinity and comparable
EE. Furthermore, this intermediate compound possessed
moderate antagonistic activity. By the introduction of a flexible
benzyl-amine linker into the 4-position (11) affinity and on-target
activity were improved drastically. The solution of the crystal
structure of 11 in complex with its bacterial target PqsR facilitated
further structure-guided optimization. Notably, the ligand
assumed an angled conformation spanning over the quinolone-
and alkylchain-pocket. Inspired by the alkyl chain of the natural
ligand HHQ, we extended the linker moiety, which led to the top
candidate 20 showing inverse agonistic/antagonistic activity in the
nanomolar range. The rather unusual approach of allowing more
flexibility instead of rigidifying the ligand might be also applicable
to a broader scope of druggable proteins. Especially in cases
where the biological function of the drug target depends on
different conformational states (e.g. inducible transcriptional
regulators), this flexible fragment-growing strategy might be a
valuable tool to rapidly generate lead-like molecules. Usually, a
selected fragment-sized hit already provides efficient directed
interactions with the target. Allowing compound flexibility during
the enlargement process could help to retain the beneficial
binding geometry of the fragment core while granting access to
Compound 11 showed low micromolar activity in the E.coli assay
(IC50=2.6±0.8 µM) and inhibited pyocyanin with an IC50 of
105±13 µM. Optimized hit 20 displayed the highest cellular
efficacy of this new class of QSI (IC50 = 5.9±0.7 µM). The
literature-known compounds R1-R2 were included as references
in Table 3. The PqsR antagonist R1 originated from an HTS
screening.[26] R2 was discovered in-house by modification of
HHQ.[22] We established a metric called PEI (pyocyanin efficiency
index; Table 3), which normalized the effect on pyocyanin for the
molecular weight of the inhibitors to enable a better comparison.
The optimized and lead-like hit 20 displayed efficiency in the
range of the benchmark compounds R1 and R2. This clearly
emphasized the potential of newly discovered QSI 20. In contrast,
slightly less active compounds 21 and 22 were not able to inhibit
pyocyanin to the same extent. This might be due to efflux or
permeation problems. The phenomenon that even small changes
within a molecule can have drastic effects on the activity in P.
aeruginosa was also observed in other projects and emphasizes
the need for further insights on the requirement for intracellular
activity, especially in the case of Gram-negative pathogens.[22,38]
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
RESEARCH ARTICLE
Michael Zender, Florian Witzgall,
Alexander Kiefer, Benjamin Kirsch,
Christine K. Maurer, Andreas M. Kany,
Ningna Xu, Stefan Schmelz, Carsten
Börger, Wulf Blankenfeldt, Martin
Empting*
Page No. – Page No.
Flexible Fragment Growing Boosts
Potency of Quorum Sensing Inhibitors
against Pseudomonas aeruginosa
Virulence
Growth vector identification and exploitation: Enthalpic efficiency-driven
fragment priorization lead to 2-amino pyridines as starting points for the generation
of novel pathoblockers against P. aeruginosa. Allowing flexibility in the hit
optimization process facilitated successful fragment growing accompanied by a
thousand-fold boost in activity as compared to the initial fragment.
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