Design, synthesis, and evaluation of compounds capable of reducing Pseudomonas aeruginosa virulence

Article abstract of DOI:10.1016/j.ejmech.2019.111800

Anti-virulence approaches in the treatment of Pseudomonas aeruginosa (PA)-induced infections have shown clinical potential in multiple in vitro and in vivo studies. However, development of these compounds is limited by several factors, including the lack of molecules capable of penetrating the membrane of gram-negative organisms. Here, we report the identification of novel structurally diverse compounds that inhibit PqsR and LasR-based signaling and diminish virulence factor production and biofilm growth in two clinically relevant strains of P. aeruginosa. It is the first report where potential anti-virulent agents were evaluated for inhibition of several virulence factors of PA. Finally, co-treatment with these inhibitors significantly reduced the production of virulence factors induced by the presence of sub-inhibitory levels of ciprofloxacin. Further, we have analyzed the drug-likeness profile of designed compounds using quantitative estimates of drug-likeness (QED) and confirmed their potential as hit molecules for further development.

Full text of DOI:10.1016/j.ejmech.2019.111800

Journal Pre-proof
Design, synthesis, and evaluation of compounds capable of reducing Pseudomonas
aeruginosa virulence
Mohammad Anwar Hossain, Narsimha Sattenapally, Hardik I. Parikh, Wei Li, Kendra
P. Rumbaugh, Nadezhda A. German
PII:
S0223-5234(19)30952-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111800
EJMECH 111800
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2019
Revised Date: 16 October 2019
Accepted Date: 17 October 2019
Please cite this article as: M.A. Hossain, N. Sattenapally, H.I. Parikh, W. Li, K.P. Rumbaugh, N.A.
German, Design, synthesis, and evaluation of compounds capable of reducing Pseudomonas
aeruginosa virulence, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111800.
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DESIGN, SYNTHESIS, AND EVALUATION OF COMPOUNDS CAPABLE OF REDUCING
PSEUDOMONAS AERUGINOSA VIRULENCE.
Mohammad Anwar Hossain1, Narsimha Sattenapally1, Hardik I. Parikh2, Wei Li3, Kendra P. Rumbaugh4,
Nadezhda A. German1.
1
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of
Pharmacy, Amarillo, TX, 79106, USA
2 Department of Medicinal Chemistry, School of Pharmacy, VCU, Richmond, VA 23284, USA
3 Department of Chemical Engineering, Texas Tech University, Lubbock, TX, 79409, USA
4 Dept of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA
*Corresponding author, Tel: 806-414-9190, email: nadezhda.german@ttuhsc.edu
Abstract:
Anti-virulence approaches in the treatment of Pseudomonas aeruginosa (PA)-induced infections have
shown clinical potential in multiple in vitro and in vivo studies. However, development of these
compounds is limited by several factors, including the lack of molecules capable of penetrating the
membrane of gram-negative organisms. Here, we report the identification of novel structurally diverse
compounds that inhibit PqsR and LasR-based signaling and diminish virulence factor production and
biofilm growth in two clinically relevant strains of P. aeruginosa. It is the first report where potential
anti-virulent agents were evaluated for inhibition of several virulence factors of PA. Finally, co-
treatment with these inhibitors significantly reduced the production of virulence factors induced by the
presence of sub-inhibitory levels of ciprofloxacin. Further, we have analyzed the drug-likeness profile of
1

designed compounds using quantitative estimates of drug-likeness (QED) and confirmed their potential
as hit molecules for further development.
1.
Introduction:
Quorum sensing is part of the bacterial network that supports many critical biological processes,
including adaptation, pathogenicity, and resistance development. Understanding of the biology of
quorum sensing in Pseudomonas aeruginosa (PA) is required to explain the versatility of this pathogen.
PA has multiple extracellular factors required for survival in the host and facilitation of the microbial
dissemination, including elastases,
O2N
Br
D
A
N
S
O
N
H
alkaline
proteases,
pyocyanin,
O
Br
HN
O
O
C-30
M64
siderophores, rhamnolipids, and
hydrogen cyanide 1-3. It uses quorum
sensing (QS), a “communication”
via small chemical molecules, to
regulate production and release of
these factors. In addition, QS
pathways control activation of cell-
associated factors required for the
host colonization, biofilm formation
and establishment of chronic
Pyoverdine: > 90% inhibition @ 10ꢀM
In vivo: 10-1000 fold increase in bacterial
clearance
Pyocyanin: 95% inhibition @ 1ꢀM
In vivo: prevents re-colonization
60% reduction in mortality
30% reduction in mortality
B
E
O
H
N
N
H
C9H19
HN
O
O
O
Cl
V-06-018
TP-5
Cl
Pyocyanin: 90% inhibition @ 100ꢀM
LasR: IC50 = 50ꢀM
O
C
Br
F
O
O2N
OH
C7H15
O
N
H
O
N
H
1
Pyocyanin: 10-30% inhibition @ 50ꢀM
Biofilm: 40% inhibition @ 50ꢀM
PqsR: 100% inhibition @ 5ꢀM
Figure 1. Examples of compounds reported as inhibitors of PqsR (A,
F) and LasR (B-E) pathways. Ligand 1 (F) was selected as a
reference compound for our studies.
infection, like flagella, pili, and lipopolysaccharides. Inhibition of specific, or a combination of, QS
pathways in PA has been long proposed to halt pathogenicity without affecting viability, hence, without
forcing bacteria to develop resistance.4 Thus, quorum sensing inhibitors (QSIs) are thought to have
therapeutic potential, especially as potentiators of existing antibiotics5-12 and their development was
2

summarized in many comprehensive reviews13-16. It has been noted13 that limited membrane
permeability, active efflux17, 18 and presence of PA produced acylases (Ntnase)19 are major obstacles for
the development of PA QSIs. Additional boundaries in the field include the limited comparative studies
of lead compounds and use of modified E.coli strains for testing molecules designed to modulate
quorum sensing in PA10. Nevertheless, several developed compounds have shown significant activity in
vivo, confirming the therapeutic potential of the “anti-virulence” concept (Fig. 1).
PA has three highly interconnected major QS signaling pathways, and their complex regulation is
described elsewhere20. These systems, Las, Rhl, and PQS, control various aspects of PA pathogenicity,
and the contribution of each to infection is influenced by the surrounding microenvironment, such as
levels of phosphates21, iron22-24, and oxygen21, 25. Furthermore, the genetic make-up of PA isolates
dictates the individual contribution of each of the three QS systems26-29. The las and rhl genes are
involved in the regulation of lipase production, synthesis of elastases, and secretion of rhamnolipids.
The Las system is activated during acute stages of an infection30, but its role is limited under chronic
conditions31.
The PqsR protein regulates the expression of several virulence factors when activated by 2-heptyl-3-
hydroxy-4(1H)-quinolone, also known as Pseudomonas Quinolone Signal (PQS, Fig.2).
O
O
CH3
N
O
Among these factors, pyocyanin (Fig 2), a 5-
methyl-1-hydroxypenazine, has the most
clinical significance due to multiple toxic
effects it produces in patients. PqsR inhibitors
(Fig. 1) are known to inhibit biofilm
F
OH
OH
N
N
N
N
H
C7H15
HN
O
PQS
ciprofloxacin
pyocyanin
Figure 2. Representative structures of Pseudomonas
Quinolone Signal PQS, antimicrobial agent ciprofloxacin,
and virulence factor pyocyanin.
formation30, and production of pyocyanin32 under in vitro an in vivo conditions. Interestingly, one of the
papers reported that PA has functionally inversed a quinolone-based inhibitor using available enzyme
3

machinery33, where quinolone-based antagonist of PqsR was enzymatically converted by bacteria to a
potentiator of QS. Availability of these enzymes is explained by the large pool of quinolone-based
molecules produced by PA, some acting via the PqsR pathway34. Similar complex responses were
observed in PA cells treated with the low levels of ciprofloxacin (Fig. 2). In the presence of sub-
inhibitory levels of this antibiotic, PA had an increased rate of biofilm growth. Thus, ciprofloxacin
promoted pathogenicity of bacteria, raising potential issues with its application34, 35. Overall,
experimental data suggest that quinolone-based QSIs may have limited therapeutic potential due to the
heterogenicity of the induced responses.
Our research group is interested in developing novel, structurally diverse, non-quinolone inhibitors of
the PqsR and LasR pathways. We envisioned that inhibitors with dual activity might weaken PA
virulence in both acute and chronic stages of infection. In addition to developing new anti-virulence
agents, we have set a goal of monitoring possible correlation between the ability of a compound to
inhibit PqsR or LasR and its effect on the production of biofilm, pyocyanin, and rhamnolipids. Previous
reports indicated connections between inhibition of a specific pathway and a selected virulence factor,
but we were not able to find reports analyzing the effect of the same compound on several virulence
factors. We envisioned that complicated regulation of quorum sensing in PA might result in the scenario
that the same compound might inhibit one virulence factor, but promote activation of another one,
reducing or even eliminating the overall therapeutic potential of a molecule.
Towards these goals, we searched for novel QSIs using virtual screening approach that utilized a
CH3
N
N
O
A
H3C
B
H3C
N
N
O2N
X
R
Cl
O
F
NH
CH3
O
O
N
NH
6a: X = C; R =
O
O
6c: X = N; R =
O
N
N
H
O
O
O
H
O
O
Cl
3
2
N
H
6b: X = N; R =
O
N
Cl
BuO
O
O
O
S
R
N
OBu
N
H
N
N
N
F
O
N
NH
7a: R =
HO
7c: R =
Cl
Figure 3. Compounds prepared in our laboratory for evaluation as anti-virulnce agents. A. Compounds identified
O
4
by target-HNbased approach. B. Compounds designed using fragment-based approach.
N
O
NH
7b: R =
O
5
N
O
4

reported crystal structure of PqsR (see Supplemental Materials) and a library of compounds available
from ZINC databases36. Using HINT score and accessibility of compounds by synthetic routes as
selecting tools, we have identified four compounds (Fig. 3) for synthesis and evaluation in our
laboratory. All four compounds are novel and possess distinct chemotypes. Further, we used fragment-
based approach to construct ligands with the potential activity at both receptors, LasR and PqsR (Fig 3).
By using both methods, target-based and fragment-based design, we expected to obtain PqsR inhibitors
with various degrees of selectivity for LasR or PqsR, a strategy that could potentially identify the
contribution of each signaling pathway in the observed activity.
2.
Chemistry:
37-41
The reference compound 1 was prepared as previously published
(see supplemental materials).
Target compounds 2-7 (Fig. 3) were not reported previously, and respective synthetic routes were
developed in our laboratory (Schemes 1-7).
Synthesis of the target compound 2a and its methyl-substituted analog 2b started with the hydrolysis of
dimethyl 2-nitroterephthalate to a carboxylic acid derivative 11 followed by its amide coupling with the
2,3-dimethyl aniline, unsubstituted or monosubstituted. Resulted nitrobenzoate 16 was reduced using
standard procedure. The final step consisted of Meerwein’s arylation42 reaction catalyzed by copper
bromide and afforded target compounds in the 10% overall yield (Scheme 2).
5

Scheme 1. Synthetic route of the target compound 2. Reagents and conditions: a) 1N NaOH (aq), 1,4-dioxane, r.t,
18 h; b) 3,4-dimethylaniline, BOP, DIPEA, DMF, r.t, 16 h; c) hydrazine hydrate, EtOH, Raney Ni, 50 °C, 2 h; d)
(i) NaNO2, HBr(aq), water, 0-5 °C, 30 min, (ii) CuBr, acetone, 4-chlorostyrene, 0-5 °C, 60 min.
Target compound 3 was synthesized in three steps starting from the commercially available 4-sec-
butylphenol as described in Scheme 2. Bromoethoxy intermediate 19 was obtained upon alkylation of
starting phenol with the 1,2-dibromoethane and further coupled with the 5-chloro-2-
hydroxybenzaldehyde to obtain compound 20. Final step employed standard reductive amination
procedure to gain final compound 3.
Scheme 2. Synthetic route of the target compound 3. Reagents and conditions: a) 1,2-dibromoethane, NaOH(aq),
reflux, 4 h; b) 5-chloro-2-hydroxybenzaldehyde, NaOH, EtOH, reflux, 6 h; c) 4-amino-1,2,4-triazole, EtOH,
reflux, 5 h
.
To access compound 4, we have coupled commercially available 4-fluorobenzaldehyde 21 with the 4-
chlorophenol. Aldehyde 22 was reduced in the presence of the lithium aluminum hydride, followed by
reaction with phosphorous tribromide to gain benzyl bromide 24. Intermediate 25 was prepared in our
laboratory by reacting 3-bromopropylamine with sodium azide (see Supplemental Materials) and further
coupled with 24 in the presence of cesium carbonate. Formation of the triazole ring in the target
compound 4 was achieved by modified azide-alkyne cycloaddition using morpholine derivative 27, also
prepared in our laboratory with a total yield of 9.4% (supplemental material scheme S6).
6

Scheme 3. Synthetic route of the target compound 4. Reagents and conditions: a) 4-chlorophenol, Cs2CO3, DMF,
85 °C, 16 h; b) (i) LiAlH4, THF, 0 °C to r.t, 4 h, (ii) PBr3, DCM, 0 °C, 30 min, r.t, 12 h; c) 3-azidopropan-1-amine
(25), Cs2CO3, DMF, 85 °C, 16 h; d) (i) 27, MeOH:H2O (5:1), Na2CO3, r.t, 30 min; (ii) DMF, r.t, 24 h.
Scheme 4 depicts the synthesis of compound 5a that starts with the nitration of 4-chlororesorcinol to
obtain the intermediate 29a. After coupling with butylbromide, nitro-group of 30a was reduced to amine
and resulted compound was condensed with 2-naphtol to access the final product 5a. Compounds 5b and
5c were prepared in a similar manner starting with acylated chlororesorcinol (see supplemental material,
scheme S8).
Scheme 4. Synthetic route of the target compounds 5a. Reagents and conditions: a) acetic acid, acetic anhydride,
ceric ammonium nitrate, 0 °C, 30 min, r.t, 30 min; b) Cs2CO3, DMF, r.t, 12 h; c) (i) N2H4, Raney Ni, EtOH,
50 °C, 1 h, (ii) 2-naphthol, NaNO2, HCl, water, 0-5 °C, 12 h.
Target compound 6a was prepared via Friedel-crafts alkylation of benzene 32 with glutaric anhydride,
followed by nitration of the intermediate 33. Final step in this scheme included reaction of the
carboxylic acid 34 with cyclopentylamine under standard amide coupling conditions.
7

Scheme 5. Synthetic route of the target compound 6a. Reagents and conditions: a) anhydrous AlCl3, DCM, r.t, 1 h,
40 °C, 6 h; b) H2SO4, -20 °C, fuming HNO3:H2SO4 (1:3), -15 °C, 15 min; c) cyclopentylamine, BOP, DIPEA,
DMF, r.t, 16 h.
To access compound 6b, we had to prepare key intermediate 38 in three steps starting with the L-(+)-
methionine (Scheme 6A). 3-nitrobenzoyl chloride 39 was condensed with the alanine and further reacted
with either intermediate 38 or with the commercially available flucytosine to gain compounds 6b and 6c
respectively (Scheme 6B).
Scheme 6. Synthetic route of the intermediate 30 (A) and target compounds 6b-c (B). Reagents and conditions: a)
L-(+)-methionine, H2O : MeOH (7:1), MeI, r.t, 48 h; b) NaHCO3, H2O, reflux, 15 h; c) 6 N HCl, 30% H2O2,
reflux, 1 h; d) β-Alanine, 0-5 °C, 1 N NaOH (aq), 30 min; e) respective amine, BOP, DIPEA, DMF, r.t, 16 h.
Our last set of derivatives was prepared using steps described in Scheme 7. Key intermediate 44 was
accessed by converting ethyl 4-bromobutyrate 41 to iodobutyrate 42 and reacting it with the sodium
benzenesulfinate. Next, target compounds 7a-c were prepared by coupling carboxylic acid 44 with the
corresponding amines in 15% overall yield.
8

Scheme 7. Synthetic route of the target compounds 7a-c. Reagents and conditions: a) NaI, acetone, reflux 18 h; b)
(i) sodium benzenesulfinate, EtOH, reflux, 12 h, (ii) 95% EtOH, LiOH, r.t, 2 h; c) respective amine, BOP, DIPEA,
DMF, r.t, 16 h.
Final compounds were characterized using nuclear magnetic resonance spectroscopy (1HNMR and
13CNMR), high-resolution mass spectrometry (HRMS), and the purity of final compounds was
confirmed by elemental analysis and/or by high-performance liquid chromatography (HPLC) (see the
Supporting Information for full characterization data). Target compounds with the purity of >95% were
subjected to biological testing.
3. Results and discussion.
All in vitro studies include a previously reported PqsR inhibitor, compound 1, as a positive control. It is
a “prove-of-concept” study, where we aim to identify inhibitors of QS in P. aeruginosa using a battery
of in vitro tests. Based on the previously published research43, 44, we have selected to screen all tested
compounds for anti-virulence activity at 10 ꢀM level. Further, the most active compounds will be
submitted for a concentration-dependent analysis of their activity, and results will be promptly reported.
Obtained data were analyzed using GraphPad Prism 8.0.1, and statistical significance was calculated
using one-way ANOVA with or without multiple comparisons with three to eight replicates for all the
experiments (n = 3-8) and at least two independent biological experiments (N ≥ 2).
3.1 Effect of selected compounds on biofilm growth of PA.
9

Firstly, we confirmed that our compounds
displayed
no
B
A
Figure 4. A panel of novel compounds and reference compound 1 have shown no cytotoxicity in PAO1 cell line at
10 ꢀM level or above (A). At the same time, these compounds interfere with the P. aeruginosa biofilm growth (B).
Biofilm biomass (OD595/OD600) was measured by crystal violet staining after 10 hours of growth in the presence or
absence (for vehicle-treated control) of 10 ꢀM QSIs. Data presented as the percent growth of biofilm-biomass in
comparison with vehicle-treated control. Data shown as mean ± SEM of at least 18 experiments. Statistical
significance was assessed using one-way analysis of variance (ANOVA) plus Dunett’s posttest for multiple
comparisons **, P < 0.01; ***, P < 0.001; ****, P 0.0001.
cytotoxicity wild-type PA strains (Fig. 4A, supplemental material Table S2.)45. Use of both PAO1 and
PA14 cell lines in cytotoxic screening was done to ensure that the observed effects were not cell-line
specific46. Next, we have assessed the effect of synthesized compounds on PA biofilm growth using two
different strains, PAO1 and PA14. Under presented experimental conditions, biofilm growth over 12
hours leads to high background noise for biofilm reading due to an aggregation of dead cells. Thus,
according to our observations and published reports47-49, in all biofilm-based assays, bacteria were
incubated in the presence of a selected compound for 12 hours at 37 °C. Final readings (OD595) were
taken after biofilm residues were stained with crystal violet (see supplemental material). To ensure the
consistency in the analysis of observed results we have normalized data as a ratio of the readings of
crystal violet staining (OD595) and optical density of growth
10

(OD600), as previously reported50, 51. Under
experimental conditions, no inhibition was
observed in the presence of all three phenylazo
analogs 5a-5c. Compound 2b, a methylated
analog of 2a, has shown some of the inhibition,
although not statistically significant due to a
larger variation in responses we have observed
(Fig. 4). All other molecules have inhibited the
biofilm growth in the range of 40% - 60%,
although none had exceeded the activity of
reference compound 1.
Figure 5. Effect of tested compounds on the production of
pyocyanin by PAO1 bacterial strain. All ligands were tested
at 10ꢀM level. Percent of pyocyanin production was
presented in comparison with the vehicle-treated control.
Shown are the average of ± standard error (SEM) of at least
32 replicates. Statistical significance was assessed using
one-way analysis of variance (ANOVA) plus Dunett’s
posttest for multiple comparisons *, P < 0.0183; **, P <
0.0036; ****, P < 0.0001; ns – not statistically significant.
3.2 Effect of selected compounds on production of pyocyanin.
PA produces multiple extracellular factors for survival in the host: elastases, alkaline proteases,
pyocyanin, siderophores, rhamnolipids, and hydrogen cyanide, which all facilitate microbial
dissemination. Among them pyocyanin plays a significant role in the establishment and progression of
infections25, 52, 53. Levels of this toxic metabolite reach micromolar levels in the sputum of patients with
cystic fibrosis, causing a wide range of side effects, including tissue inflammation, suppression of the
immune system and others. We have determined the IC50 of pyocyanin to be 16.8 μM in mouse 3T3
cells (see supplemental material, Fig. S4). To investigate the ability of pyocyanin to modulate the host
environment via G-protein coupled receptors (GPCRs)- induced pathways we have obtained receptor
binding profile of this compound from the National Institute of Mental Health’s Psychoactive Drug
Screening Program, where primary binding assay was used to monitor GPCR inhibition effect, if any, at
11

the level of 10 μM.
No significant
A
B
inhibition of tested
GPCRs by pyocyanin
was observed (see the supplemental material, Fig. S5).
To measure levels of produced pyocyanin, we have used reported spectrometry-based quantification
method. Among tested compounds, a statistically significant reduction in the production of pyocyanin
was observed in PAO1 strain incubated with compounds 2a, 3, 4, 6a and 7a. Synthesized analogs 5a-5c
have shown either no effect, or changes in pyocyanin levels were not statistically significant. Since this
class of molecules had no effect on biofilm growth and pyocyanin production (Fig 4, 5), we have
omitted phenyldiazo series from further studies. Similarly, compound 2b also was not included in the
further biological testing. Compounds 6c, 7b, and 7c have potentiated the production of pyocyanin in the
range of 20-90% and were omitted from the study. Interestingly, all three compounds caused reduced
biofilm growth when incubated with PAO1 at the level of 10 μM. These data suggest that there is no
strong relationship between compound’s ability to inhibit biofilm growth and to alter the production of
pyocyanin.
3.3 Effect of synthesized analogs on PqsR and LasR systems in selected PA strains.
Further, we evaluated the ability of selected compounds to inhibit the PqsR and LasR QS pathways at
the concentration level of 10 ꢀM using well-established gene reporter assays (PA14 pAW1::GFP54, and
PAO1 lasB::GFP55) (Fig. 6). Among tested compounds, only 6c demonstrated relative selectivity for
PqsR, whereas the rest of the molecules had shown dual inhibition of both QS pathways (Fig. 6). PqsR
ligands
12

2-4
that
were
Figure 6. Reference compound 1 and selected novel ligands reduced the QS-induced
signaling in P. aeruginosa. (A) P. aeruginosa PAO1 lasB::GFP expressing LasR, (B) P.
aeruginosa PA14 pAW1::GFP expressing PqsR after 12 hours of incubation in presence
and absence of 10 µM of tested compounds. P. aeruginosa GFP reporters were grown for
12 h in LB broth supplemented with 5% BSA, monitored for their level of GFP
fluorescence, and data were normalized to growth (OD600). Data shown the average of at
least 32 experiments. Statistical significance was assessed using one-way analysis of
variance (ANOVA) plus Dunett’s posttest for multiple comparisons *, P < 0.05; **, P <
0.0022; ****, P 0.0001.
identified using the
target-based
approach
demonstrated no selectivity for PqsR over LasR, possibly due to the similarity of binding pockets of
both of these proteins. This statement can be further supported by the lack of reported selective PqsR
inhibitors. As shown in Fig. 6, active compounds inhibited quorum sensing-induced signaling up to 30%,
whereas compound 6c and 7c have shown no significant effect in blocking LasR-induced signaling. It
has to be noted that in our hands, positive control 1 (Fig. 6) was not as effective in inhibition of PqsR as
it was reported previously (Fig. 1). This observation once again highlights the ultimate need for the use
of unified protocols allowing cross-referencing of results obtained in different laboratories. We saw no
correlation between the level of QS inhibition and the level of inhibition of biofilm growth for any tested
compounds.
3.4 The effect of the selected compound on production of rhamnolipids.
Next, we evaluated the ability of our compounds to modulate the production of rhamnolipids, another
virulence factor produced by PA. Rhamnolipids are known to modulate bacterial motility and
development of biofilm and to promote bacterial invasion by disrupting the epithelial cell barrier.
Although there is no clear indication that inhibition of rhamnolipids will result in therapeutically
significant outcome, we were interested to see if inhibition of PqsR and LasR by our compounds will
13

affect the production of rhamnolipids in PA.
As indicated in Fig. 7 our compounds 3, 6a, 6b,
and 7a have been able to reduce levels of
rhamnolipids by 30-50%. At the same time,
compound 4 increased levels of rhamnolipids,
although results were not statistically significant.
During this study, we have evaluated our
compounds for their ability to inhibit the production
of three virulence factors: biofilm, pyocyanin, and
rhamnolipids (Fig 8). We have noted that a given
compound may cause different, sometimes
Figure 7. Changes in the production of rhamnolipids in
the presence of tested compounds, including reference
compound 1. Quantification of rhamnolipids was done
using methylene-blue-based assay after 24 hours of P.
aeruginosa growth in the presence or absence of 10
µM of compounds. The amount of produced
rhamnolipids was identified using standard curve
constructed with six concentrations of commercially
available rhamnolipids. Data shown the average of at
least 12 experiments. Statistical significance was
assessed using one-way analysis of variance (ANOVA)
plus Dunett’s posttest for multiple comparisons *, P <
opposite, responses in these assays. For example, compound 6b inhibited biofilm growth by a little over
40%, and inhibited pyocyanin
formation up to 40%.
At the same time, compound 4 had
caused inhibition of both biofilm and
pyocyanin production, but stimulated
the production of rhamnolipids.
Perhaps, a wide variety of potential
Figure 8. Observed trends in the anti-virulence activity of selected
compounds.
responses produced by the same compound is another factor that limits
14

translation of in vitro
A
B
results to in vivo settings.
Therefore, we envision that
full characterization of
QSIs
requires
an
Figure 9. Presence of selected compounds (10ꢀM) interferes with the production of
biofilm (A) and pyocyanin (B) by PA14. Effects were measured after 10 hours (A)
and 24 hours (B). Shown are the average of +/- standard error (SEM) of at least 20 (A)
and 12 (B) replicates. Statistical significance was assessed using one- way analysis of
variance (ANOVA) plus Dunnett’s posttest for multiple comparison to PA14, ****, P
< 0.0001.
understanding of the effect
of these molecules on a
panel of
virulence factors.
In our study, we have
identified compounds 2a, 3,
and 7a as the ones with the
most optimal anti-virulence
B
A
profile
that
includes
Figure 10. Treatment of PA14 with the combination of tested compounds and sub-
inhibitory level of ciprofloxacin reduces pro-virulence activity of sub-inhibitory
level of ciprofloxacin. All tested compounds, including reference compound 1, were
used at 10ꢀM. Anti-virulence properties of compounds were evaluated using
biofilm-based assay (A) and pyocyanin production test (B). Data presented as the
percent of production in comparison with vehicle-treated control. Shown are the
average of +/- standard error (SEM) of at least 20 replicates. Statistical significance
was assessed using one- way analysis of variance (ANOVA) plus Dunnett’s posttest
for multiple comparison to PA14, ****, P < 0.0001
reduced production of
biofilm, pyocyanin, and
rhamnolipids in PA.
3.5 Effect of selected
compounds on the pro-virulence effect of sub-inhibitory levels of ciprofloxacin.
Sub-inhibitory levels of the antibiotic ciprofloxacin have previously been reported to activate the
virulence
program of PA56, 57. Similarly, we have observed that ciprofloxacin at the level of ½ of MIC did not
prevent the formation of biofilm. Moreover, the levels of pyocyanin were increased by two-fold in
15

response to the sub-inhibitory concentration of ciprofloxacin when compared to untreated bacteria (Fig.
8). This observation is in line with the previously reported data58, 59, and raises serious concerns
regarding ciprofloxacin-based therapy. Thus, we have screened the selected QSIs 2a, 3 and 7a for their
potential ability to reduce effects caused by sub-inhibitory levels of ciprofloxacin. In this experiment, we
have evaluated our compounds using PA14 bacterial strain to mimic infection state. As seen in Fig 9,
compounds 2a, 3 and 7a reduced biofilm growth by 5-25% and pyocyanin levels by 15-80%. Further,
these compound significantly reduced the ability of ciprofloxacin (1/2 MIC) to affect the production of
virulence factors (Fig 10). This inhibition was more pronounced than the one caused by an inhibitor
alone (Fig 9), suggesting a synergistic character of such combination.
Thus, biological experiments have identified three compounds, 2a, 3 and 7a, with the ability to inhibit
biofilm development, production of pyocyanin and rhamnolipids in PA strains. To ensure that these
molecules can be used for further development we evaluated their drug-like potential using quantitative
estimates of drug-likeness (QED)60,where our ligands had shown QED of 0.4, an optimal value for a
starting hit molecule60-62supporting the developmental potential of the identified compounds (see
supplemental material).
4.
Conclusion.
Overall, we have used target-based and fragment-based approaches to identify seven chemically distinct
compounds with the ability to inhibit QS. These compounds and their analogs were prepared using the
methodology developed in our laboratory and evaluated as potential QSIs using two different clinically
relevant strains of PA. This paper, to our knowledge, is the first to report evaluation of potential QSIs
using several PA-related virulence factors: inhibition of PqsR and LasR, biofilm formation, inhibition of
pyocyanin production and production of rhamnolipids. Interestingly, we have noted that some of our
tested compounds had mixed effect on production of virulence factors by PA, causing inhibition of one
16

factor and potentiation of the others. These mixed profiling of molecules, if confirmed for other
previously reported QSIs, may explain loss of efficacy of some compounds when translating from in
vitro settings to in vivo studies.
As a result of this study, we have selected the top three compounds, 2a, 3 and 7a, where compounds 2a
and 3 represent novel scaffolds of QSIs. As anti-virulence agents, these compounds were shown to
reduce the production of all tested virulence factors of PA: biofilm, pyocyanin, and rhamnolipids.
Moreover, these compounds were shown to alleviate the pro-virulence effect of sub-inhibitory
concentrations of ciprofloxacin.
Further, we have evaluated these ligands as potential drug candidates using QED parameter. Thus, we
envision that these novel compounds could represent a new class of QSIs. Ongoing studies are focused
on the evaluation of selected compounds in vivo using acute and chronic PA infection models, as well as
further optimization of their potency.
17

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21

Highlights
•
•
•
•
Novel structurally diverse inhibitors of PqsR and LasR systems in P. aeruginosa were
designed using target-based and fragment-based approaches
Synthetic pathways were developed and implemented in our laboratory to prepare
designed analogs
All compounds were evaluated in vitro (P. aeruginosa-based assay) for their ability to
modulate the production of several virulence factors, and observed trends were compared
Selected compounds were shown to alleviate the pro-virulence effect of ciprofloxacin at
sub-inhibitory levels

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: