Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen Oxide and Its Analogues

Article abstract of DOI:10.1021/acsinfecdis.1c00045

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently found in the airways of cystic fibrosis (CF) patients due to the dehydrated mucus that collapses the underlying cilia and prevents mucociliary clearance. During this life-long chronic infection, P. aeruginosa cell accumulates mutations that lead to inactivation of the mucA gene that results in the constitutive expression of algD-algA operon and the production of alginate exopolysaccharide. The viscous alginate polysaccharide further occludes the airways of CF patients and serves as a protective matrix to shield P. aeruginosa from host immune cells and antibiotic therapy. Development of inhibitors of alginate production by P. aeruginosa would reduce the negative impact from this viscous polysaccharide. In addition to transcriptional regulation, alginate biosynthesis requires allosteric activation by bis (3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO) inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind c-di-GMP. However, P. aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P. aeruginosa. Future development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P. aeruginosa.

Full text of DOI:10.1021/acsinfecdis.1c00045

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Article
Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen
Oxide and Its Analogues
Soo-Kyoung Kim, Huy X. Ngo, Emily K. Dennis, Nishad Thamban Chandrika, Philip DeShong,
Sylvie Garneau-Tsodikova,* and Vincent T. Lee*
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ABSTRACT: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen
that is frequently found in the airways of cystic fibrosis (CF) patients due to the
dehydrated mucus that collapses the underlying cilia and prevents mucociliary
clearance. During this life-long chronic infection, P. aeruginosa cell accumulates
mutations that lead to inactivation of the mucA gene that results in the
constitutive expression of algD-algA operon and the production of alginate
exopolysaccharide. The viscous alginate polysaccharide further occludes the
airways of CF patients and serves as a protective matrix to shield P. aeruginosa
from host immune cells and antibiotic therapy. Development of inhibitors of
alginate production by P. aeruginosa would reduce the negative impact from this
viscous polysaccharide. In addition to transcriptional regulation, alginate
biosynthesis requires allosteric activation by bis (3′-5′)-cyclic dimeric guanosine
monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO)
inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues
inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind
c-di-GMP. However, P. aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb
and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P. aeruginosa. Future
development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P. aeruginosa.
KEYWORDS: Alg44, alginate, cystic fibrosis, inhibitors, Pseudomonas aeruginosa
from individual CF patients.11−14 Even more problematic is
seudomonas aeruginosa is an opportunistic pathogen that
Pcapitalizes on deficiencies in the host immune response to that, over time in the patient, P. aeruginosa undergoes
cause acute and chronic infections.1 Despite not being one of
mutagenesis through mutation in the DNA repair gene
mutS,11,13 resulting in accumulation of additional mutations
the abundant commensal bacteria in the normal microflora of
typical healthy humans,2,3 P. aeruginosa has a tremendous
in the P. aeruginosa genome. One of the genes often mutated is
mucA,15 which leads to mucoid conversion. P. aeruginosa
ability to cause opportunistic infections as demonstrated by the
strains with mucA mutations lack the MucA protein and release
AlgT/U sigma factor.15−17 The AlgT/U activates the
constitutive expression of the algD-algA operon that encodes
the alginate biosynthesis proteins.15,18 The overexpression of
the alginate biosynthesis proteins leads to the constitutive
production of the mucoid alginate polysaccharide. The
combination of dehydrated mucus and the mucoid alginate
polysaccharide leads to further occlusion of the airway and loss
of lung function. Since P. aeruginosa producing alginate has
enhanced resistance to antibiotics, inhibition of alginate
prevalence of this bacteria in healthcare-associated infections
(HAIs).4 In addition to these infections, P. aeruginosa is often
found in the airways of cystic fibrosis (CF) patients.5 CF
patients have a defect in cystic fibrosis transmembrane
regulator (CFTR)6 that is a symporter for water and chloride
ions7 to maintain the appropriate hydration of the airway
surface liquid.8,9 In CF patients, the dehydrated mucus
collapses on the cilia leading to decreased mucociliary
escalatory clearance of mucus and trapped microbes.8,9 As a
consequence, CF airways have a suboptimal immune response
and an increased microbial load.10 Airway infections have
decreased the lifespan of CF patients and led to aggressive
antibiotic therapies that have greatly extended life expectancies.
However, P. aeruginosa is one bacterium that is not easily
eliminated by antibiotics. Despite these treatments, P. aerugi-
nosa has formed lifelong associations with patients as
determined by sequencing of genomes of longitudinal isolates
Received: January 25, 2021
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production may reduce airway blockage and antibiotic
resistance.
In addition to transcriptional regulation, alginate production
also requires allosteric activation by bis (3′-5′)-cyclic dimeric
guanosine monophosphate (c-di-GMP) through binding to the
PilZ domain of the Alg44 protein (Alg44PilZ), a component of
the biosynthesis complex in the cytoplasmic membrane.19−21
c-di-GMP is a signaling nucleotide widely used by bacteria.22
The molecule is synthesized from two guanosine triphosphate
(GTP) molecules by diguanylate cyclases, which are regulated
by the product through binding to the allosteric inhibitory site
(I-site).23,24 Similar to other secondary signaling molecules, c-
di-GMP binds receptors to allosterically alter protein
function.25 In the case of Alg44, ablation of the c-di-GMP
binding residues in the PilZ domain results in strains that fail
to produce alginate polysaccharide.20,21 On the basis of
analogy to the bacterial cellulose synthase (Bcs) system, c-di-
GMP binding to the PilZ domain is thought to shift the PilZ
domain that occludes the catalytic site in the glycosyltransfer-
ase.26 As a result, nucleotide activated monosaccharides can
enter the active site, polymerize, and be exported out of the
bacterial cell envelope.27 So, c-di-GMP synthesis and its
binding to Alg44 serve as potential targets of inhibition to
prevent alginate biosynthesis.
Different strategies have been taken to ameliorate the
negative effect of alginate production. One is using enzymes to
degrade alginate polymers.28,29 Another is to identify small
molecule inhibitors that either prevent expression of the algD-
algA operon, synthesis of c-di-GMP signaling, or c-di-GMP
binding to Alg44. Previous studies have identified two classes
of inhibitors that interfere with c-di-GMP activation of
alginate. One class is thiol-benzo-triazolo-quinazolinone that
interacts with the PilZ domain of Alg44 and prevents this
domain from binding c-di-GMP.30 A second class of inhibitor
is ebselen (Eb), which occupies the I-site of diguanylate
cyclases and inhibits the biosynthesis of c-di-GMP.31 Since Eb
is a compound that has undergone phase III clinical trials for
ischemic stroke32 and acute hearing loss,33 it was anticipated
that Eb analogues would have a fast-track to application as a
chemotherapeutic for treatment of CF. Because of its safety
profile and its effectiveness at inhibiting c-di-GMP, we tested
whether Eb can inhibit alginate production, which is regulated
by c-di-GMP. Here we show that Eb, ebselen oxide (EbO),
ebsulfur (EbS), and their analogues (Figure 1) can inhibit
alginate production by P. aeruginosa. From this small
structure−activity relationship (SAR) study, we show that
specific positions of Eb can be modified without affecting
inhibitory activity toward alginate. One particular modification,
EbO, improved the selectivity index for inhibition of alginate
production by about 20-fold. We show that Eb can covalently
modify PilZ domain of Alg44 using cysteine 98 (C98) and that
this modification prevents c-di-GMP binding. However, in
P. aeruginosa cells, modification of Alg44 at C98 is not required
for alginate inhibition. Together, these results indicate that
EbO, Eb, EbS, and their analogues can inhibit alginate
production by P. aeruginosa and may provide a strategy to
reduce disease burden by mucoid bacteria in the CF airway.
Figure 1. Chemical structures of compounds used in this study.
out in two steps as previously reported36 for analogous
compounds (Scheme S1). The first step involved the coupling
of 2-iodobenzoyl chloride with various amines in the presence
of a base to afford intermediates 4−8 in 25%-quantitative
yields. The resulting 2-iodobenzamides, when subjected to a
combination of CuI, 1,10-phenanthroline, and KSeCN in the
presence of a base (Cs2CO3) underwent cyclization to provide
Eb, 3b, 3g, and 3h in 4−13% yields.36
EbO, Eb, and Their Analogues Inhibit Alginate
Secretion by P. aeruginosa. Prior studies have shown that
Eb and its oxidized analogue, EbO, interfere with the
diguanylate cyclase WspR by covalent modification of cysteine
and by occupying the I-site to inhibit its activity.31 Since c-di-
GMP binding to Alg44 is required for alginate production, we
tested whether Eb and EbO can reduce alginate secretion by
P. aeruginosa. To assess inhibition of alginate secretion, we
employed the P. aeruginosa PA14 strain harboring pMMB-
algU.20,30 The parental PA14 strain with pMMB vector control
produced little alginate (11.96 1.12 μg/mL/OD600) (Figure
2).30 Induction of algU expression with 200 μM IPTG led to
copious production and export of alginate polysaccharide
(304.2 10.8 μg/mL/OD600) (Figure 2A; DMSO control).
When treated with 100 μM of Eb or EbO, alginate production
by P. aeruginosa PA14 pMMB-algU was reduced by ∼50% and
∼70%, respectively (Figure 2A). To confirm the potency of Eb
and EbO for alginate inhibition, P. aeruginosa PA14 was treated
with varying concentrations of Eb or EbO (0, 25, 50, and 100
μM). Eb inhibition only occurred at 100 μM concentration
(Figure 2B; green circles). In contrast, 25 μM of EbO was
sufficient to inhibit alginate secretion (Figure 2B; yellow
circles). From these data, the IC50 values were calculated to be
80 μM for Eb and 14 μM for EbO (Table 1). Addition of
compounds to extracts of alginate did not interfere with the
assay (Figure S17), suggesting that the observed inhibition
occurs during the growth of the bacterial cultures. Treatment
with 100 μM of Eb derivatives 3b, 3g, and 3h resulted in
inhibition of alginate production by P. aeruginosa, albeit to
different degrees (Figure 2C). The greater inhibition by 3g and
3h with planar aromatic groups as compared to 3b with the
isobutyl group suggest that properties such as size and
geometry of this side chain impacts interaction with the
cellular target. Taken together, these data indicated that Eb
RESULTS
■
Preparation of EbO, Eb, EbS, and Their Analogues.
We have previously reported the synthesis and characterization
of EbS and its analogues 1a−h, and 2a,b.34,35 Herein, the
synthesis of Eb and its analogues 3b, 3g, and 3h was carried
B
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Figure 2. Ebselen (Eb) and ebselen oxide (EbO) inhibit alginate production by P. aeruginosa. P. aeruginosa PA14 pMMB-algU for alginate
production by the addition of 200 μM IPTG was quantified in the presence of (A) 100 μM Eb (green), EbO (yellow), DMSO (white); or (B)
varying concentration of Eb (green circles) or EbO (yellow circles); or (C) 100 μM of Eb analogues 3b (gray), 3g (crimson), and 3h (brown). The
graph indicates the average alginate production per OD600 for six independent experiments. *, **, and *** indicate p values <0.05, 0.005, and
0.0005, respectively, by t-test.
and EbS are acting specifically to inhibit alginate production by
P. aeruginosa.
Table 1. Selectivity Index (SI) for Alginate Inhibition
a
b
c
Cpd
IC50 (μM)
CC50 (μM)
SI (CC50/IC50)
EbS and Its Analogues Potentiate Alginate Inhibition
in P. aeruginosa. Eb is a small molecule with a selenium atom
that serves as the active moiety for inhibition.37 Previous
studies have shown that EbS, the sulfur analogue of Eb, act to
inhibit growth of eukaryotic parasites,38 yeast,34 and broad
spectrum antibacterial activity35,39 and has IC50 similar or
better than Eb. To identify Eb analogues that have greater
potency for inhibition of alginate production, a SAR was
established for compounds that include the replacement of the
Se atom with sulfur, alteration of the phenyl ring, or a
combination of both types of modifications (Figure 1).
Induction of pMMB-algU by 200 μM IPTG in the presence
of the DMSO solvent carrier led to the production of alginate
(Figure 4). When treated with 100 μM EbS or EbS analogues
1a−1h, alginate production was reduced by approximately 3-
to 25-fold (Figure 4A). When treated with oxidized versions of
EbS (EbSO) analogues 2a and 2b, alginate production was
either not affected (2b) or, interestingly, increased by
approximately 1.4-fold (2a) (Figure 4B). The potency of
EbS and EbS analogues 1a−1h was determined by treatment
with varying concentrations (25, 50, or 100 μM) of EbS or EbS
analogues (Figure 4C). Treatment with EbS reduced alginate
secretion (102, 80, or 58 μg/mL/OD600) at 25, 50, or 100 μM
of concentration, respectively, compared to 533 μg/mL/OD600
of DMSO control (Figure 4C). Alginate production by
P. aeruginosa PA14 pMMB-algU was inhibited by EbS
analogues 1a−1h by 70% to 90%. The maximal inhibition of
alginate production was greater than 90% for EbS analogues,
1g and 1h, even at 25 μM concentration (Figure 4C). The
IC50 value was from 22 to 24 μM for EbS and EbS analogues
(Table 1). These results indicated that EbS and EbS analogues
are also potent inhibitors of alginate production by
P. aeruginosa compared to Eb. Prior studies showed that an
oxidized version of EbS (EbSO), the sulfoxide form of EbS, is
inactive against other bacteria.35 At 100 μM, the two EbSO
analogues, 2a and 2b, failed to inhibit alginate production
indicating that the reduced sulfur atom is required for alginate
inhibition (Figure 4B).
Eb
EbO
EbS
1a
1b
1c
1d
1e
1f
80
14
11
10
14
11
10.5
18
32
7
109.1
413.5
79
1.36
29.5
7.18
2.98
1.31
2.39
9.03
3.08
3.98
4.67
5.18
29.8
18.4
26.3
94.8
55.5
127.3
32.7
41.4
1g
1h
8
a
The half-maximal inhibitory concentration (IC50) values were
calculated using data from Figures 2 and 4 for 50% reduction in
alginate production. Cytotoxicity concentration 50% (CC50) values
were calculated using data from Figure 6 for 50% cell death.
b
c
Selectivity index (SI) CC50/IC50.
and EbO and their analogues are able to inhibit alginate
production by P. aeruginosa.
Eb, EbO, and EbS Do Not Inhibit Growth of
P. aeruginosa. One possibility for the observed inhibition
of alginate production is that Eb, EbO, and related compounds
are inhibiting growth of P. aeruginosa. If this is the case, then
addition of these compounds to cultures of parental PA14
should alter the growth curve. When Eb, EbO, and EbS were
tested at 100 μM, the PA14 growth curve were mostly
unaffected (Figures 3A and S18). When the colony forming
unit (CFU) of these cultures were enumerated by serial
dilution and plating, the number of CFUs were all between 0.4
× 109 and 1.1 × 109 per optical density at 600 nm (OD600
)
(Figure 3B). These results indicate that Eb, EbO, and EbS are
not causing toxicity to P. aeruginosa. For the P. aeruginosa
PA14 pMMB-algU strain, induction of alginate production by
the addition of IPTG shifts the metabolism to alginate
production. As a consequence, the OD600 of the bacteria
cultures drops noticeably. When Eb, EbO, and EbS are added
at the same time as the IPTG inducer, the growth of the
bacteria is partially restored (Figure 3C). At the end of the
growth curve, the CFU per OD600 of bacteria treated with Eb,
EbO, and EbS is indeed higher than the DMSO control
(Figure 3D). Together, these results indicate that Eb, EbO,
Eb, EbS, and Their Analogues Promote Formation of
Disulfide/Selenylsulfide Bond with PilZ Domain of
Alg44. Eb and EbS perform their inhibitory activity through
the formation of Se−S or S−S adducts, respectively, on
C
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Figure 3. Effect of compounds on bacterial growth. The cell growth of P. aeruginosa (A) PA14 wild-type or (C) PA14 pMMB-algU in the presence
of indicated compounds at 100 μM was measured by OD at 600 nm. In a separate experiment, the colony forming unit (CFU) per OD600 was
determined by measuring OD600 and CFU by serial dilution for (B) PA14 wild-type or (D) PA14 pMMB-algU treated with 100 μM of indicated
compounds for 6 h. All experiments were performed in triplicate. Error bars indicate standard deviation from the mean.
Figure 4. Inhibition of alginate production by EbS and EbS analogues 1a−1h. Quantification of alginate secretion by P. aeruginosa PA14 pMMB-
algU treated with 100 μM of (A) EbS, EbS analogues 1a−1h, or DMSO; or (B) EbSO analogues 2a and 2b. (C) Alginate production in the
presence of varying concentration of EbS or EbS analogues 1a−1h. P. aeruginosa PA14 pMMB-algU was induced by the addition of 200 μM IPTG
for alginate production and normalized by the optical density in the unit μg/mL/OD600 of bacterial culture. The graph average and SD show six
independent experiments. *** indicates p value <0.0005, by one-way ANOVA.
cysteines of target proteins.35,40 Previous studies in P. aerugi-
nosa have shown that Eb inhibits WspR activity by modifying
the N-terminal cysteine.31 This cysteine modification by Eb
occupies the inhibitory site (I-site) on WspR31 that normally
allows feedback inhibition of diguanylate cyclase by the c-di-
GMP product.23 The alginate biosynthesis pathway encodes
alg44, a PilZ domain c-di-GMP binding protein, that is
required for alginate production. Furthermore, Alg44 protein
encodes one cysteine residue (C98) that has previously been
shown to be modified by thiol-benzo-triazolo-quinazolinone.30
We hypothesized that Eb, EbO, and/or EbS could form a
covalent bond between selenium or sulfur and the C98 of
Alg44 to prevent c-di-GMP binding, thereby reducing alginate
production. To test if the covalent modification of Alg44
occurred, the molecular weights of the PilZ domain of Alg44
(Alg44PilZ) treated with compounds or DMSO were
determined by mass spectrometry. Purified MBP-Alg44PilZ
protein treated with DMSO showed the expected molecular
weight of 58 384 Da (Figure 5A). When Alg44PilZ was
incubated with Eb, EbO, or EbS for 1 h, the molecular weight
was increased by 274, 273, and 228 Da, respectively, as
compared to DMSO control (Figure 5A). The increase in
molecular weights for Eb and EbS matched the expected mass
of these two compounds, indicating that the protein is
covalently modified by these compounds. However, when
treated with EbO the molecular weight of MBP-Alg44PilZ was
D
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Figure 5. Eb, EbO, and EbS can covalently modify Alg44PilZ on cysteine at position 98 to prevent binding to c-di-GMP, but inhibition of alginate
production is independent of this covalent modification. (A) Mass spectra of His-MBP-Alg44PilZ after incubation in the presence of Eb, EbS, EbO,
or DMSO. (B) Fraction bound of 32P-c-di-GMP to His-MBP-Alg44PilZ after treatment with DMSO, 100 μM Eb or EbS followed by additional
incubation without DTT (white bars) or with 1 mM DTT (colored bars). (C) Alginate production by P. aeruginosa PA14 alg44 (C98A and C98S)
pMMB-algU was induced with 0.2 mM IPTG treated with 100 μM Eb, EbS, or DMSO. (D) Fraction bound of 32P-c-di-GMP to His-MBP-
Alg44PilZ, Alg44PilZ (C98A) or Alg44PilZ (C98S) in the presence of 100 μM Eb, EbS, or DMSO (indicated as D). Graphs show average of alginate
produced from three independent experiments. **p < 0.005; ***p < 0.0005 by one-way ANOVA.
increased by the same amount as Eb (Figure 5A), suggesting
that EbO is first reduced to Eb prior to modification of the
protein.
(C98A) or (C98S) proteins lacking a sulfhydryl group were
tested for their binding to 32P-c-di-GMP in the presence of Eb
or EbS. Both Alg44PilZ (C98A) and (C98S) proteins were able
to bind 32P-c-di-GMP (Figure 5C). As expected, both proteins
lacking C98 were not inhibited by Eb and EbS (Figure 5C),
suggesting that C98 of Alg44 is the target of Eb and EbS.
Together these studies show that, in vitro, the Alg44 protein
can be modified at C98 by Eb, EbS, and their analogues, which
in turn inhibit the ability of Alg44 to bind c-di-GMP.
Eb and EbS Modification of Alg44 Is Not Required for
Alginate Inhibition. In order to test whether the only
mechanism of action by which Eb and EbS inhibit alginate
production is by modifying C98 of Alg44, the P. aeruginosa
PA14 strains with chromosome replacements of alg44 with
alg44 (C98A) or (C98S) were used in the alginate inhibition
assay.30 Induction of pMMB-algU with IPTG allowed for
increased alginate production (Figure 5D) indicating that C98
is not required for alginate production.30 When treated with
100 μM Eb or EbS, the PA14 strains with alg44 (C98A) and
alg44 (C98S) were also inhibited by Eb or EbS at a level
similar to wild-type P. aeruginosa (Figures 2, 4, and 5D). These
results indicate that Eb and EbS were able to inhibit alginate
Given that the molecular weight of MBP-Alg44PilZ increased
by molecular weight of the compounds, we postulated that the
modification of Alg44PilZ by Eb, EbO, or EbS inhibits c-di-
GMP that is required for alginate production. To investigate
this possibility, we tested the ability of compounds to inhibit c-
di-GMP binding to Alg44PilZ. Addition of 100 μM of Eb and
EbS reduced 32P-c-di-GMP binding to Alg44PilZ by 90 and
75%, respectively, compared to DMSO control (Figure 5B).
The covalent selenylsulfide (Se−S) and disulfide bonds
between the protein and Eb/EbS are reversed by a reducing
agent such as dithiothreitol (DTT) and should restore
Alg44PilZ binding to c-di-GMP. After incubation of Alg44PilZ
with compounds for 1 h, the addition of 1 mM DTT restored
the ability of Alg44PilZ to bind c-di-GMP by 8-fold for Eb and
1.7-fold for EbS (Figure 5B). To determine the residue in
Alg44PilZ that is modified by Eb or EbS, the one cysteine (C98)
in the PilZ domain of Alg44 was tested since previous studies
have shown that it is modified by thiol-benzo-triazolo-
quinazolinone through a disulfide bond.30 The Alg44PilZ
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Figure 6. Evaluation of cytotoxicity for (A) EbS and EbS analogues 1a−1h against A549 cell line; (B) EbS (same data as in panel A for ease of
comparison), oxidized version of EbS (EbSO) analogues 2a and 2b, Eb, and Eb analogues 3b, 3g, and 3h against A549 cell line; (C) EbS and EbS
analogues 1a−1h against J774A.1 cell line; and (D) EbS (same data as in panel B for ease of comparison), EbSO analogues 2a and 2b, Eb, and Eb
analogues 3b, 3g, and 3h against J774A.1 cell line. Controls include treatment with Triton-X (TX, 1% v/v, positive control) and 0.5% DMSO
(negative control). In instances where >100% cell survival was observed, we displayed the data as 100% cell survival (normalized data).
Experiments were performed in two independent samples. The average and standard deviation are shown. Note: The corresponding non-
normalized data are presented in Figure S19.
F
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secretion by P. aeruginosa through a cellular target that is
independent of C98 in Alg44.
Cytotoxicity of Eb and EbS Analogues on Mamma-
lian Cells. For Eb and analogues to be utilized as alginate
inhibitors in CF patients, the compounds have to be tested for
safety toward mammalian cells. All Eb and EbS analogues were
tested against A549 human adenocarcinoma cells and J774A.1
mouse macrophage cells to determine the cytotoxic effect in
the range of 0.13−32 μg/mL (Figures 6 and S19; Table S1).
Both lung and macrophage cell lines were chosen as
Pseudomonas causes chronic lung infections in CF patients
and as macrophages are the first responders to the infection, a
chemotherapeutic should not be toxic to these cells. Against
the A549 cells, we observed that EbS had an CC50 of ∼4 μg/
mL and EbS analogues 1a−1h also displayed similar CC50
values. Only one EbS analogue, 1f, was less cytotoxic with
more than 90% cell survival at 8 μg/mL. The EbSO analogues
exhibited greatly improved profiles with both 2a and 2b having
full cell survival at 32 μg/mL. Eb had an approximately 4-fold
better CC50 value than EbS, which matched reports of
cytotoxicity from HEK-293 cell studies.34,35 For the Eb
analogues, CC50 values only varied by 2-fold from Eb with
CC50 values of about 8, 16, and 32 μg/mL for 3b, 3g, and 3h,
respectively. Overall, EbSO analogues displayed the best CC50
values followed by the Eb analogues and then the EbS
analogues and this pattern was observed when comparing
analogues with the same R groups. The EbSO analogues, 2a
and 2b (CC50 >32 μg/mL), had at least an 8-fold
improvement in CC50 value compared to the EbS analogues,
1a and 1b (CC50 ∼4 μg/mL). In addition, the corresponding
Eb analogue, 3b (CC50 ∼8 μg/mL), exhibited 2-fold
improvement compared to 1b. Similarly, Eb analogues 3g
(CC50 ∼16 μg/mL) and 3h (CC50 >32 μg/mL) displayed 4-
fold and at least 8-fold improvement over the corresponding
EbS analogues, respectively. The trends observed with the
A549 cells remained consistent with the J774A.1 macrophages,
but with somewhat greater cell survival. EbS displayed 76% cell
survival at 16 μg/mL (∼4-fold greater CC50 than with A549).
EbS analogues 1d and 1f exhibited the least cytotoxicity of the
EbS analogues, 1a−1h, with greater than 80% cell survival at
16 μg/mL. All other Eb analogues displayed ≥60% cell survival
at 4 μg/mL and less than 40% survival at 16 μg/mL. The
EbSO analogues, 2a and 2b, again displayed total cell survival
at the highest concentration, 32 μg/mL. Full cell survival was
also observed for Eb at 16 μg/mL and Eb derivatives 3g and
3h at 32 μg/mL.
Figure 7. Evaluation of cytotoxicity for EbO against HEK-293, A549,
and J774A.1 cell lines. The positive control used was treatment with
Triton-X (TX, 1% v/v). In instances where >100% cell survival was
observed, we displayed the data as 100% cell survival (normalized
data). Experiments were performed in four independent samples. The
average and standard deviation are shown. Note: The corresponding
non-normalized data are presented in Figure S20.
synthesis of alginate. The results from this current study
indicate that Eb, EbO, EbS, and their analogues are effective in
reducing the production of alginate by P. aeruginosa. On the
basis of the compounds tested, the Se/S atom in the molecule
is the active moiety. In the case of EbS, the sulfur atom must
be in the reduced state since EbSO are inactive. In contrast,
EbO appears to be active, but is reduced to Eb prior to activity.
Whether EbO can serve as a pro-drug requires further studies.
The Eb, EbO, and EbS scaffolds appear to tolerate a wide
variety of modifications and may allow future development of
compounds with greater potency or chemical probes. The
results presented in this limited SAR indicate that the phenyl
ring enhances activity in comparison to the isobutyl group.
Eb is a compound that has undergone phase III clinical trials
for ischemic stroke and acute hearing loss as well as a phase II
clinical trial for bipolar/manic disorder. Eb was proven to be
safe through oral administration during clinical trials,
suggesting it is well tolerated. We calculated the selectivity
index (SI) values of these compounds for alginate inhibition
(Table 1). The SI for a compound is defined as the ratio of its
cytotoxicity concentration 50% (CC50) value against half-
maximal inhibitory concentration (IC50) value. It is important
to note that differences in inhibitors binding to proteins
present in the different assays were not accounted for in the
selectivity discussion to follow. The analysis shown here
suggests that Eb, while able to inhibit alginate production, has
a narrow therapeutic window (SI value of 1.36). EbS has a
similar toxicity profile to mammalian cells, but has improved
inhibitory activity toward alginate production and has an
improved therapeutic index with SI values ranging from 1.31 to
9.03. EbO, which is typically considered inactive, is indeed less
toxic to A549, HEK-293, and J774A.1 cells, but yet has similar
inhibitory activity as EbS. Thus, EbO represents a lead
compound for further development for compounds with
enhanced therapeutic index with an SI value of 29.5.
Finally, we tested EbO in a concentration range of 1 to 256
μg/mL against HEK-293, A549, and J774A.1 cell lines to
establish its toxicity (Figures 7 and S20; Table S1). EbO
displayed 100% cell survival at 64 μg/mL against the J774A.1
cell line. At the same concentration (64 μg/mL) EbO
exhibited 60% and >30% cell survival against the HEK-293
and A549 cell lines. Compared to other Eb derivatives, EbO
displayed even better safety profile against mammalian cell
lines.
DISCUSSION
■
P. aeruginosa is a leading cause of infection of CF patients.
Mucoid conversion of P. aeruginosa through mutational
inactivation of the mucA gene is correlated with increased
production of alginate and the concomitant decline in lung
function, leading to morbidity and mortality. One possible
approach to dealing with the mutant bacteria is to inhibit the
The alginate assay is based on induction of a plasmid
encoded algU/T. The observed inhibition of alginate
production by Eb, EbS, and their analogues likely occurs
downstream of transcription of the algD-algA alginate biosyn-
thesis operon. Targets upstream of algD-algA transcription are
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Figure 8. Model for inhibition of P. aeruginosa alginate production by EbO and analogues. Schematic diagram for the alginate biosynthetic proteins
encoded in the algD-algA operon and the algC gene. The protein structures are placed in the cellular location (outer membrane, periplasm, and
inner membrane) based on previous literature including AlgD (PDB ID: 1MUU),41 Alg44 (PDB ID: 4RT0),21 AlgK (PDB ID: 3E4B),42 AlgE
(PDB ID: 3RBH),43 AlgG (PDB ID: 4NK6),44 AlgX (PDB ID: 4KNC),45 AlgL (PDB ID: 4OZV; unpublished), and AlgJ (PDB ID: 4O8V).46
Structures for Alg8, AlgI, AlgF, and AlgA are not yet available. The number of conserved cysteine residues is indicated in parentheses.
unlikely since these compounds did not cause a general growth
defect in P. aeruginosa. Furthermore, the alginate assay
corrected for cell number through OD600 measurements. The
obvious target for inhibition is Alg44 since c-di-GMP is
required for alginate production.20,31 When tested in vitro, Eb,
EbO, and EbS can interfere with c-di-GMP binding to Alg44
supporting the idea that Alg44 is the target (Figure 5A−C).
However, P. aeruginosa with alg44 (C98A) or alg44 (C98S) are
still inhibited by Eb and EbS despite not having a cysteine
required for cross-linking (Figure 5D), suggesting that
inhibition by Eb and EbS must target another aspect of
alginate production. Analyzing the genes in the algD-algA
operon and the algC gene, that is required for generating the
mannuronic acid precursor, only a subset of genes have
cysteines including AlgD, Alg8, Alg44, AlgK, AlgX, AlgL, AlgI,
and AlgG (Figure 8). In this manuscript, we have
demonstrated that Alg44 is not the sole target for EbO and
analogues. In contrast, AlgE, AlgG, AlgJ, and AlgA do not have
cysteine residues and are likely not the target of inhibition.
Future studies will reveal the target of inhibition by EbO (Eb
and EbS), perhaps using EbO probes with modifications that
target proteins through covalent bonds. In addition, studies
using clinical isolates of P. aeruginosa from CF patients will
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enable determination whether the observed inhibition can act
to broadly inhibit alginate production.
have a greater selectivity index for inhibiting alginate
production by P. aeruginosa. From this study, it could be
concluded that EbO represents an interesting avenue for the
future development of chemical inhibitors for alginate
secretion by P. aeruginosa, which could in turn reduce the
adverse side effects of alginate production on CF patients.
If these efforts are successful in developing alginate
inhibitors, how does this fit into current treatment for CF
patients? The most direct approach is to restore the function of
CFTR through correcting the mutation in the gene. While
gene therapy has yet to be effective for CF patients,47
a
METHODS
different approach has been taken to restore CFTR protein
function.48 Many classes of the CFTR mutations produce the
CFTR protein; however, the protein fails to reach the plasma
membrane or has a defect in channel function.49 In the past
decades, CFTR correctors were developed through screening
for small molecules that restored activity of G551D-CFTR
alleles in cell-based assays.50,51 In 2012, Vertex Pharmaceut-
icals received FDA approval for ivacaftor, which was shown to
improve lung function in patients with G551D allele.52−54
However, the majority of the CF patients (>90%) have the
Δ508 allele that does not respond to ivacaftor.55 Subsequent
screening identified compounds that corrected the Δ508-
CFTR56,57 and when used in combination with modulators are
highly effective for improving lung function and reducing
microbial load in CF patients with Δ508-CFTR.58,59 Orkambi,
a combination of ivacaftor/lumacaftor, and trikafta, comprised
of elexacaftor/tezacaftor/ivacaftor, were approved by the FDA
in 2015 and 2019, respectively. For those CF alleles that do
not respond to the treatment or for patients that have serious
complications as a result of the above treatment, infection by
P. aeruginosa and mucoid conversion still represent a serious
condition. Reduction of alginate produced by P. aeruginosa,
while not curative, would reduce the impact of mucoid
conversion. The results described here represent one approach
to reduce the alginate in the airway and prevent the negative
impact of mucoid P. aeruginosa on lung function. Future
development of EbO analogues will lead to the development of
compounds with improved therapeutic index.
■
Materials and Instrumentation. All chemicals were
purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar
(Ward Hill, MA), and AK scientific (Union City, CA), and
used without further purification. Chemical reactions were
monitored by thin layer chromatography (TLC) using Merck
Silica gel 60 F254 plates and visualization was achieved using
1
UV light. H and 13C NMR spectra were recorded at 400 and
100 MHz (or 125 MHz), respectively, on a Varian 400 MHz
spectrometer (MR400) (or a Varian 500 MHz spectrometer;
VNMRS 500), using the indicated deuterated solvents.
Chemical shifts (δ) are given in parts per million (ppm).
Coupling constants (J) are given in Hertz (Hz), and
conventional abbreviations used for signal shape are as follows:
s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet;
tt = triplet of triplets.
Synthesis and Characterization of Compounds in
This Study. We previously synthesized and characterized EbS
as well as compounds 1a−h and 2a,b.34,35 The previously
reported synthetic scheme36 was used to prepare Eb and its
analogues 3b, 3g, and 3h (Scheme S1). All the compounds
synthesized are presented in Figure 1.
Preparation of Compound 5. To a solution of aniline
(0.40 mL, 4.50 mmol) and Et3N (0.90 mL, 9 mmol) in
anhydrous CH2Cl2(20 mL), 2-iodobenzoyl chloride (0.5 mL,
3.75 mmol) was added dropwise. The product was washed
with 1 N HCl (50 mL) and a saturated solution of sodium
bicarbonate (50 mL) and dried with MgSO4. The organic layer
was removed in vacuo and the crude product was purified via
recrystallization in i-PrOH to afford the known compound 536
CONCLUSIONS
■
1
In summary, we have identified novel EbO, Eb, and EbS
analogues that can inhibit alginate production by P. aeruginosa.
From the limited SAR performed we could emphasize that
specific positions of Eb and EbS can be modified without
affecting inhibitory activity toward alginate. Both Eb and EbS
analogues were able to covalently modify PilZ domain of Alg44
using cysteine 98 (C98) and this modification prevented c-di-
GMP from binding to Alg44. The Eb and its oxidized version
(EbO) were shown to inhibit alginate production by
P. aeruginosa. EbO exhibited better potency at inhibiting
Alg44 with lower micromolar value when compared to Eb. The
Eb analogues 3g and 3h performed better than Eb in alginate
inhibition assays, indicating the possibilities for more vigorous
scaffold expansion. In the case of EbS, the parent compound
and its analogues displayed better inhibition of alginate
production compared to Eb. However, contrary to the result
observed for EbO, the oxidized version of EbS (EbSO) failed
to inhibit alginate production. The results from mass
spectrometry studies revealed that Eb, EbS, and their analogues
form selenylsulfide and disulfide bonds with the PilZ domain
of Alg44. Even though an obvious target for inhibition is Alg44,
the inhibition of alginate production in P. aeruginosa PA14
strains with alg44 (C98A) or alg44 (C98S) indicate another
cellular target that is independent of C98 of Alg44. Finally,
when tested against A549 and J774A.1 cell lines for their
toxicity, EbO analogues displayed better safety profiles and
(299 mg, 25%) as a pink solid: H NMR (400 MHz, CDCl3,
Figure S1) δ 7.89 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.0 Hz, 2H),
7.51 (d, J = 6.8 Hz, 1H), 7.48−7.30 (m, 4H), 7.20−7.10 (m,
2H); 13C NMR (100 MHz, CDCl3, Figure S2) δ 167.4, 142.3,
140.2, 137.7, 131.6, 129.3, 128.7, 128.5, 125.1, 120.3, 92.6.
Preparation of Compound 6. To a solution of isoamyl-
amine (1.0 mL, 9.0 mmol) and Et3N (2.0 mL, 15.0 mmol) in
anhydrous CH2Cl2(20 mL), 2-iodobenzoyl chloride (1.0 mL,
3.75 mmol) was added dropwise. The product was washed
with 1 N HCl (50 mL) and a saturated solution of sodium
bicarbonate (50 mL) and dried with MgSO4. The organic layer
was removed in vacuo to afford the known compound 660 (2.4
1
g, quantitative yield) as a yellow solid: H NMR (400 MHz,
CDCl3, Figure S3) δ 7.81 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 3.6
Hz, 2H), 7.10−7.00 (m, 1H), 5.78 (s, 1H), 3.43 (q, J = 7.6 Hz,
2H), 1.69 (nonet, J = 6.4 Hz, 1H), 1.49 (q, J = 7.6 Hz, 2H),
0.93 (d, J = 6.4 Hz, 6H); 13C NMR (100 MHz, CDCl3, Figure
S4) δ 169.5, 142.7, 139.9, 131.1, 128.4, 128.3, 92.6, 38.6, 38.4,
26.1, 22.6.
Preparation of Compound 7. To a solution of benzyl-
amine hydrochloride (0.65 g, 4.50 mmol) and Et3N (0.90 mL,
9 mmol) in anhydrous CH2Cl2(20 mL), 2-iodobenzoyl
chloride (0.5 mL, 3.75 mmol) was added dropwise. The
product was washed with 1 N HCl (50 mL) and a saturated
solution of sodium bicarbonate (50 mL) and dried with
MgSO4. The organic layer was removed in vacuo and the crude
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product was purified via recrystallization in i-PrOH to afford
the known compound 736 (639 mg, 51%) as a white solid: 1H
NMR (400 MHz, CD3OD, Figure S5) δ 7.88 (d, J = 8.0 Hz,
1H), 7.46−7.40 (m, 3H), 7.30−7.37 (m, 3H), 7.24−7.28 (m,
1H), 7.14 (app. tt, J = 7.6, 2.0 Hz, 1H), 4.53 (s, 2H); 13C
NMR (100 MHz, CDCl3, Figure S6) δ 169.4, 142.2, 140.1,
137.7, 131.4, 129.0, 128.5, 128.4 (2 carbons), 127.9, 92.6, 44.4.
Preparation of Compound 8. To a solution of
phenethylamine (1.1 mL, 9.0 mmol) and Et3N (2.0 mL, 15.0
mmol) in anhydrous CH2Cl2(20 mL), 2-iodobenzoyl chloride
(1.0 mL, 3.75 mmol) was added dropwise. The product was
washed with 1 N HCl (50 mL) and a saturated solution of
sodium bicarbonate (50 mL) and dried with MgSO4. The
organic layer was removed in vacuo to afford the known
Compound 7 (0.34 g, 1 mmol), copper(I) iodide (0.19 g, 1
mmol), 1,10-phenanthroline (0.18 g, 1 mmol), cesium
carbonate (0.70 g, 2 mmol), and potassium selenocyanate
(0.16 g, 1.2 mmol) were suspended in DMF (5 mL). The
mixture turned red and was heated to 100 °C for 12 h. The
reaction mixture was cooled to room temperature, diluted with
EtOAc (40 mL), and filtered through Celite. The filtrate was
washed with cold H2O (2 × 20 mL) and brine (2 × 20 mL),
and dried with MgSO4. The organic layer was removed in
vacuo and the crude product was purified via recrystallization
from i-PrOH and flash column chromatography (SiO2, 49:1/
CH2Cl2:MeOH then 19:1/CH2Cl2:MeOH) to afford the
known compound 3g36 (38 mg, 13%) as white solid: H
1
NMR (400 MHz, CDCl3, Figure S13) δ 8.04 (d, J = 7.6 Hz,
1H), 7.91 (d, J = 6.4 Hz, 1H), 7.76−7.64 (m, 2H), 7.39 (d, J =
7.2 Hz, 2H), 7.36−7.24 (m, 3H), 5.36 (d, J = 14.8 Hz, 1H),
4.60 (d, J = 14.8 Hz, 1H); 13C NMR (100 MHz, CDCl3, which
matches the literature,36 Figure S14) δ 168.2, 145.1, 136.7,
134.4, 133.3, 130.8, 129.3, 129.0, 128.7, 128.6, 126.6, 45.4.
Preparation of Compound 3h. Compound 8 (0.35 g, 1
mmol), copper(I) iodide (0.19 g, 1 mmol), 1,10-phenanthro-
line (0.18 g, 1 mmol), cesium carbonate (0.70 g, 2 mmol), and
potassium selenocyanate (0.16 g, 1.2 mmol) were suspended in
DMF (5 mL). The mixture turned red and was heated to 100
°C for 3 h. The reaction mixture was cooled to room
temperature, diluted with EtOAc (40 mL), and filtered
through Celite. The filtrate was washed with cold H2O (2 ×
20 mL) and brine (2 × 20 mL), and dried with MgSO4. The
organic layer was removed in vacuo and the crude product was
purified via recrystallization from i-PrOH and twice via flash
column chromatography (SiO2, 49:1/CH2Cl2:MeOH then
19:1/CH2Cl2:MeOH) to afford the known compound 3h62
compound 861 (2.6 g, quantitative yield) as a yellow solid: H
1
NMR (400 MHz, CD3OD, Figure S7) δ 7.86 (d, J = 8.0 Hz,
1H), 7.38 (app. tt, J = 8.0, 1.2 Hz, 1H), 7.32−7.24 (m, 4H),
7.24−7.16 (m, 2H), 7.20−6.80 (m, 1H), 3.56 (t, J = 8.0 Hz,
2H), 2.92 (t, J = 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3,
Figure S8) δ 169.6, 142.4, 140.0, 138.8, 131.2, 129.0, 128.9,
128.31, 128.30, 126.8, 92.6, 41.3, 35.6.
Preparation of Compound Eb. The known compound
Eb was prepared using a previously published protocol.36
Compound 5 (0.299 g, 0.93 mmol), copper(I) iodide (0.18 g,
0.93 mmol), 1,10-phenanthroline (0.17 g, 0.93 mmol), cesium
carbonate (0.81 g, 2.5 mmol), and potassium selenocyanate
(0.16 g, 1.2 mmol) were suspended in DMF (5 mL). The
mixture turned red and was heated to 100 °C for 12 h. The
reaction mixture was cooled to room temperature, diluted with
EtOAc (40 mL), and filtered through Celite. The filtrate was
washed with cold H2O (2 × 20 mL) and brine (2 × 20 mL),
and dried with MgSO4. The organic layer was removed in
vacuo and the crude product was purified via recrystallization
from EtOH and flash column chromatography (SiO2, 49:1/
CH2Cl2:MeOH) to afford the known compound Eb36 (14 mg,
1
(12 mg, 4%) as white solid: H NMR (400 MHz, CDCl3,
which matches the literature,62 Figure S15) δ 8.03 (d, J = 8.0
Hz, 1H), 7.56 (d, J = 3.6 Hz, 2H), 7.42−7.36 (m, 1H), 7.32−
7.20 (m, 6H), 4.09 (t, J = 6.8 Hz, 2H), 3.02 (t, J = 7.2 Hz,
2H); 13C NMR (100 MHz, CDCl3, Figure S16) δ 167.2, 148.3,
138.0, 137.9, 131.9, 129.02, 128.97, 128.8, 128.6, 127.2, 126.7,
126.1, 123.9, 46.2, 36.4.
1
6%) as colorless needles: H NMR (400 MHz, CDCl3, Figure
S9) δ 8.10 (d, J = 7.6 Hz, 1H), 7.68−7.56 (m, 4H), 7.48−7.43
(m, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.26 (t, J = 7.6 Hz, 1H); 13C
NMR (125 MHz, CDCl3, Figure S10) δ 169.6, 139.3, 137.8,
132.7, 129.6, 129.5, 127.0, 126.8, 125.6, 123.9, 121.4.
Bacterial Strains, Plasmids, Cells, and Growth
Conditions. For the alginate production assay, P. aeruginosa
strains PA14, PA14 algU (C98A), or algU (C98S) harboring
pMMB-algU or pMMB were grown in Luria−Bertani (LB)
broth at 37 °C supplemented with 50 μg/mL carbenicillin and
0.2 mM IPTG. For protein purification, E. coli strains
harboring pVL847 were grown in LB M9 medium (5 g/L
yeast extract, 10 g/L tryptophan, 2 g/L glucose, 1 g/L sodium
succinate hexahydrate, 1 g/L NH4SO4, 0.5 g/L NaCl, 2 g/L
KH2PO4, 7 g/L anhydrous Na2HPO4, 3 mM MgSO4) at 30 °C
supplemented with 15 μg/mL gentamicin and 1 mM IPTG.
Growth was determined by measuring optical density at 600
nm (OD600) using a SpectraMax M5 plate reader. For the
cytotoxicity assays, the human lung carcinoma cell line A549
and the mouse macrophage cell line J774A.1 that were utilized
were a kind gift from Prof. David K. Orren (University of
Kentucky, Lexington, KY) and Prof. David J. Feola (University
of Kentucky, Lexington, KY), respectively. Cells were cultured
in Dulbecco’s Modified Eagle’s Medium (DMEM; catalog #
VWRL0100, VWR, Chicago, IL) supplemented with 10% fetal
bovine serum (FBS; from ATCC) and 1% penicillin/
streptomycin (from ATCC) at 37 °C with 5% CO2.
Preparation of Compound 3b. Compound 6 (0.32 g, 1
mmol), copper(I) iodide (0.19 g, 1 mmol), 1,10-phenanthro-
line (0.18 g, 1 mmol), cesium carbonate (0.70 g, 2 mmol), and
potassium selenocyanate (0.16 g, 1.2 mmol) were suspended in
DMF (5 mL). The mixture turned red and was heated to 100
°C for 3 h. The reaction mixture was cooled to room
temperature, diluted with EtOAc (40 mL), and filtered
through Celite. The filtrate was washed with cold H2O (2 ×
20 mL) and brine (2 × 20 mL), and dried with MgSO4. The
organic layer was removed in vacuo and the crude product was
purified via recrystallization from EtOAc and twice via flash
column chromatography (SiO2, 49:1/CH2Cl2:MeOH then
19:1/CH2Cl2:MeOH) to afford the known compound 3b60
1
(26 mg, 10%) as white solid: H NMR (400 MHz, CDCl3,
which matches the literature,60 Figure S11) δ 8.01 (d, J = 7.6
Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H),
7.39 (t, J = 7.6 Hz, 1H), 3.86 (t, J = 6.8 Hz, 2H), 1.67 (nonet, J
= 6.4 Hz, 1H), 1.59 (q, J = 6.4 Hz, 2H), 0.95 (d, J = 6.8 Hz,
6H); 13C NMR (100 MHz, CDCl3, which matches the
literature,60 Figure S12) δ 167.3, 137.8, 132.0, 129.0, 127.9,
126.4, 124.1, 43.4, 39.6, 25.9, 22.7.
Preparation of Compound 3g. The known compound
Quantification of Alginate Secretion by P. aeruginosa.
P. aeruginosa PA14 harboring pMMB-algU were grown
3g was prepared using a previously published protocol.36
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overnight in LB broth, subcultured with 50 μg/mL
carbenicillin at 37 °C and 0.2 mM IPTG for 6 h. Induced
bacteria were collected by centrifuge; the supernatant
containing alginate was precipitated by the addition of equal
volume of isopropanol at −20 °C. After precipitation by
centrifuge for 20 min, the alginate pellet is resuspended in
distilled water and assay by the addition of 0.1% carbazole in
absolute ethanol and borate-sulfuric acid reagent.63 The
reaction was incubated at 55 °C for 30 min. These samples
were measured at 530 nm using the SpectraMax M5 plate
reader. This is a spectrometric assay in which the uronic acid
sugars were modified by carbazole to produce a compound
that absorbs at 530 nm. The values were calculated and
converted to unit of alginate production using commercially
available alginate to generate a standard curve. Alginate
concentration was normalized by alginate/mL/OD600 of
bacterial culture (Figures 2 and 4). Addition of compounds
to extracts of alginate did not interfere with the assay (Figure
S17).
Effect of Compounds on Bacterial Growth. P. aeruginosa
PA14 was subcultured 1:50 dilution in LB broth containing 0,
25, 50, and 100 μM concentration of compounds and
monitored by measuring OD600 values for 15 h at 30 or 37
°C, as indicated (Figures 3 and S18). In addition, the colony
forming units (CFU), which reflects the number of viable
bacteria, was enumerated by counting colonies formed after
serial dilution and plating on LB agar plates. The experiments
were performed in triplicate.
and imaged using Fujifilm phosphorimager.64 The intensity of
radiolabeled signal was quantified by Fujifilm Multi Gauge
software v3.0. These data are presented in Figure 5.
Determination of Molecular Weight of Alg44PilZ by
Liquid Chromatography Mass Spectrometry (LC-MS).
Ten μM Alg44PilZ were incubated at room temperature in the
presence of 100 μM Eb, EbO, or EbS for 1 h. After incubation,
protein samples were acidified with 1 μL 10% TFA and
analyzed by LC-MS. Protein was eluted with a linear gradient
of 15−90% solvent B in 20 min, followed by 5 min wash at
90% B and equilibration at 15% B for 10 min. Solvent A is
2.5% MeCN in water with 0.1% formic acid, and solvent B is
75% MeCN in water with 0.1% formic acid. Spectra of m/z
500−1700 were acquired with an Orbitrap Fusion Lumos mass
spectrometer with resolution of 15 000 at m/z 200. Source
fragmentation of 30% was applied to disrupt potential clusters.
Five spectra (microscans) were averaged for each spectrum
recorded. After acquisition, mass spectra over the chromatog-
raphy peak were averaged. Averaged spectrum was deconvo-
luted using MagTran program65 (a universal algorithm for fast
and automated charge state deconvolution of electrospray
mass-to-charge (m/z) ratio spectra). These data are presented
in Figure 5.
Mammalian Cytotoxicity Assays for Eb, EbS, and
Their Analogues. Ebselen (Eb), ebsulfur (EbS), and all
analogues were tested against the A549 and J774A.1 cell lines
to evaluate mammalian cytotoxicity. A resazurin cell viability
assay was used as previously described with minor
modifications.66 A549 and J774A.1 cells were counted using
a hemacytometer and plated into 96-well plates at a
concentration of 4 × 103 cells/mL and 1 × 104 cells/mL,
respectively. After 24 h incubation, cells were treated with
compound. The compounds were tested in concentrations
ranging from 0.13 to 32 μg/mL with final concentration of
DMSO at 0.5%. 1% Triton-X was used as a positive control.
After 24 h treatment, 10 μL of 2 mM resazurin was added to
the plates and incubated at 37 °C in the dark for 5 h. Plates
were read with an excitation of 360 nm and emission of 390
nm and normalized to no drug (DMSO only) control (Figure
6). It is important to note that testing xenobiotics at sub-IC50
concentrations can result in an increase in cell growth,
resulting in >100% cell survival in the treatment groups.67 In
instances where >100% cell survival was observed, we
displayed the data as 100% cell survival in Figure 6. We are
providing the data with the non-normalized observed % in
Figure S19. All compounds were tested in duplicate. The
corresponding values of the concentrations of Eb and EbS
analogues 1a−1h, 2a, 2b, 3b, 3g, 3h, Eb, and EbS in
micromolar used for cytotoxicity experiments are reported in
Table S1. In addition, EbO was tested against the A549, HEK-
293, and J774A.1 cell lines in a concentration range of 1 to 256
μg/mL. Experiments were done in quadruplicate and the 100%
normalized data are reported in Figure 7. We are providing the
data with the non-normalized observed % in Figure S20.
Purification of His-MBP-Alg44PilZ (C98A) and (C98S).
E. coli T7Iq strains harboring pVL847 His-MBP-Alg44PilZ
(C98A) and (C98S) were grown overnight, subcultured in
LB M9 medium supplemented with 15 μg/mL gentamicin for
4 h at 30 °C, then the addition of 1 mM IPTG for 4 h. Induced
cells were collected by centrifuge and resuspended in 10 mM
Tris, pH 8, 100 mM NaCl, and 25 mM imidazole. Bacteria
were lysed by the addition of 10 μg/mL DNase, 25 μg/mL
lysozyme, and 1 mM PMSF. Insoluble material was removed
by centrifuge. His-MBP-Alg44PilZ (C98A) and (C98S) in
lysates were purified by a NiII−NTA column using an AKTA
fast protein liquid chromatography (FPLC) in 10 mM Tris, pH
8, and 100 mM NaCl using an imidazole gradient from 25 mM
to 250 mM. The purification of protein was dialyzed for 1 h
and overnight against 10 mM Tris, pH 8, 100 mM NaCl. After
then, protein was dialyzed for 4 h against 10 mM Tris, pH 8,
100 mM NaCl, and 50% glycerol. The purification of protein
was aliquoted, frozen with liquid nitrogen, and stored at −80
°C.
Synthesis of 32P-c-di-GMP. Radiolabeled GTP (α-32P-
GTP) was mixed with WspR D70E encoding diguanylate
cyclase in c-di-GMP binding buffer (10 mM Tris, pH 8, 100
mM NaCl, and 5 mM MgSO4) and incubated for 2 h at 37 °C.
After incubation, the mixture was performed for WspR
separation from 32P-c-di-GMP using 3 kDa molecular weight
cutoff filter.
DRaCALA. To confirm binding of purified His-MBP-
Alg44PilZ, Alg44PilZ (C98A) and (C98S) to 32P-c-di-GMP, 10
μM purified protein was incubated with Eb, EbS (100 μM), or
DMSO for 1 h. The mixture was incubated again in the
presence of 1 mM DTT for 1 h. The total 10 μL of each
sample was incubated with 10 μL of 0.8 nM 32P-c-di-GMP in
c-di-GMP binding buffer (10 mM Tris, pH 8, 100 mM NaCl,
and 5 mM MgSO4). The mixture was shaken for 1 min at
room temperature, spotted on a nitrocellulose sheets, dried,
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00045.
A synthetic scheme for the synthesis of the compounds
presented (Scheme S1); a table of the data used to
prepare Figures 6 and 7 (Table S1); figures presenting
K
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1H and 13C spectra for molecules synthesized (Figures
S1−16); a figure demonstrating that addition of
compounds does not interfere with detection by the
alginate assay (Figure S17); a figure showing that
compounds have no effect on the growth of P. aeruginosa
PA14 when incubated in LB containing varying
concentrations of compounds for 15 h at 30 °C (Figure
S18); as well as non-normalized cytotoxicity bar graphs
(Figure S19 and S20) (PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
Sylvie Garneau-Tsodikova − Department of Pharmaceutical
Sciences, College of Pharmacy, University of Kentucky,
Lexington, Kentucky 40536, United States; orcid.org/
0000-0002-7961-5555; Email: sylviegtsodikova@uky.edu
Vincent T. Lee − Department of Cell Biology and Molecular
Genetics, University of Maryland at College Park, College
Park, Maryland 20742, United States; orcid.org/0000-
0002-3593-0318; Email: vtlee@umd.edu
Authors
Soo-Kyoung Kim − Department of Cell Biology and
Molecular Genetics, University of Maryland at College Park,
College Park, Maryland 20742, United States
Huy X. Ngo − Department of Pharmaceutical Sciences, College
of Pharmacy, University of Kentucky, Lexington, Kentucky
40536, United States
Emily K. Dennis − Department of Pharmaceutical Sciences,
College of Pharmacy, University of Kentucky, Lexington,
Kentucky 40536, United States
Nishad Thamban Chandrika − Department of
Pharmaceutical Sciences, College of Pharmacy, University of
Kentucky, Lexington, Kentucky 40536, United States;
orcid.org/0000-0002-3605-169X
Philip DeShong − Department of Chemistry and
Biochemistry, University of Maryland at College Park, College
Park, Maryland 20742, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.1c00045
(14) Bianconi, I., D’Arcangelo, S., Esposito, A., Benedet, M., Piffer,
E., Dinnella, G., Gualdi, P., Schinella, M., Baldo, E., Donati, C., and
Jousson, O. (2019) Persistence and microevolution of Pseudomonas
aeruginosa in the cystic fibrosis lung: A single-patient longitudinal
genomic study. Front. Microbiol. 9, 3242.
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Holloway, B. W., and Deretic, V. (1993) Mechanism of conversion to
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mucoid Pseudomonas aeruginosa which control spontaneous con-
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Characterization of a locus determining the mucoid status of
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by startup funds from the College of
Pharmacy at the University of Kentucky (S.G.-T.). S.-K.K.
acknowledges support from the National Research Foundation
of Korea (2018R1A6A3A03011278). V.T.L. acknowledges
support from the Cystic Fibrosis Foundation (Lee16G0) and
National Institutes of Health (R01-AI110740).
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