Mechanistic Duality of Bacterial Efflux Substrates and Inhibitors: Example of Simple Substituted Cinnamoyl and Naphthyl Amides

Article abstract of DOI:10.1021/acsinfecdis.1c00100

Antibiotic resistance poses an immediate and growing threat to human health. Multidrug efflux pumps are promising targets for overcoming antibiotic resistance with small-molecule therapeutics. Previously, we identified a diaminoquinoline acrylamide, NSC-3

Full text of DOI:10.1021/acsinfecdis.1c00100

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Article
Mechanistic Duality of Bacterial Efflux Substrates and Inhibitors:
Example of Simple Substituted Cinnamoyl and Naphthyl Amides
Napoleon D’Cunha,∇ Mohammad Moniruzzaman,∇ Keith Haynes, Giuliano Malloci, Connor J. Cooper,
Enrico Margiotta, Attilio V. Vargiu, Muhammad R. Uddin, Inga V. Leus, Feng Cao, Jerry M. Parks,
Valentin V. Rybenkov, Paolo Ruggerone, Helen I. Zgurskaya,* and John K. Walker*
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ABSTRACT: Antibiotic resistance poses an immediate and growing threat to
human health. Multidrug efflux pumps are promising targets for overcoming
antibiotic resistance with small-molecule therapeutics. Previously, we identified a
diaminoquinoline acrylamide, NSC-33353, as a potent inhibitor of the AcrAB−
TolC efflux pump in Escherichia coli. This inhibitor potentiates the antibacterial
activities of novobiocin and erythromycin upon binding to the membrane fusion
protein AcrA. It is also a substrate for efflux and lacks appreciable intrinsic
antibacterial activity of its own in wild-type cells. Here, we have modified the
substituents of the cinnamoyl group of NSC-33353, giving rise to analogs that
retain the ability to inhibit efflux, lost the features of the efflux substrates, and
gained antibacterial activity in wild-type cells. The replacement of the cinnamoyl
group with naphthyl isosteres generated compounds that lack antibacterial activity
but are both excellent efflux pump inhibitors and substrates. Surprisingly, these
inhibitors potentiate the antibacterial activity of novobiocin but not erythromycin.
Surface plasmon resonance experiments and molecular docking suggest that the replacement of the cinnamoyl group with naphthyl
shifts the affinity of the compounds away from AcrA to the AcrB transporter, making them better efflux substrates and changing their
mechanism of inhibition. These results provide new insights into the duality of efflux substrate/inhibitor features in chemical
scaffolds that will facilitate the development of new efflux pump inhibitors.
KEYWORDS: efflux pump inhibitors, AcrAB−TolC, Escherichia coli, antibiotic permeation, antibiotic potentiation
osocomial infections caused by Gram-negative (GN)
Npathogens continue to rise, constituting a serious threat to
public health and safety as well as having a tremendous
economic impact.1−3 The ability of many of these pathogens to
develop additional drug resistance against frontline antibiotics
further increases the threat.3,4 In 2013, the Centers for Disease
Control (CDC) reported approximately 51,000 infections from
Pseudomonas aeruginosa with roughly 6,000 of those attributed
to drug-resistant strains.5 By 2019, the CDC reported that the
number of drug-resistant infections had increased to an
estimated 32,600, resulting in approximately 2,700 deaths.2 As
a result, there is an urgent need to not only discover new
antibiotics but also develop new strategies to combat drug-
resistant GN bacteria.6,7
The search for new antibiotics against GN bacteria has proven
extremely challenging.8,9 The difficulty in finding new lead
matter for GN bacteria is borne out by the poor hit rates typically
seen in high-throughput screening campaigns performed by Big
Pharma,10,11 which can be traced to the rarity of chemical
scaffolds that accumulate in GN bacteria. This is a consequence
of compounds having poor permeation across the outer
membrane (OM)12 or being extruded from the cell by efflux
pumps after crossing the OM or a combination of both.13 The
OM permeation barrier and active efflux often work together
synergistically, severely limiting the chemical space able to target
GN pathogens.14−16
The OM of GN bacteria is made up of an asymmetric bilayer
comprising a highly charged lipopolysaccharide (LPS)-rich
outer leaflet and a phospholipid-based inner leaflet.17 As a result
of the distinctly different chemical makeup of the outer and
inner leaflets, it is very challenging to identify molecules with
chemical properties suitable for passive permeation through
both leaflets.18,19 Some compounds can cross the OM by
diffusing slowly through porins, which are trimeric β-barrel
proteins that control the uptake of small molecules.20 However,
compounds able to use porins to cross the OM tend to be highly
Received: February 24, 2021
https://doi.org/10.1021/acsinfecdis.1c00100
© XXXX American Chemical Society
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Figure 1. Previously identified compounds that bind to the membrane fusion protein AcrA of E. coli and inhibit efflux.38,39
i
Table 1. Antibacterial and Potentiation Activity for Substituted Cinnamoyl Analogs
a
b
MIC (μM)
potentiation (μM)
f
MPC4
(NOV)
MPC4
(ERY)
CC
WT/WT-
WT-pore/
e
efflux inhibition, SS ratio 32
μM/0 μM
50c
d
#
R
WT
WT-pore
ΔtolC
ΔtolC-pore
(μM)
pore
ΔtolC-pore
g
N
128
64
200
25
100
50
>400
25
32−64
4−8
100
25
100
25
>400
25
12.5
>200
25
0.5
2
12.5
12.5
50
0.125
0.125
12.5
12.5
50
NA
NA
NA
NA
3.1
3.1
12.5
12.5
>200
6.25
6.25
200
12.5
200
NA
NA
NA
NA
2
1
1
2
1
1
2
NA
NA
8
2
2
0.5
1
4
2
>16
1
NA
NA
6.5
7.2
3.9
5.9
1
8.3
11.2
12.3
5.5
1
h
E
2
6
7
8
9
2-Cl
2-Br
2-F
2-Me
2-NO2
1.56
12.5
12.5
6.25
>200
6.25
6.25
200
10.0
11.7
28.3
ND
ND
10.1
11.2
ND
13.3
ND
25
50
>400
12.5
12.5
25
25
12.5
>400
6.25
6.25
12.5
25
10 3-Cl
11 4-Cl
12 4-Br
13 4-iPr
14 2,3-
25
>200
50
1
2
1
12.5
400
>200
400
6.25
64
diCl
a
b
MIC values, determined as the average of two replicates. Minimum concentration of test compound required to reduce the MIC of novobiocin
c
d
(NOV) or erythromycin (ERY) by 4-fold measured in WT-pore cells. Measured in A549 cells; values are the average of two replicates. The ratio
of MIC values for compounds in WT cells vs permeated WT-pore cells. The ratio of MIC values for compounds in WT-pore cells vs ΔtolC-pore
cells. Accumulation of Nile Red fluorescent probe in WT-pore cells in the presence (32 μM) and absence (0 μM) of each compound. Values for
novobiocin. Values for erythromycin. MIC = minimum inhibitory concentration; WT = wild-type; ND = not determined; SS = steady state; NA =
not applicable.
e
f
g
h
i
polar and therefore may be less likely to permeate the more
lipophilic inner membrane (IM) to reach their cytoplasmic
targets.8,18
transporter, AcrA is the MFP, and TolC is the OM channel.23,24
The substrate binds to the AcrB trimer located in the IM while
the TolC trimer forms a channel in the OM allowing molecules
to be extruded out of the cell. The MFP, AcrA, assembles as a
trimer of dimers and serves to span the gap between AcrB and
TolC, forming the pump complex. The structural asymmetry of
the AcrB protomers revealed a “functional rotation” mechanism
in which each of the three monomers constituting the IM
transporter cyclically assumes three conformations, referred to
as loose, tight, and open (or access, binding, and extrusion,
respectively). Only the fully assembled AcrAB−TolC system is
able to extrude substrates.25−27
The other major challenge associated with GN bacteria is the
extrusion of xenobiotics via efflux pumps. GN pathogens express
multidrug efflux pumps that are able to extrude a wide range of
antibiotics. Across different GN bacterial strains, the type and
number of pumps expressed as well as their drug specificity can
vary significantly, though some homology also exists.21,22
In GN bacteria, the major efflux pumps belong to the
resistance nodulation cell division (RND) superfamily.23 The
RND transporter associates with a specific OM channel and a
membrane fusion protein (MFP) to form a tripartite complex
that exploits proton gradients across the IM to shuttle substrates
out of the cell. In E. coli, the primary tripartite efflux pump
system is the AcrAB−TolC complex in which AcrB is the RND
Because so many different classes of antibiotics are affected by
efflux, one strategy that has emerged to combat drug resistance is
to target bacterial efflux pumps with small-molecule efflux pump
inhibitors (EPIs). Inhibition of these complex molecular
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a
Scheme 1. Synthetic Routes and Conditions for the Preparation of New Analogs
a
(a) HOAc (gl), then add cinnamoyl chloride, rt, 12 h. (b) HOAc (gl), then add naphthyl chloride, rt, 12 h. (c) LiOH, THF-H2O (9:1). (d)
TBTU, CH2Cl2, 4-Me piperazine. (e) 15 or 16, Pd(PPh3)4, K2CO3, microwave, ArB(OH)2.
machines should restore the effectiveness of antibiotics that are
susceptible to efflux activity. In general, this EPI strategy has
focused mainly on identifying compounds that bind to and
inhibit RND transporters such as AcrB in E. coli or the
homologous MexB in Pseudomonas aeruginosa.28,29 Although
several novel EPIs have been reported over the years, only a few
of these compounds have reached clinical trials and none have
reached commercial development.30−35
At least two classes of EPIs, pyridopyrimidines and
pyranopyridines, have been shown to bind in a particular
hydrophobic pocket (often referred to as the “hydrophobic
trap”), which is located within the distal substrate-binding site of
AcrB (hereafter referred to as the deep binding pocket,
DBP).28,29,36 The hydrophobic trap is lined with phenylalanine
residues that can stabilize inhibitors mainly through π-stacking
interactions.37 The mechanism of inhibition, as suggested by
structural analyses of inhibitor-bound AcrB and MexB
structures, involves the stabilization of the transporter in the
ligand-bound conformation and the failure to “functionally
rotate” out of this state. The binding sites and mechanisms of
other EPIs that are proposed to target RND pumps remain to be
identified.
Recently, we disclosed a new strategy for identifying EPIs that
focuses on targeting the MFP (AcrA) component of the
tripartite efflux pumps in E. coli as opposed to the RND
transporter, AcrB.38 Screening the NCI Diversity Set V, a
collection of freely available compounds (1593) from the NCI
and the Developmental Therapeutics Program (DTP), led to
the identification of seven apparent AcrA binders that improved
the potency of the antibiotics novobiocin (NOV) and
erythromycin (ERY) in E. coli. Two compounds, NSC-60339
(1) and NSC-33353 (2), had attractive profiles as potential EPIs
(Figure 1). They exhibited only weak antibacterial activity and
were shown to bind to AcrA using surface plasmon resonance
(SPR) experiments. These compounds inhibited efflux in a
biochemical assay that measures intracellular accumulation of
the fluorescent bisbenzimide probe, Hoechst 33342.38 This
probe is a good substrate of the AcrAB−TolC pump and is
unable to accumulate in the cytoplasm of E. coli in the absence of
an EPI.
Medicinal chemistry efforts to elaborate NSC-60339 (1) led
to the identification of compounds such as 3 and 4 (Figure 1),39
which show modest improvements in terms of potentiation,
exhibit stronger binding to AcrA, and feature increased
permeation in wild-type (WT) E. coli. Interestingly, 1 and 2
differ in their mechanism of action, as they inhibit Hoechst
33342 efflux with different kinetics and only 1 alters the
proteolytic profile of intracellular AcrA.
NSC-33353 (2) is a 4,6-diaminoquinoline substituted with a
cinnamoyl amide attached through the C6 amine (Table 1). It
was originally studied as a potential chemokine receptor
modulator40 and later found to be an inhibitor of condensins.41
It was shown to potentiate the antibacterial activities of NOV
and ERY with minimum potentiation concentration (MPC4)
values of 1.56 and 3.1 μM, respectively (Table 1). The MPC4
value is the lowest concentration of test compound, in this case
2, that potentiates (lowers) the MIC of the corresponding
antibiotic by 4-fold. These antibiotics were chosen because they
operate through different antibacterial mechanisms (NOV
targets DNA replication and transcription while ERY targets
the translation machinery) and are unable to effectively
accumulate in E. coli due to a combination of efflux by the
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AcrAB−TolC pump and/or permeation across the OM.38 NOV
effectively permeates the OM but is an excellent substrate of
AcrAB−TolC and is efficiently pumped out (Table 1). In
contrast, ERY is a poor substrate of AcrAB−TolC and its activity
is mostly diminished due to slow permeation across the OM
(Table 1).42
Several other analogs in this series displayed antibacterial
activity (≤50 μM) in E. coli and appeared to be only weak
substrates for efflux (Table 1). However, substitution with the
electron-withdrawing 2-NO2 group (9) led to a complete loss of
antibacterial activity, while replacing Cl with Br (12) at the 4-
position or the dichloro- substituted phenyl (14) led to a loss of
antibacterial activity in WT and WT-pore cells. These
compounds did show activity in the efflux-deficient ΔtolC cells
with MICs ≤ 25 μM, suggesting that these substitutions
promote efflux by AcrAB−TolC (Table 1).
Potentiation for NOV and ERY was also evaluated. Several
compounds (6, 7, 8, 10, 11, 13) were able to potentiate NOV
with MPC4 values in the range of 6.25−12.5 μM, which is
slightly higher than what was observed with 2 (1.56 μM, Table
1). These compounds also potentiated ERY with MPC4 values
similar to their NOV potentiation. One exception was bromo
analog, 6, which potentiated ERY (MPC4 = 3.1 μM) about 4×
better than it potentiated NOV (MPC4 = 12.5 μM). Three
analogs (9, 12, and 14) failed to potentiate the activities of either
NOV or ERY. These were the same analogs that also lacked
antibacterial activity in the WT cells. In general, analogs
displayed similar cytotoxicity to that observed with 2, except for
fluoro analog, 7, which gave a CC50 (28.3 μM) about 3-fold
higher than the original lead, 2.
Compound 2 was also tested for its intrinsic antibacterial
activity in a set of four E. coli strains: WT and ΔtolC where the
AcrAB−TolC pump has been inactivated and their hyper-
porinated derivatives, WT-pore and ΔtolC-pore cells.42 The
MICs for 2 were 200 and 100 μM in the WT and WT-pore cells,
respectively. However, in both efflux-deficient cell types, 2
displayed strong antibacterial activity with an MIC of 12.5 μM.
These findings suggest that 2 is likely a substrate of the efflux
pump, rendering it ineffective as an antibacterial agent. It also
displayed cytotoxicity in A549 cells with a CC50 of 10 μM.
Herein, we describe follow-up medicinal chemistry efforts as
well as biochemical and in silico-based mechanistic evaluation on
the second screening hit, NSC-33353 (2), as a potential EPI of
AcrAB−TolC. Modifications of the cinnamoyl group in 2 led to
analogs with both potent antibacterial and EPI properties. The
replacement of the cinnamoyl group with naphthyl isosteres
gave compounds with very different EPI profiles, likely due to
changes in how they bind to and inhibit AcrAB−TolC.
Because most of these analogs possess intrinsic fluorescence
that increases when compounds are bound to lipids or nucleic
acids,43 we also analyzed their intracellular accumulation and the
effect of efflux and permeation across the OM using an activity-
independent fluorescence assay. In this assay, increasing
concentrations of compounds were incubated with WT and
WT-pore cells and the efflux-deficient derivatives ΔtolC and
ΔtolC-pore. The change in fluorescence was then monitored as a
function of time (Figure 2). In agreement with their respective
antibacterial activities, the cinnamoyl analogs 7, 8, 10, and 11
accumulated with similar kinetics in the efflux-proficient and
efflux-deficient cells, suggesting that these compounds avoid
efflux by AcrAB−TolC (Figure S1). However, these compounds
slowly permeated the OM as observed from the increased rates
of their intracellular accumulation in the hyperporinated strains.
Antibacterial and Potentiation Activities of the
Naphthyl Derivatives. Attention was next shifted to analogs
where the cinnamoyl group was replaced with various
substituted naphthyl groups, as in previous efforts going from
1 to 4 (Table 2). Bromo (15 and 16) and methoxy-substituted
(17 and 18) analogs displayed antibacterial activity in ΔtolC and
ΔtolC-pore cells with only 15 giving any appreciable activity in
the WT-pore cells (MIC = 50 μM). Thus, all compounds are
substrates for efflux. Analog 15 potentiated the activities of both
NOV and ERY with identical MPC4 values (12.5 μM), while
16−18 potentiated NOV (MPC4 = 12.5−25 μM) but
surprisingly did not potentiate ERY (MPC4 ≥ 200 μM).
These naphthyl analogs gave only slightly higher CC50’s (∼20
μM) than the corresponding cinnamoyl compounds.
RESULTS
■
In our previous efforts to optimize 1, we found that the
cinnamoyl analog, 3 (Figure 1), displayed a strong affinity for
AcrA.39 The presence of the same cinnamoyl group in both 2
and 3 suggested that the quinolone ring of 2 and the phenyl
dihydroimidazoline ring of 3 might bind to AcrA in a similar
manner. This observation further suggested that we might follow
similar structure−activity relationships (SAR) developed with 1
to explore modifications of 2. We therefore sought to investigate
additional substitutions in the phenyl ring of the cinnamoyl
group to further probe the SAR in this region and assess the
cinnamoyl group as a possible contributor to the observed
cytotoxicity of 2. To mitigate this potential liability, the
replacement of the cinnamoyl moiety with substituted naphthyl
groups was also explored. In the earlier SAR study of 1, naphthyl
analog 4 (Figure 1) was found to greatly potentiate NOV and
bind strongly to AcrA.
Chemistry. The synthesis of all new compounds was carried
out according to Scheme 1. Selective acylation of the 6-amino
group in 2-methylquinoline-4,6-diamine (5) was achieved via
the procedure described by Hagmann and Springer.40 Diamine
5 was dissolved in glacial acetic acid, and the resulting solution
was added to the requisite acid chloride, which was dissolved in
acetic acid to exclusively give the desired 6-substituted amides.
The acid chlorides were typically prepared in situ from the
corresponding carboxylic acids with either oxalyl chloride or
thionyl chloride. Further elaboration of bromo-substituted
naphthyl analogs (15 and 16) via a standard Suzuki coupling
reaction with various arylboronic acids led to the aryl-
substituted naphthyl analogs (24−36).
Substitution with electron-withdrawing groups such as cyano
(19), carbomethoxy (22), or an amide moiety (23) led to a
complete loss of antibacterial and potentiation activity. The
unsubstituted 20 displayed some antibacterial activity (MIC =
50 μM) in ΔtolC cells and potentiated both NOV (MPC4 = 25
μM) and ERY (MPC4 = 50 μM). The cyclopropyl analog, 21,
lacked antibacterial activity in WT cells but was active in
hyperporinated WT-pore cells (MIC = 50 μM), making it one of
the few analogs affected by permeation. It also had a CC50 of 31
μM, which was the lowest observed in this series.
Antibacterial and Potentiation Activities of the
Cinnamoyl Derivatives. Substitution on the phenyl ring of
the cinnamoyl group was initially explored (Table 1). The
replacement of the chlorine with bromine (6) at the 2-position
yielded an analog with antibacterial activity (MIC = 25 μM) in
WT cells, representing a 8-fold decrease in the MIC compared to
2. The MIC was unchanged in the hyperporinated cells and only
slightly lower in the efflux deficient cells (MIC = 12.5 μM).
D
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Previous docking calculations suggested that adding aromatic
rings at either the C5- or C6-positions might increase the affinity
for AcrA (vide infra).44 As a result, a set of aromatic substituted
naphthyl groups was prepared. The corresponding C6-
substituted analogs (24−27) failed to show any antibacterial
activity, and only phenyl-substituted analog 24 exhibited any
potentiation (Table S1). Following the trend observed with
other naphthyl compounds, 24 only potentiated NOV (MPC4 =
25 μM) and not ERY.
The C5-aryl-substituted analogs are shown in Table 3. Most
of these analogs (28, 29, 31, 34, and 36) had similar profiles.
They showed no antibacterial activity in either WT or WT-pore
cells but did exhibit similar antibacterial activity in the efflux-
deficient ΔtolC and ΔtolC-pore cells. They potentiated NOV
(MPC4 = 6.25−12.5 μM) but not ERY. Two analogs, the
dimethylphenyl, 30, and 3-hydroxyphenyl analogs, 32, had no
antibacterial activity in any of the cell types. Compound 30
potentiated NOV (MPC4 = 25 μM) but not ERY, while 32 failed
to potentiate either antibiotic. Three analogs (31, 33, and 36)
were able to potentiate ERY in addition to NOV. The 3-
aminosubstituted analog, 31, was the weakest of the three in
terms of potentiation values while the 2-hydroxyphenyl analog,
33, was the best, potentiating both NOV and ERY with MPC4 =
12.5 μM. Compound 33 was the only naphthyl analog that
possessed antibacterial activity even in the WT cells (MIC = 50
μM). The CC50 value for 33 was 27.3 μM. The replacement of
hydroxy or methoxy with an electron-withdrawing cyano group
(35) led to a loss of both antibacterial and potentiation activity.
Taken together, these results show that, unlike the cinnamoyl-
derived analogs, the majority of the naphthyl analogs lacked
antibacterial activity in the WT or WT-pore cells. Several
compounds exhibited antibacterial activity in ΔtolC cells,
suggesting that the naphthyl analogs were more susceptible to
Figure 2. Intracellular accumulation of compounds (A) 7, (B) 8, (C)
10, and (D) 11 in E. coli cells with different genetic backgrounds.
Doubling concentrations of compounds from 1 to 32 μM were added to
WT (blue), WT-pore (yellow), ΔtolC (gray), and ΔtolC-pore
(orange); cells were incubated at room temperature for indicated
periods of time, and fluorescence was measured in real time. Data from
at least two independent experiments were fitted to a two-exponential
equation to extract the steady-state intracellular concentrations. Error
bars are SD (n = 2).
g
Table 2. Antibacterial and Potentiation Activity for Substituted Naphthyl Analogs
a
b
MIC values, determined as the average of two replicates. Minimum concentration of test compound required to reduce the MIC of novobiocin
c
d
(NOV) or erythromycin (ERY) by 4-fold measured in WT-pore cells. Measured in A549 cells; values are the average of two replicates. The ratio
of MIC values for compounds in WT cells vs permeated WT-pore cells. The ratio of MIC values for compounds in WT-pore cells vs ΔtolC-pore
cells. Accumulation of Nile Red fluorescent probe in WT-pore cells in the presence (32 μM) and absence (0 μM) of each compound. MIC =
e
f
g
minimum inhibitory concentration; WT = wild-type; ND = not determined; SS = steady state.
E
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g
Table 3. Antibacterial and Potentiation Activity for C(5)-Aryl Substituted Naphthyl Analogs
a
b
MIC values, determined as the average of two replicates. Minimum concentration of test compound required to reduce the MIC of novobiocin
c
d
(NOV) or erythromycin (ERY) by 4-fold measured in WT-pore cells. Measured in A549 cells; values are the average of two replicates. The ratio
of MIC values for compounds in WT cells vs permeated WT-pore cells. The ratio of MIC values for compounds in WT-pore cells vs ΔtolC-pore
cells. Accumulation of Nile Red fluorescent probe in WT-pore cells in the presence (32 μM) and absence (0 μM) of each compound. MIC =
e
f
g
minimum inhibitory concentration; WT = wild-type; ND = not determined; SS = steady state.
efflux than the cinnamoyl analogs. Another difference between
the two sets of compounds was the finding that, with only a few
exceptions (15, 20, 31, 33, and 36), the naphthyl analogs could
potentiate only NOV and not ERY. Aryl substitution at the C5
position of the naphthyl ring yielded the best results in terms of
NOV potentiation.
Efflux Inhibition in Bacterial Growth-Independent
Assays. Intrinsic antibacterial activities of compounds can
contribute to the observed MPC values, and in the case of the
cinnamoyl derivatives, the MICs are very close to the observed
MPCs (Table 1). To establish whether the potentiation of
antibiotic activities is indeed due to the inhibition of efflux, we
first analyzed MPCs in efflux-deficient ΔtolC-pore cells. We
found no potentiation of NOV or ERY activities in these cells
(Table S2).
fluorescence properties distinct from those of the compounds.
We chose the Nile Red probe, which is not fluorescent in water
solutions but emits red light in hydrophobic environments such
as lipid membranes. Cells were incubated with increasing
concentrations of compounds, and the kinetics of the change in
fluorescence was analyzed (Tables 1−3 and Figure S2). Most of
the tested cinnamoyl derivatives inhibited efflux of Nile Red with
efficiency similar to that of 2. Compounds such as 6 and 10−12
were more effective than 2 at the highest concentration (32
μM), whereas 9 and 14 failed to inhibit Nile Red efflux (Table
1).
Similarly, naphthyl analogs were comparable to 2 as inhibitors
of Nile Red efflux. Among this series, 17 and 36 were more
effective and 16, 18, and 20 were all less effective than 2 (Figure
S2 and Tables 2 and 3). Thus, compounds inhibited efflux in
both bacterial growth-dependent assays (i.e., by potentiating the
activities of the antibiotics) and in growth-independent assays.
The inhibition of Nile Red efflux and potentiation of antibiotic
activities were observed in WT-pore cells but not in efflux-
We next analyzed the ability of compounds to inhibit efflux in
a bacterial growth-independent assay using fluorescent probes
that are substrates of AcrAB−TolC. Since these compounds are
intrinsically fluorescent (Figure 2), we needed a probe with
F
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deficient ΔtolC-pore cells, further confirming the dependence of
these activities of compounds on efflux.
residues K46-K396 and K46-T296, respectively. The proteolytic
profiles of the untreated cells are dominated by these two AcrA
fragments (Figure 3). In contrast, an additional K46-K346
fragment is accumulated when AcrA fails to assemble into a
complex. Although 2 and most of its derivatives generated in this
study did not affect the proteolytic profiles of AcrA in the
periplasm of WT-pore cells, 12 and 17 and, to a lesser extent, 31
and 36 altered the AcrA profiles, as seen from the accumulation
of the characteristic K46-K346 fragment. This result suggests
that treatment of cells with these compounds interfered with the
assembly of the AcrAB−TolC complex (Figure 3). All four
compounds bound the purified AcrA with relatively high
affinities, with 17 and 36 being the strongest binders (Table
4). This result further demonstrated that, although many
compounds bind the purified AcrA, their specific interactions
can lead to different outcomes and certain interactions may
affect the assembly of the AcrAB−TolC pump.
We next used ensemble docking to gain insight into the
molecular mechanism underlying the interactions of com-
pounds with AcrA. The unbound AcrA monomer is quite
flexible and samples a wide range of conformations among its
four structural domains: α-hairpin, lipoyl, β-barrel, and
membrane proximal (MP).45 The two-predominant cis and
trans conformations of AcrA differ in their inter-residue contacts
between the β-barrel and MP domains. In the complex with
AcrB and TolC, AcrA adopts a trans conformation. Previous
studies have shown that 1 binds predominantly at site IV, which
is located between the lipoyl and β-barrel domains of AcrA
(Figure 4A).44
To analyze AcrA interactions with 2 and its analogs, we used
an ensemble of 29 conformations of full-length monomeric AcrA
structures obtained from molecular dynamics simulations.
Compounds were docked to four previously identified sites on
AcrA (Figure 4A), and the top pose was selected on the basis of
the Vina docking score (Table 4).44
For most of the cinnamoyl and naphthyl analogs, the top
docking score corresponded to binding at site III, which is
located at the flexible interface of the β-barrel and MP domains.
The overall most favorable docking score for the cinnamoyl
analogs was −9.5 kcal mol−1, which was obtained for 10, and
−9.6 kcal mol−1, obtained for naphthyl analogs 25, 30, and 35
(Table 4). For site III, the top docking scores were all within 1.6
kcal mol−1 of each other, which is within the expected error
range for docking calculations of this type.46 Thus, we cannot
reliably distinguish between the predicted binding affinities of
the cinnamoyl and naphthyl analogs.
Interactions with AcrA. To gain insight into the
mechanism of antibiotic potentiation by 2 and its analogs, we
next analyzed how these compounds interact with AcrA. In SPR-
based assays with purified AcrA protein, we found that all
cinnamoyl analogs bound AcrA with equilibrium dissociation
constants (KD) ranging from 0.02 to 0.3 mM (Table 4). In
Table 4. Interactions with AcrA and AcrB Determined by
d
SPR and Molecular Docking
AcrA docking score
a
b
KD (mM)
(kcal mol−1
)
AcrB docking
site
II
site
III
site
IV
score
b
#
AcrA
AcrB site I
(kcal mol−1
)
c
NOV
MC
0.04
0.28
+
0.53 NA
0.11 NA
NA
NA
NA
NA
NA
NA
NA
NA
c
2
ND
−7.3 −7.3 −8.1 −7.5
−11.9
−12.1
−12.1
−12.5
−11.5
−12.0
−11.2
−11.6
−12.6
−12.3
−13.3
−13.5
−12.9
−12.8
ND
−13.1
ND
ND
−14.6
ND
ND
−14.2
ND
−14.5
−15.6
ND
−15.3
−15.3
−15.3
−14.2
−16.3
−15.1
6
7
8
9
0.02
0.28
0.26
0.05
0.02
0.01
0.08
0.05
0.02
ND
0.25
0.001
ND
ND
ND
ND
ND
NB
ND
ND
0.02
ND
ND
1.18
ND
0.06
ND
ND
ND
ND
0.002
0.02 −7.0 −7.4 −7.9 −7.3
0.03 −7.6 −7.7 −8.1 −7.5
ND
−7.3 −7.5 −8.0 −7.6
0.04 −6.9 −7.1 −7.9 −7.7
0.06 −8.3 −8.8 −9.5 −8.4
0.17 −6.8 −7.3 −7.9 −7.4
0.01 −7.0 −7.5 −7.9 −7.6
0.06 −7.2 −7.5 −8.1 −7.9
0.05 −7.3 −7.4 −8.1 −7.5
0.01 −7.6 −8.2 −8.7 −8.3
0.25 −7.8 −8.2 −8.8 −8.1
0.02 −7.6 −8.4 −8.6 −8.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
ND
ND
−7.6 −7.7 −8.5 −7.9
−8.6 −8.7 −9.0 −8.4
0.07 −7.8 −8.1 −8.7 −8.1
ND
ND
NB
ND
ND
−7.8 −8.2 −8.7 −8.3
−7.8 −8.5 −8.5 −8.4
−8.5 −8.9 −9.3 −9.2
−8.3 −9.0 −9.2 −9.2
−8.6 −9.3 −9.6 −9.6
0.05 −8.3 −9.0 −8.9 −8.9
ND −7.8 −8.4 −9.1 −8.9
4.63 −8.3 −8.1 −9.0 −8.1
0.05 −8.3 −8.8 −9.5 −8.4
ND
0.02 −9.1 −8.9 −9.4 −8.4
ND −8.8 −8.7 −9.2 −8.2
4.92 −8.6 −8.9 −9.4 −8.3
ND −8.0 −8.8 −8.9 −8.1
−8.6 −9.2 −9.6 −8.6
Regardless of the substituent position, the R groups do not
participate in any polar interactions with the protein, and these
analogs have similar docking poses at this site (Figure 4). For
example, both the ortho-substituted compound 2 (Figure 4B)
and the para-substituted analog 11 (Figure 4C) form polar
contacts with T53 and are near Q51, T54, E55, R294, Q341, and
I343. Importantly, these residues are known to be critical for the
cis-to-trans conformational transitions in AcrA and for the
functional activity of the efflux pump.45 All C(6)-substituted
analogs as well as the nonaryl-substituted C(5)-analogs were
also predicted to bind in a similar position regardless of
substitution (Figure 5E−G) with the exception of 23, which was
predicted to bind elsewhere in site III. For the C5-substituted
aryl compounds, the naphthyl ring is flipped ∼180° in its
predicted binding pose (Figure 5H).
0.07 −8.6 −9.1 −9.6 −8.6
0.04 −8.2 −9.2 −9.0 −7.9
a
b
Data were fitted into a two-state reaction model. Selected on the
c
d
basis of the Vina docking score. See refs 38 and 47. NOV =
novobiocin; MC = MC-207,110; ND = no data; NB = no binding;
NA = not applicable.
contrast, naphthyl substituents that bound AcrA varied
considerably in their affinities. Among all tested compounds,
17 and 36 were found to bind AcrA with the highest affinity (KD
= 1−2 μM).
For compounds with EPI activities, we also analyzed their
interactions with AcrA in vivo using a proteolysis assay.38 In this
assay, AcrA assembled into the AcrAB−TolC complex is cleaved
into two major proteolytic fragments comprising amino acid
Therefore, SPR experiments suggest that compounds from
both the cinnamoyl and naphthyl series interact with AcrA.
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Figure 3. Proteolytic profiles of periplasmic AcrA in the presence and absence of (A) 12, (B) 17, (C) 36, and (D) 31. Cells were osmotically shocked
with sucrose and treated with the indicated concentrations of trypsin for 30 min at 37 °C. Reactions were terminated by SDS sample buffer and boiling
the samples for 5 min. Proteins and proteolytic fragments were separated by 12% SDS-PAGE, and AcrA fragments were visualized using polyclonal
anti-AcrA antibody. The characteristic proteolytic fragments of AcrA as identified in ref 70 are indicated.
Figure 4. (A) Cryo-EM structure of AcrAB−TolC (PDB ID: 5NG5).71 Individual subunits of TolC (purple), AcrA (green), and AcrB (blue) are
shown in a surface representation. A single monomer of AcrA is shown as a cartoon and colored by domain (α-hairpin = green, lipoyl = orange, β-barrel
= yellow, membrane proximal (MP) = red). Sites I−IV used for docking are labeled. Top binding poses of (B) o-substituted cinnamoyl compound 2
(green) and (C) p-substituted cinnamoyl derivative 11 (gray) from AcrA docking. AcrA is shown as a cartoon and colored by domain with nearby
residues shown as sticks and polar interactions represented by dashed yellow lines. Top site III docking poses of (D) o-substituted and (E) p-
substituted cinnamoyl derivatives. Analogs are shown as sticks, and AcrA is shown as a surface and colored by residue type (hydrophobic = white, polar
= cyan, positive = blue, negative = red, aromatic = magenta, proline = light green, glycine = dark green).
These interactions apparently are not sensitive to chemical
modifications and cannot explain the observed differences in the
activities of the compounds. Compounds 12, 17, and 36, which
display high affinities for purified AcrA, affect its proteolytic
profiles and possibly the assembly of the AcrAB−TolC complex.
However, we note that these ligands share similar docking scores
and poses with other compounds in the AcrA model. Thus,
additional factors besides interactions with AcrA alone likely
contribute to the observed activities of these compounds.
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Figure 5. Top poses from docking naphthyl analogs to AcrA. Example top binding poses for (A) C6, (B) C5, (C) C6 aryl, and (D) C5 aryl-substituted
analogs are shown as sticks. Polar interactions are represented by dashed yellow lines, and residues that form polar interactions are also shown as sticks
and colored by domain (β-barrel = yellow, membrane proximal (MP) = red). (E−H) Top-scoring poses of naphthyl analogs grouped on the basis of
substitution. Analogs are shown as sticks, and AcrA is shown as a surface and colored by residue type (hydrophobic = white, polar = cyan, positive =
blue, negative = red, aromatic = magenta, proline = light green, glycine = dark green).
Figure 6. (A) Top docking pose and ligand surface of o-substituted cinnamoyl derivatives 6−9 and 14 at the DBP of AcrB; π−π-stacking interactions
are shown as magenta dotted lines (ligand, yellow sticks; protein, gray sticks). (B) Top docking pose and ligand surface of p-substituted cinnamoyl
derivatives 11−13 at the same site (ligand, cyan sticks; protein, gray sticks).
Interactions with AcrB. The analogs with cinnamoyl and
naphthyl substitutions were notably different in both their
growth-inhibitory and EPI activities. In particular, the naphthyl
group increased the propensity of the compounds to be expelled
by the AcrAB−TolC pump and reduced their potency in
combination with ERY but not with NOV. These results
suggested either changes in how the compounds interact with
the efflux pump components, or in their mechanism of action, or
both. Using SPR-based assays with purified AcrB protein, we
found that the binding affinities of all cinnamoyl and a few
naphthyl substituents toward AcrB varied from low micromolar
range to low millimolar range and were comparable to those
toward AcrA (Table 4). These affinities were also similar to the
previously reported affinities of AcrB to antibiotics NOV,
doxorubicin, and minocycline and such EPIs as MC207,110.47,48
However, for several naphthyl-containing compounds such as
20, 28, 33, and 35, a binding signal was detected for the
immobilized AcrB but not for AcrA, suggesting that these
naphthyl substituents have higher affinity for the AcrB
transporter.
from P. aeruginosa tentatively assigns a density to NOV in the
DBP of MexB.49 Additionally, small molecule substrates such as
minocycline and some previously characterized EPIs have been
crystallized within the DBP of AcrB.28,29,50 Specifically, EPIs
were found to bind AcrB in the hydrophobic trap. We therefore
carried out molecular docking runs of 2 and its analogs to the
DBP of AcrB (Figure 6). The best docking pose of compound 2
(Vina docking score of −11.9 kcal mol−1) is more favorable than
the one calculated for AcrA. The ligand binds in the DBP with
the cinnamoyl scaffold in the hydrophobic trap and the
quinolinium heterocycle in the most hydrophilic portion of
the pocket (Figure 6A). Two parallel displaced π−π-stacking
interactions were observed, one involving the o-chloro-
cinnamoyl moiety engaged by F178 and the other being the
peptidyl quinolinium π-system with F628. No hydrogen bonds
were observed, pointing to hydrophobic contacts as the main
source of affinity. Indeed, the main residues contributing to
binding are F610, V612, V139, V571, Y327, P326, and I277. The
role of the chloro substituent at the ortho position of the phenyl
ring is unclear upon first inspection, but it could be important for
affecting directionality and bonding with F178.
No crystal structure of NOV bound to AcrB is available, but
the recent cryo-EM study of the MexAB−OprM tripartite pump
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Figure 7. Docking poses and ligand surfaces of naphthyl AcrB binder inhibitors (green), nonbinders (light gray) and substrates (orange) in the DBP of
AcrB; π−π-stacking interactions are reported as magenta dotted lines, and protein residues are depicted as gray sticks.
Cinnamoyl Derivatives. The cinnamoyl derivatives display
the same chemical interaction pattern (Figure 6A) with similar
average docking scores (−12.1 0.4 kcal mol−1, Table 4) than
their parent compound 2. Consistent with the experimental
data, these results indicate that all tested ligands can be
effectively accommodated within the DBP of AcrB by exploiting
two important chemical features: aromaticity and amphiphi-
licity. Both features contribute to the optimal fit within the
protein cavity, dictating ligand orientation and defining the
interaction pattern.
extended conformations than the latter, which instead are more
bent and are all superposable (Figure 6B). Compound 14,
characterized by a dichlorobenzene moiety, is a substrate of
AcrB (Table 1), but in contrast to monosubstituted inhibitors
10 and 2, it lacks EPI activity in both bacterial growth-
dependent and -independent assays. Both computational and
experimental data suggest that the 2,3-dichloro substitution
negatively affects the interaction with F178 by altering
electrostatic and/or local steric effects. F178, in fact, is one of
the most important phenylalanine residues in the hydrophobic
trap for binding both inhibitors and substrates, on the basis of X-
ray experiments and previous computational studies.36,51,52
Upon visual inspection, p-substituted ligand poses differ
subtly from o/m-analogues: Specifically, they assume more
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d
Table 5. MIC and Potentiation Data for Selected EPIs in E. cloacae and K. pneumoniae
E. cloacae ATCC13047
K. pneumoniae ATCC13883
fold
fold
fold
fold
fold
fold
c
b
c
b
c
b
c
b
c
b
c
b
MPC4
MIC
MPC4
MIC
MPC4
MIC
MPC4
MIC
MPC4
MIC
MPC4
MIC
a
a
#
MIC
(NOV)
(NOV)
(ERY)
(ERY)
(CIP)
(CIP)
MIC
(NOV)
(NOV)
(ERY)
(ERY)
(CIP)
(CIP)
NOV
ERY
CIP
2
6
7
400
400
0.031
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NA
NA
NA
12.5
25
NA
NA
NA
64
64
4
32
32
32
1
2
1
1
1
1
1
1
NA
NA
NA
12.5
50
>100
25
6.25
>100
50
100
>100
>100
>100
>100
25
NA
NA
NA
8
2
1
8
8
1
1
1
1
1
1
1
64
1
NA
NA
NA
NA
NA
NA
1
1
1
1
1
1
1
ND
ND
ND
ND
ND
ND
ND
50
400
NA
NA
NA
12.5
>100
25
NA
NA
NA
64
1
NA
NA
NA
>100
>100
100
100
50
NA
NA
NA
2
1
1
1
1
1
1
1
1
1
1
1
1
1
NA
NA
NA
>100
25
25
25
25
12.5
25
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
1
4
4
0.031
>100
>100
100
>100
25
>100
>100
>100
>100
>100
>100
>100
ND
ND
ND
ND
ND
25
25
8
8
25
64
NA
64
16
1
1
1
1
64
1
4
10
11
13
15
16
21
28
33
34
36
12.5
12.5
50
100
>100
25
>100
>100
>100
>100
6.25
12.5
25
100
>100
>100
>100
12.5
>100
50
NA
NA
8
ND
ND
ND
ND
ND
ND
ND
50
100
50
100
>100
>100
>100
>100
50
>100
>100
>100
>100
100
ND
ND
>100
>100
>100
>100
>100
1
a
b
MIC values (μM for test compounds, mg/L for antibiotics), determined as the average of two replicates. Minimum concentration of test
compounds required to reduce the MIC of novobiocin (NOV), erythromycin (ERY), or ciprofloxacin (CIP) by 4-fold measured in the indicated
c
d
strains. The ratio of MIC values for novobiocin, erythromycin, and ciprofloxacin in the absence and presence of 25 μM of test compounds. ND =
no data; NA = not applicable.
The o-nitro compound 9 constitutes a special case. Although
retaining the above-mentioned interaction pattern, the nitro
group provides polarity to the most hydrophobic portion of the
ligand, such that amphiphilicity is reduced and the interaction
with the hydrophobic trap is less favorable (−11.5 kcal mol−1).
In agreement, 9 lacks EPI activity.
interaction pattern observed, while amphiphilicity sets the
molecular orientation. Differing from the cinnamoyl analogues,
F628 further stabilizes the ligand in the pocket by means of two
aromatic interactions (one parallel-displaced stacking and one
sandwich stacking). The stacking interaction with F178 is
preserved with the quinolinium ring interacting with F615 as
well. However, F615 does not seem relevant for other
compounds. The amino acids V139, G179, I277, A279, P326,
Y327, V571, F610, and V612 are the most contacted residues,
contributing primarily through hydrophobic interactions, even
if, in some cases, Q151 and N274 are also involved.
As illustrated in Figure 6, both inhibitors and substrates follow
the binding trend described so far, but peculiarities can be
detected, depending on functionalization of the naphthalene
ring at position 5 or 6. Specifically, C5-substituted ligands
feature an overall higher affinity than the unsubstituted
compound 20 or the related C6-substituted complexes (−15.1
vs −13.2 kcal mol−1 according to the docking scores).
The computational data are consistent with the experimental
results. Specifically, functionalization with a phenyl moiety at the
5-position in 29 allows for the formation of an additional π−π
sandwich stacking with the Y327 phenolic side chain, preserved
for all the related C5-substituted compounds but absent in the
C6-subsituted ligand top poses. For the latter, in fact, the ligand
is hindered and forced into its own extended orientation so that
it cannot interact by the same mode. Moreover, large
substituents could lead to unfavorable contacts compared to
the less hindered ligand 17. Compound 35 is characterized by
the presence of an acetonitrile group, which might give rise to
unfavorable electrostatic interactions with M573. For the
docking pose of substrate 26, stacking between the quinolinium
heterocycle and F178 is lacking, which could partially explain its
activity profile. Compound 23, which has a saturated piperazine
amide substituent, is unable to engage in π-stacking interactions
with Y327 and suffers from additional unfavorable steric clashes
The preferred conformation of the three p-substituted
cinnamoyl analogues within the DBP is linear and extended
with the main substituent oriented toward Y327 and P326
(Figure 6B). These ligands share the same interaction pattern
reported for the o-substituted analogues with some distinctive
exceptions represented by Q151, G179, A279, and N274, and
the latter interaction observed only in the docking pose of 12.
The binding modes of 11 and 13 are superimposable with each
other but not with 12. In 11 and 13, the anilinic amine of the
quinolinium is oriented toward Q151, while in 12, it is oriented
toward N274 on the opposite side. The brominated ligand binds
less deeply in the DBP than the chlorinated and isopropyl
derivatives, probably due to the greater size of the bromo
substituent, which affects ligand directionality to some extent.
Due to its positioning, the quinolinium group of 12 is too far
from F628 to establish substantial parallel-displaced stacking.
The interaction with F628 is preserved by the peptide bond π-
system, but it is likely weaker in strength than when it is made
with the quinolinium group. Consistent with this observation,
the o-substituted derivatives show on average better docking
scores than the corresponding p-substituted ones (Table 4).
Naphthyl Derivatives. The docking scores for selected
naphthyl derivatives are generally more favorable than
cinnamoyl derivatives (Table 4). The introduction of the
naphthyl ring isostere in place of the cinnamoyl moiety leads to a
more favorable interaction within the DBP of AcrB. This
difference is especially evident in the interaction pattern of 20
with the protein, in which the naphthalene is unsubstituted and
therefore represents a good reference molecule for this series
(Figure 7). Even in this case, π−π bonds appear to determine the
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supporting the observed experimental data that the ligand does
not bind to AcrB.
bound compounds with the access of ERY into the PBP. Note
that, according to a previous docking study,54 NOV appeared to
bind preferentially to the region of the DBP, including some of
the phenylalanine residues lining the hydrophobic trap (F178,
F610, F615). This binding mode is very similar to that
tentatively assigned to NOV in MexB where it interacts with
residues F178, F615, and F617.49 Our results therefore suggest
that the observed antibacterial potentiation of NOV and Nile
Red efflux inhibition activities are enabled by the interactions of
the compounds with the hydrophobic trap within the DBP of
AcrB.
Previous mutagenesis studies on AcrB showed that Y327A
substitution decreased the MICs (i.e., reduced efflux activity) of
NOV but not ERY, whereas F178A and F628A substitutions in
AcrB decreased the MICs of both antibiotics.55−57 In agreement,
our molecular docking analyses further support the role of F178
and F628 for the binding of both substrate and inhibitor
compounds to AcrB because these residues are involved in well-
ordered π-stacking interactions of differing stability. We also
identified Y327 as a crucial residue for both recognition and
activity of the ligands, especially of naphthyl derivatives, and
other contact residues (polar and apolar) that contribute to the
general interaction pattern observed.
The results presented here expand on our previous efforts to
develop novel EPIs targeting the MFP AcrA of the AcrAB−TolC
efflux pump of E. coli. By following up on a novel hit from the
NCI collection, we have identified structure−activity relation-
ships for compounds that bind to AcrA and prevent efflux of
both NOV and ERY. Additional modifications led to
compounds with stronger affinities to AcrB and thus changed
the mechanism of inhibition relative to our previously reported
AcrA inhibitors. Future efforts will be directed toward further
exploring the mechanism of potentiation of NOV for the
naphthyl compounds and expanding the SAR around this series
to optimize binding to AcrA.
Potentiation of Antibacterial Activities in Other Gram-
Negative Bacteria. We previously showed that 2 can
potentiate the activities of NOV and ERY in other enter-
obacteria such as Enterobacter cloacae and Klebsiella pneumoniae
but not Pseudomonas aeruginosa.38 In agreement with these
findings, most of the cinnamoyl derivatives with EPI activities in
E. coli also potentiated the activities of NOV, ERY, or both in
E. cloacea and K. pneumoniae (Table 5). Compounds 6, 7, and 8
also potentiated the activity of ciprofloxacin (CIP) but only
against K. pneumoniae. In contrast, the naphthyl derivatives were
notably less potent in these bacteria, likely due to their low
permeation resulting from the synergistic action of active efflux
and the OM barrier. Only 10 and 13 showed EPI activity in
P. aeruginosa but only in combination with NOV and not with
ERY or CIP (Table S3). The reasons for species- and antibiotic-
specific differences in EPI activities are not immediately clear,
but these could be the result of species-specific differences in the
intracellular permeation of EPIs and antibiotics.
DISCUSSION
■
We have prepared a series of cinnamoyl- and naphthyl-derived
amides of the 4,6-diamino-2-methylquinoline scaffold as
potential EPIs. The cinnamoyl analogs profiled very similar to
the original hit. Many of them were shown to bind strongly to
AcrA, potentiate NOV and ERY in E. coli, and inhibit efflux in a
fluorescent probe accumulation assay. A number of these
analogs also exhibited strong antibacterial activity in addition to
efflux inhibition and avoided TolC-dependent efflux. Unfortu-
nately, they also demonstrated cytotoxicity that limits their
utility. The replacement of the cinnamoyl group with a set of
corresponding naphthyl-based analogs led to compounds with
different profiles. In general, these analogs did not bind to AcrA
but did potentiate NOV. However, most of these analogs did not
potentiate ERY in E. coli. They appear to permeate the OM of
E. coli and show antibacterial activity but are likely substrates of
the pump, which limits their antibiotic effects. The naphthyl
analogs did show lower cytotoxicity, typically 2- to 3-fold relative
to the cinnamoyl compounds.
METHODS
■
MTS Cytotoxicity Assay (CC50). A549 cells (human
alveolar basal epithelial cells, 1.0 × 104 cells/well) were seeded
in 96-well plates and incubated in Dulbecco’s Modified Eagle
Medium with 10% fetal bovine serum plus 1% penicillin/
streptomycin solution, 1% nonessential amino acids, and 1%
glutamine. The compounds were diluted in the medium to the
indicated concentrations to a final concentration of 1% DMSO
and added to the cells 48 h after plating, with each concentration
tested in triplicate. Soluble MTS reagent [3-(4,5-dimethylth-
iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium, Promega] was added 72 h after incubation; the
cultures were incubated for 90 min, and absorbance was read at
490 nm. The CC50 was calculated as the concentration of the
inhibitor required to reduce cell viability 50% relative to
untreated cells. The data are plotted as log[inhibitor] versus
response and fit to a variable slope model using Graph Pad Prism
(v6; GraphPad Software, Inc.).58
NOV and ERY differ in their interactions with AcrA and AcrB
and in their propensity to be expelled by the AcrAB−TolC
pump. The antibacterial activity of ERY is strongly enhanced by
hyperporination of the OM, despite fully active efflux (Table
1).42 In contrast, the activity of NOV is almost exclusively
affected by AcrAB−TolC. Our previous studies also showed that
NOV but not ERY binds to both AcrA and AcrB in the SPR
assays.38,48 The results of this study further demonstrate how
specific chemical modifications govern the effect of EPIs on
antibacterial activities of these antibiotics. Most of the analyzed
compounds are likely to interact with both AcrA and AcrB,
although the naphthyl derivatives have higher affinities in
docking to the DBP of AcrB. In the crystal structure of the ERY-
bound AcrB, this large antibiotic is located in a proximal
multisite drug-binding pocket of AcrB (known as the proximal
binding pocket, PBP) that extends farther into the interface with
DBP.53 Hence, the PBP is believed to be the primary recognition
and binding site of high-molecular mass compounds such as
ERY (∼735 Da). Interestingly, in the assembled AcrAB−TolC
complex (Figure 4A), site III of AcrA, which is the preferred
binding site of 2 and its analogs, is adjacent to and faces the PBP
of AcrB. Hence, the potentiation of ERY by cinnamoyl
derivatives could be due to the direct interference of AcrA-
Microbiological Assay. E. coli BW25113 (WT, Δ(araD-
araB)567 Δ(rhaD-rhaB)568 ΔlacZ4787 (::rrnB3) hsdR514 rph-
1), and ΔtolC (BW25113 ΔtolC ygiBC) strains of E. coli and
their hyperporinated WT-pore (BW25113 attTn7::mini-Tn7T
Kmr araC ParaBAD fhuAΔC/Δ4L) and ΔtolC-pore (ΔtolC ygiBC
attTn7::mini-Tn7T Kmr araC ParaBAD fhuAΔC/Δ4L) variants
used in this study are derivatives of BW 25113 and GD102,
respectively.42 The E. coli BL21(DE3) strain was used for
overexpression and purification of AcrA,59 and the AG100AX
L
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Article
strain was used for overexpression and purification of AcrB.48
MICs were analyzed using a 2-fold broth dilution method.60 For
the checkerboard assay, an antibiotic and a test compound were
serially diluted into 96-well plates as described previously.61
Surface Plasmon Resonance. For the amino coupling of
AcrA and AcrB, CM5 chip surfaces were activated with 0.05 M
N-hydroxysuccinimide and 0.2 M N-ethyl-N-(3-
diethylaminopropyl)carbodiimide (BIAcore). AcrA and AcrB
were injected over surfaces immediately after activation. After
immobilization, the excess of reactive groups was blocked by
injecting 0.5 M ethanolamine HCl (pH 8.0). The immobiliza-
tion and subsequent binding experiments were conducted in
running buffer containing 20 mM HEPES-KOH (pH 7.0), 150
mM NaCl, and 0.03% DDM supplemented with 5% DMSO.
The CM5 chip contains four chambers, whereas the first
(control surface) was activated and processed in the same way
but the protein was omitted during the immobilization step. The
second and third chambers contained the immobilized AcrA and
AcrB (ligand). The immobilized densities of both proteins
(ligand) were 7167 and 7072 response units (RU), respectively.
For kinetic modeling, we considered only the simplest models
that would be compatible with one or two distinct events during
both inhibitor binding and dissociation. These four models are
(i) a simple 1:1 binding model; (ii) a heterogeneous ligand
(HL), in which different protein populations on the on-chip
surface have different kinetic properties; (iii) two-state reaction
or ligand-induced conformational change, wherein conforma-
tional change occurs on the same time scale as ligand binding;
(iv) bivalent analyte, where multiple analytes bind independ-
ently at nonidentical sites.62,63
prepared with AutoDock Tools.65 Protein flexibility was
considered indirectly using the ensemble of conformations.66
For AcrA, we used 29 previously identified conformations
extracted from a 50 ns molecular dynamics simulation of a
model of the full-length AcrA monomer.44 In the case of AcrB,
we employed 10 available X-ray conformations (PDB_IDs:
2DHH, 2GIF, 2J8S, 3W9H, 4DX5, 4DX7, 4U8V, 4U8Y, 4U95,
4U96).67 Guided docking runs were performed in both cases.
For AcrA, we adopted a 25 Å × 25 Å × 25 Å box centered at four
different sites defined on the basis of the center of mass of
selected residues: site I (E67), site II (K241), site III (I343,
I252), and site IV (F81, F254).68 For AcrB, we used a 40 Å × 40
Å × 40 Å box centered in the center of mass of the DBP. The top
10 docking poses were retained for each protein−ligand pair,
and the top ones were selected for further analysis. Molecular
graphics were rendered using UCSF Chimera69 or PyMOL 2.4
(https://pymol.org).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00100.
C(6)-Aryl substituted naphthyl analogs, MIC and
potentiation data, fluorescence vs time plots, activities
of compounds, abbreviation list, and chemistry proce-
dures with analytical data for all new synthetic
1
compounds including H NMR, 13C NMR, and mass
spectra data (PDF)
Fluorescence Accumulation Assay. An accumulation
assay was performed in a temperature-controlled microplate
reader (Tecan Spark 10M multimode microplate reader
equipped with a sample injector) in fluorescence mode.14
Cells from frozen stocks were inoculated into LB medium and
incubated for 16 h at 37 °C. Cells were then subcultured into a
fresh 30 mL volume of LB medium and grown at 37 °C to an
optical density at 600 nm (OD600) of 0.3. The cells were then
induced with 0.1% arabinose and grown to an OD600 of 1.0,
collected by centrifugation at 4000 rpm for 20 min at room
temperature, and washed in 25 mL of HEPES-KOH buffer (50
mM; pH 7.0) containing 1 mM magnesium sulfate and 0.4 mM
glucose (HMG buffer). The cells in HMG buffer were adjusted
to an OD600 of ∼1.0 and kept at room temperature during the
experiment. Fluorescence intensities of Nile Red accumulated in
the cells were plotted as a function of time and normalized to the
emission of the dye before the cells were added. The data were
imported into MatLab (MathWorks, Inc.) to be fitted to a
simple exponential equation in the form of F = A1 + A2 [1 −
exp(kt)], where A1 represents the amplitude of the initial fast
uptake of a dye, and A2 and kt are the amplitude and rate,
respectively, that are associated with the subsequent slower
uptake.14
Ensemble Docking to AcrA and AcrB. Molecular docking
calculations targeting both AcrA and AcrB were performed using
AutoDock Vina,46 which implements a stochastic global
optimization approach. The program was used with default
settings but for the exhaustiveness parameter (giving a measure
of the exhaustiveness of the local search), which was set to 1024
(default 8). Ligand protonation states were calculated using
Marvin calculator plugins,64 and compounds were considered
flexible during docking (number of rotatable bonds in the range
of 2 to 5). Protein and ligand input files in PDBQT format were
AUTHOR INFORMATION
■
Corresponding Authors
Helen I. Zgurskaya − Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma
73072, United States; orcid.org/0000-0001-8929-4727;
Email: elanaz@ou.edu
John K. Walker − Department of Pharmacology and Physiology,
Saint Louis University School of Medicine, St. Louis, Missouri
63110, United States; orcid.org/0000-0002-8683-0026;
Email: john.walker@health.slu.edu
Authors
Napoleon D’Cunha − Department of Pharmacology and
Physiology, Saint Louis University School of Medicine, St.
Louis, Missouri 63110, United States
Mohammad Moniruzzaman − Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma
73072, United States
Keith Haynes − Department of Pharmacology and Physiology,
Saint Louis University School of Medicine, St. Louis, Missouri
63110, United States
Giuliano Malloci − Department of Physics, University of
Cagliari, Cagliari 09042, Italy; orcid.org/0000-0002-
5985-257X
Connor J. Cooper − Graduate School of Genome Science and
Technology, University of Tennessee, Knoxville, Tennessee
37996, United States; Biosciences Division, Oak Ridge
National Laboratory, Oak Ridge, Tennessee 37831, United
States; orcid.org/0000-0002-5527-9948
Enrico Margiotta − Department of Physics, University of
Cagliari, Cagliari 09042, Italy; orcid.org/0000-0003-
4790-021X
M
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Article
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Attilio V. Vargiu − Department of Physics, University of
Cagliari, Cagliari 09042, Italy; orcid.org/0000-0003-
4013-8867
Muhammad R. Uddin − Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma
73072, United States
Inga V. Leus − Department of Chemistry and Biochemistry,
University of Oklahoma, Norman, Oklahoma 73072, United
States
Feng Cao − John Cochran Division, Department of Veteran
Affairs Medical Center, St. Louis, Missouri 63106, United
States; orcid.org/0000-0002-4820-6628
Jerry M. Parks − Biosciences Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee 37831, United States;
orcid.org/0000-0002-3103-9333
Valentin V. Rybenkov − Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma
73072, United States; orcid.org/0000-0002-5300-4369
Paolo Ruggerone − Department of Physics, University of
Cagliari, Cagliari 09042, Italy; orcid.org/0000-0003-
0825-0824
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.1c00100
Author Contributions
∇N.D. and M.M. contributed equally to this work. G.M., J.M.P.,
P.R., A.V.V., V.V.R., J.K.W., and H.I.Z. designed the research
and wrote the manuscript. N.D. and K.H. synthesized the
compounds. M.M., I.V.L., and M.R.U. characterized compound
properties and activities. F.C. analyzed cytotoxicity of the
compounds. C.J.C., G.M., and E.M. performed docking and
computational analyses. All authors contributed to writing and
editing the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the National Institute of Health, AI052293
(H.I.Z.) and AI136799 (H.I.Z., G.M., E.M., A.V.V., P.R., V.V.R.,
and J.K.W.), who supported this work. G.M., E.M., A.V.V., and
P.R. acknowledge technical support by Giovanni Serra and
Andrea Bosin (University of Cagliari). C.J.C. was supported by a
National Science Foundation Graduate Research Fellowship
under Grant No. 2017219379. This research used resources of
the Compute and Data Environment for Science (CADES) at
Oak Ridge National Laboratory, which is supported by the
Office of Science of the U.S. Department of Energy under
Contract No. DE-AC05-00OR22725. We also gratefully
acknowledge Dr. Fahu He and the St. Louis University NMR
facility for acquiring 13C samples on the 700 MHz NMR.
(21) Levy, S. B.; Marshall, B. Antibacterial resistance worldwide:
causes, challenges and responses. Nat. Med. 2004, 10, S122−S129.
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Mediated Antibiotic Resistance in Gram-Negative Bacteria. Clin.
Microbiol. Rev. 2015, 28, 337.
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(24) Krishnamoorthy, G.; Tikhonova, E. B.; Dhamdhere, G.;
Zgurskaya, H. I. On the role of TolC in multidrug efflux: the function
and assembly of AcrAB−TolC tolerate significant depletion of
intracellular TolC protein. Mol. Microbiol. 2013, 87, 982−97.
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